B idging Nano echnology and Neu obiology:
Vol age-Sensing and Pho o he mal Con ol o
Neu al Ac i i y Using Semiconduc o and
Me allic Nanoc ys als
Thesis by
Zuzanna Lawe a
o he deg ee o
Doc o o Philosophy in Physics
Uni e sidad del País Vasco
Euskal He iko Unibe si a ea
Supe ised by
Ma ek G zelczak and Ra ael Yus e
Donos ia, No embe 2025
(cc) 2025 Zuzanna Lawe a (cc by-sa 4.0)
1
Abs ac
D i en by he need o minimally in asi e, high- esolu ion neu al in e aces, his disse a ion
b idges nano echnology and neu obiology by de eloping nanoscale ools o op ical de ec ion
and modula ion o neu al ac i i y. Wi h a ocus on semiconduc o quan um do s and plasmonic
gold nanoc ys als, his disse a ion p esen s e o s o de elop complemen a y pla o ms o
"Read" and "W i e" neu al signals. Designed o ei he epo changes in memb ane po en ial o
deli e ligh -d i en s imuli, hese nanoma e ials o e unc ional ad an ages a dimensions
inaccessible o con en ional bioelec onic app oaches.
Chap e 1 p o ides an in e disciplina y li e a u e e iew, in oducing he NanoNeu o, ou lining
how quan um do s and plasmonic nanopa icles can in e ac wi h neu al sys ems and
highligh ing key challenges such as a ge ed deli e y, biocompa ibili y, and in eg a ion wi h
li ing cells. I p o ides an o e iew o he cu en s a e o he a and si ua es he objec i e o
he p esen ed esea ch wi hin his amewo k.
Chap e 2 desc ibes an expe imen al pla o m o gene ically enginee ed “sel -spiking” HEK293
cells ha i e spon aneous, synch onized ac ion po en ials. This in i o wo kho se model,
alida ed ia calcium imaging, p o ided a simpli ied ye consis en es bed o e alua ing
nanoscale ol age-sensi i e p obes.
A he hea o he disse a ion, Chap e 3 de ails he de elopmen o a quan um do -based
ol age senso . Sphe ical quan um well nanoc ys als we e syn hesized and cha ac e ized using
spec oscopic and elec on mic oscopy echniques o con i m hei physicochemical p ope ies.
To adap he nanoc ys als o biological en i onmen s, hei su aces we e enginee ed o
aqueous s abili y and inco po a ed in o cell memb anes using usogenic liposomes. Thei abili y
o uni o mly label he plasma memb ane was assessed by luo escence mic oscopy in sel -
spiking HEK293 monolaye s. Al hough he quan um do s emained pho os able, luo escence
changes du ing ac ion po en ials emained below he h eshold equi ed o eliable de ec ion.
The absence o clea spike-co ela ed signals unde sco ed limi a ions in sensi i i y and empo al
esolu ion o came a, poin ing o he need o b igh e p obes, as e imaging, and imp o ed
signal- o-noise pe o mance.
In pa allel, Chap e 4 in es iga es pho o he mal modula ion o neu ons wi h plasmonic
nanoc ys als. Gold nanopa icles o con olled size and mo phology (sphe es and bipy amids)
we e syn hesized and cha ac e ized by spec oscopy and ansmission elec on mic oscopy o
2
de e mine hei op ical p ope ies and s uc u al uni o mi y. Following su ace unc ionaliza ion
o ensu e biological compa ibili y, nanoc ys als we e in oduced in o cell cul u es (SH-SY5Y and
co ex p ima y neu ons). Thei associa ion wi h cells and in e naliza ion was assessed using
con ocal luo escence mic oscopy and elec on mic oscopy. Upon op ical s imula ion, he
nanopa icles con e ed inciden ligh in o localized hea ing. In a p oo -o -concep expe imen
pe o med in b ain slices, his e ec was su icien o elici neu onal i ing: single ac ion
po en ials we e induced a mode a e exci a ion powe , and bu s s o ac i i y a highe
in ensi ies. These indings suppo he easibili y o plasmonic nanoc ys als as op ically
add essable ac ua o s o con olled neu omodula ion.
Chap e 5 summa izes he main indings and e lec s on echnological limi a ions and u u e
di ec ions o he wo k. The expe imen al esea ch p esen ed in his disse a ion was ca ied ou
by he au ho a he Cen o de Física de Ma e iales in Donos ia, Spain (Ma ek G zelczak’s g oup),
and complemen ed by esea ch s ays a he Neu o echnology Cen e a Columbia Uni e si y in
New Yo k (Ra ael Yus e’s g oup), he Depa men o Chemis y a Columbia Uni e si y (Jona han
Owen’s g oup), and he Fluo oNanoTools Labo a o y a he Bio isika Ins i u e in Bilbao, Spain
(Monica Ca il’s g oup). These esea ch s ays o med he ounda ion o he expe imen al wo k
desc ibed in his hesis. The b oade collabo a i e amewo k ex ended beyond hese ou
labo a o ies and included membe s o he IKUR Basque NanoNeu o Ne wo k (B3N), ein o cing
he in e disciplina y na u e o he p ojec . Al hough some ou comes, pa icula ly in ol age
sensing, did no ully mee ini ial expec a ions, he wo k con ibu ed o a deepe unde s anding
o nanoma e ial-cell in e ac ions and cla i ied key design pa ame e s o u u e ool
de elopmen . The insigh s gained p o ide a ounda ion o mo e a ge ed and in o med
ad ancemen o neu al in e aces based on nanoc ys als and nanopa icles.
3
Table o Con en s
CHAPTER 1 – Nano Neu o
Objec i e ....................................................................................................................................... 8
1. Neu obiology ......................................................................................................................... 9
1.1. Neu al Ne wo ks and B ain Func ion ............................................................................ 9
1.2. Cu en Me hods o Bioimaging .................................................................................. 14
1.3. Expe imen al Models o he Human Ne ous Sys em ................................................ 18
2. Nano echnology .................................................................................................................. 22
2.1. Semiconduc o Nanoc ys als....................................................................................... 24
2.2. Plasmonic Nanopa icles ............................................................................................. 28
3. Nano Neu o ......................................................................................................................... 34
4. Re e ences o Chap e 1 .................................................................................................... 47
CHAPTER 2 – Sel -spiking HEK Cells
1. C ea ion and Main enance o he Cell Line ......................................................................... 62
1.1. Cell Cul u e .................................................................................................................. 65
2. Calcium Imaging o Spiking HEK Cells .................................................................................. 67
2.1. Fluo escence Mic oscope Se up ................................................................................. 67
2.2. Resul s and Discussion ................................................................................................ 70
3. Re e ences o Chap e 2 .................................................................................................... 78
CHAPTER 3 – Quan um Do s o De ec ing Neu onal Ac i i y
1. Sphe ical Quan um Wells .................................................................................................... 82
1.1. O e iew o Quan um-do -based Pla o ms ............................................................... 86
1.2. Syn he ic App oaches .................................................................................................. 88
1.3. Su ace Chemis y on CdS/CdSe/CdS Nanoc ys als .................................................... 94
2. S a egies o Wa e T ans e .............................................................................................. 96
2.1. OCG Micelles ............................................................................................................... 97
2.2. Lipid Vesicles ............................................................................................................. 102
3. Cell Memb ane Inse ion .................................................................................................. 120
3.1. OCG Micelles ............................................................................................................. 120
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3.2. Fusosomes – Fusogenic Lipid Vesicles ...................................................................... 127
4. Reco dings o Spiking HEK Cells wi h Sphe ical Quan um Do s ........................................ 153
5. Chap e Summa y.............................................................................................................. 187
6. Re e ences o Chap e 3 .................................................................................................. 188
CHAPTER 4 – Plasmonic Nanoc ys als o Pho o he mal Modula ion o Neu onal Ac i i y
1. Syn hesis o Gold Nanoc ys als ......................................................................................... 196
1.1. Syn hesis o Ci a e-S abilized Gold Nanopa icles (Au NPs@ci a e) ...................... 197
1.2. Syn hesis o Size-Con olled Sphe ical Gold Nanopa icles (Au NPs@CTAB) ........... 199
1.3. Seed-Media ed Syn hesis o Gold Bipy amids .......................................................... 202
2. Bio unc ionaliza ion o Gold Nanoc ys als ........................................................................ 205
3. Gold Nanoc ys als in Neu obiological Resea ch ............................................................... 209
3.1. Physicochemical Cha ac e iza ion o NPs in Biological Media .................................. 209
3.2. In Vi o In e naliza ion S udies.................................................................................. 214
3.3. In Vi o Op ical Modula ion o Neu onal Ac i i y wi h Au NPs ................................. 220
4. Re e ences o Chap e 4 .................................................................................................. 223
CHAPTER 5 – Summa y and Ou look
1. Summa y o Majo Findings and Conclusions ................................................................... 228
2. Me hodological Re inemen o Fu u e S udies ............................................................... 229
3. Closing Rema ks and Field Ou look ................................................................................... 238
4. Re e ences o Chap e 5 .................................................................................................. 239
APPENDIX
1. Lis o Abb e ia ions .......................................................................................................... 242
2. Supplemen a y Ma e ial o Chap e #2 ........................................................................... 244
2.1. Ma e ials ................................................................................................................... 244
2.1.1. Reagen P epa a ion - Recipes .............................................................................. 245
2.2. Me hods .................................................................................................................... 246
2.2.1. Cell Cul u e P o ocols ............................................................................................ 246
2.2.2. Calcium Imaging .................................................................................................... 247
2.2.3. Fluo escence Mic oscope Se up ........................................................................... 247
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2.3. Addi ional Figu es...................................................................................................... 249
3. Supplemen a y Ma e ial o Chap e #3 ........................................................................... 252
3.1. Ma e ials ................................................................................................................... 252
3.1.1. Reagen P epa a ion - Recipes .............................................................................. 252
3.2. Me hods .................................................................................................................... 254
3.2.1. Ins umen a ion .................................................................................................... 254
3.2.2. C yoTEM ................................................................................................................ 254
3.2.3. Con ocal Mic oscope ............................................................................................. 254
3.2.4. Fluo escence Mic oscope ...................................................................................... 255
3.3. Addi ional Figu es...................................................................................................... 256
4. Supplemen a y Ma e ial o Chap e #4 ........................................................................... 263
4.1. Ma e ials ................................................................................................................... 263
4.2. Me hods .................................................................................................................... 263
4.2.1. P o ocols................................................................................................................ 263
4.2.2. Ins umen a ion ...................................................................................................... 266
4.2.3. Con ocal Imaging o Fixed Cells ............................................................................. 266
4.2.4. T ansmission Elec on Mic oscopy (TEM) o Fixed Cells ....................................... 267
4.3. Addi ional Figu es...................................................................................................... 267
5. Da a and Code Reposi o y ................................................................................................. 272
6. Documen a ion o Digi al Tools ........................................................................................ 272
6.1. Da a Analysis ............................................................................................................. 272
6.2. Figu e P epa a ion ..................................................................................................... 272
6.3. Assis ed Edi ing and Li e a u e Sea ch ...................................................................... 272
Acknowledgmen s ..................................................................................................................... 273
6
7
Chap e 1
Nano Neu o
Chap e 1. Nano Neu o
8
Objec i e
The human b ain, he mos complex o gan known, emains one o he g ea es scien i ic
challenges. To un a el he p ocesses ha go e n beha io and cogni ion, i is essen ial o
deepen ou unde s anding o hei p ima y d i e : he cen al ne ous sys em. Neu obiology,
which in es iga es he s uc u e and unc ion o neu al sys ems, has yielded ounda ional
insigh s in o pe cep ion, memo y, and disease, ca alyzing b eak h oughs in he diagnosis and
ea men o neu ological diso de s. The impac o hese disco e ies ex ends a beyond
medicine, shaping ields as di e se as a i icial in elligence and public policy. Ye , as ou scien i ic
ques ions g ow inc easingly p ecise, so oo mus he ools we employ.1
The compu a ional capabili ies o b ain is d i en by nanoscale machine y: ion channels jus a
ew nanome es wide ha ga e millisecond elec ical spikes, and synapses ha comp ess
housands o molecula componen s in o olumes smalle han a em oli e.2,3 Con en ional
ools such as elec odes and luo escen dyes lack he empo al esolu ion and speci ici y o ully
access o modula e hese mic oscopic dynamics, lea ing key biological p ocesses obscu ed. This
gap ma ks he eme gence o Nano Neu o, a ield a he in e sec ion o nanoscience and
neu obiology, which o e s unp eceden ed oppo uni ies o p ecision neu al in e acing.
Le e aging he dis inc i e elec onic, op ical, and biochemical p ope ies o nanoma e ials,
esea che s can now sense, manipula e, and s udy neu al ci cui s a scales p e iously
inaccessible.1
The p omise o Nano Neu o is p o ound: o e eal hidden pa e ns o neu al ac i i y, enable
localized neu omodula ion, and d i e inno a ion in bo h he apeu ic in e en ion and neu al
enhancemen . None heless, he ield aces o midable challenges, including he need o p ecise
a ge ing, long- e m biocompa ibili y, and minimally in asi e in eg a ion wi h li ing issue.
Chap e 1 es ablishes he concep ual and echnical ounda ion o in oducing Nano Neu o. I
begins wi h a e iew o essen ial neu obiological p inciples, ollowed by an in oduc ion o
nano echnology and he ma e ials mos ele an o neu al applica ions. Wi h his g oundwo k
in place, we examine he con e gence o hese disciplines, highligh ing cu en echnologies,
landma k s udies, and eme ging di ec ions. The goal is o si ua e he wo k p esen ed in his
disse a ion wi hin i s b oade scien i ic con ex and highligh he ounda ional ques ions i
seeks o add ess: eading and w i ing neu al ac i i y.
Chap e 1. Nano Neu o
15
bioimaging and elec ophysiological me hods ha con inue o expand ou abili y o obse e and
in e p e neu al ac i i y. As unde s anding he b ain equi es no only iden i ying i s componen s
bu also eco ding and isualizing hei dynamic in e ac ions, he de elopmen o imaging
echnologies has become cen al o neu oscience – ans o ming i om a desc ip i e discipline
in o one capable o eal- ime, mul iscale in e oga ion o neu al ac i i y.30
Cu en me hods o eco ding b ain ac i i y all in o wo main ca ego ies: elec ical and op ical
echniques, each wi h dis inc ad an ages in empo al and spa ial esolu ion (Figu e 1.3).31
Elec ical app oaches di ec ly measu e neu onal ol age changes. A he single-cell le el, pa ch-
clamp eco ding o e s high empo al p ecision and is used o s udy ion channel ac i i y.32 Fo
popula ion-le el eco dings, mul ielec ode a ays (MEAs) cap u e signals om dozens o
hund eds o neu ons simul aneously,33,34 while high-densi y p obes such as Neu opixels allow
eco dings om housands o si es ac oss b ain egions in beha ing animals.35,36 Op ical me hods
ely on ansla ing neu al signals in o ligh , using ei he chemical dyes o gene ically encoded
indica o s. Calcium imaging, one o he mos widely used echniques, de ec s in acellula
calcium ansien s associa ed wi h neu onal i ing.37 Gene ically encoded calcium indica o s
(e.g., GCaMP a ian s) enable long- e m imaging in speci ic cell ypes wi h cellula esolu ion.37
Vol age imaging, hough echnically mo e challenging, uses indica o s such as ASAP (Accele a ed
Senso o Ac ion Po en ial) o A chon o di ec ly ack changes in memb ane po en ial wi h
imp o ed empo al ideli y.38–40 Addi ionally, luo escen biosenso s ha e been de eloped o
isualize neu o ansmi e elease (e.g., iGluSnFR o glu ama e) and in acellula signaling
pa hways.41–44 These echniques can be in eg a ed wi h ad anced mic oscopy me hods, o
achie e deep issue access and high- esolu ion eco dings. On he sys ems le el, unc ional
magne ic esonance imaging ( MRI), elec oencephalog aphy (EEG) and
magne oencephalog aphy (MEG) p o ide non-in asi e measu emen s o b ain ac i i y wi h
whole-b ain co e age, o e ing impo an con ex o linking cellula ac i i y o beha io .45,46
Chap e 1. Nano Neu o
16
Figu e 1.3 O e iew o elec ical and op ical neu onal eco ding echniques. Elec ical me hods ( op):
Pa ch-clamp – high‐p ecision, single‐cell measu emen s o ion channel and memb ane dynamics;
Mul ielec ode a ays (MEAs) – simul aneous eco dings om ens o hund eds o neu ons in i o o ex
i o; Neu opixels p obes – high‐densi y, housands‐si e in i o eco dings ac oss b ain egions. Op ical
me hods (bo om): Chemical indica o s (e.g., Ca²⁺ dyes) – epo in acellula calcium ansien s linked o
spiking; Gene ically encoded indica o s – cell ype-speci ic luo escence epo e s o calcium (GCaMP),
memb ane ol age (ASAP/A chon), and neu o ansmi e elease (e.g., iGluSnFR).
Calcium Imaging
Calcium imaging is a cen al echnique in mode n neu oscience, p o iding means o moni o
neu onal ac i i y by isualizing in acellula calcium dynamics ha a e igh ly coupled o ac ion
po en ial i ing and a as a ay o cellula p ocesses.47 Cells a es main ain a e y low
in acellula calcium concen a ion o a ound 50-100 nM, bu his le el can ansien ly inc ease
C C C
C C
C C C C C C
C
Elec ode
C
C C
C C
Synapse
Molecula
Cage
Depola iza on
Ca2
Chap e 1. Nano Neu o
17
o app oxima ely 1 µM upon ac i a ion, wi h localized "ho -spo s" nea ac i e channels
po en ially eaching ens o mic omola .48 The di e si y o cellula p ocesses egula ed by
calcium a ises om he e sa ili y o i s signaling dynamics, anging in speed, ampli ude, and
spa ial o empo al dis ibu ion. As a esul , he choice o calcium imaging ools mus be ca e ully
ma ched o he speci ic cha ac e is ics o he p ocess unde s udy, as di e en indica o s a y in
hei kine ics, sensi i i y, and spa ial esolu ion.48 The unde lying p inciple o he echnique
in ol es de ec ing ee in acellula calcium ions h ough hei binding o senso molecules ha
unde go a measu able change in luo escence upon calcium binding.49
Calcium indica o s a e b oadly classi ied in o wo ypes: syn he ic small-molecule dyes (chemical
indica o s) and gene ically encoded calcium indica o s (GECIs).50 GECIs, such as he commonly
used GCaMP amily, a e p o ein-based senso s ha can be in oduced ia i al ec o s o
exp essed in ansgenic animals. The basic design o en in ol es a luo escen p o ein used o
he calcium-binding p o ein calmodulin and a calmodulin-binding pep ide (M13).31 They enable
long- e m imaging in speci ic cell ypes, o e ing excellen spa ial esolu ion and compa ibili y
wi h in i o mic oscopy.31,51 Thei main limi a ions include slowe kine ics compa ed o chemical
dyes and po en ial bu e ing e ec s on in acellula calcium, which can a ec he ideli y o spike
de ec ion – pa icula ly du ing high- equency ac i i y.49 Addi ionally, deli e y ia i al
ansduc ion o in u e o elec opo a ion is in asi e and can lead o he e ogeneous cellula
labeling o issue damage.49
Chemical calcium indica o s o e a aluable al e na i e o gene ically encoded senso s,
pa icula ly in expe imen s ha demand apid kine ics and high empo al esolu ion.52 These
syn he ic luo escen dyes a e ypically de i ed om calcium chela o s such as EGTA o BAPTA,
chemically modi ied o include luo escen epo e g oups.47 Thei as -binding kine ics and
s ong luo escence esponses p o ide highe signal- o-noise a ios, making hem well-sui ed o
de ec ing b ie calcium ansien s, such as hose associa ed wi h high- equency neu onal i ing
o localized e en s in dend i ic spines. Unlike GECIs, chemical indica o s do no equi e gene ic
manipula ion; ins ead, hey a e in oduced in o cells as memb ane-pe meable ace oxyme hyl
(AM) es e s, which a e ac i a ed by in acellula es e ases o elease he ac i e dye.50 Ea ly
examples such as Fu a-2 allowed o a iome ic calcium measu emen s, while single-
wa eleng h indica o s like Fluo-4 and Calcium G een-1 became widely used due o hei s ong
luo escence changes upon calcium binding and compa ibili y wi h s anda d exci a ion
wa eleng hs.47 Al hough his me hod allows o apid and widesp ead labeling, i lacks cell- ype
speci ici y and can lead o a iabili y in loading, dye ex usion, o compa men aliza ion. Despi e
hese limi a ions, chemical calcium indica o s emain essen ial o acu e p epa a ions whe e
Chap e 1. Nano Neu o
18
high-speed, high- esolu ion imaging is equi ed, including s udies in b ain slices and a
subcellula compa men s such as synap ic e minals.51
These indica o s comp ise a la ge oolki o e ing a b oad ange o calcium a ini ies and
exci a ion/emission spec a, enabling esea che s o ailo imaging p o ocols o speci ic
expe imen al needs, om low- o high-calcium en i onmen s and ac oss a ious op ical se ups.
While such ad ances con inue o expand he capabili ies o calcium imaging, he ocus o his
disse a ion is on chemical calcium imaging using Calcium G een-1, chosen o i s sensi i i y, as
kine ics, and sui abili y o high- esolu ion imaging in i o.
1.3. Expe imen al Models o he Human Ne ous Sys em
The human ne ous sys em is complex, wi h billions o neu ons in e connec ed by illions o
synapses.53 Di ec s udy o his sys em in humans is cons ained by signi ican e hical and
echnical challenges, limi ing he ex en o which we can expe imen ally manipula e o obse e
neu al p ocesses in i o.54 To add ess hese cons ain s, esea che s employ model sys ems ha
cap u e essen ial ea u es o he ne ous sys em while o e ing g ea e accessibili y and
expe imen al con ol.
Many ea u es o neu ons and ne wo ks a e conse ed ac oss species. Fundamen al p ope ies
o neu ons and ne wo ks obse ed in humans a e o en mi o ed in model o ganisms such as
non-human p ima es,55–57 oden s (mice, a s and squi els),58–60 zeb a ish,61 ui ly62,63 and
e en hyd ozoans like Hyd a.64,65 Relying on animal s and-ins o explo e human physiology is a
adi ion oo ed in ancien G eek medicine and ca ied in o mode n esea ch.66 Each occupies a
dis inc in es iga i e niche: p ima es enable analyses o highe -o de cogni ion and ine mo o
con ol; oden s p o ide excep ional gene ic ac abili y o co ical and beha io al s udies;
zeb a ish allow whole-b ain op ical access h oughou de elopmen ; and he simple di use
ne e ne o Hyd a o e s a minimalis amewo k o explo ing he e olu iona y o igins o
coo dina ed neu al ac i i y.54,67–69
Beyond e olu iona y inqui y, model sys ems se e as essen ial pla o ms o ansla ional
esea ch.70 Many neu ological diso de s – anging om neu odegene a i e diseases like
Alzheime 's and Pa kinson's o condi ions in ol ing abe an exci abili y such as epilepsy –
canno be ully unde s ood h ough human clinical da a alone. An ex ensi e epe oi e o
expe imen al models, om cul u ed neu onal cells o gene ically modi ied animals, p o ides he
ounda ional ools o explo ing he s uc u e and unc ion o he human ne ous sys em. These
models no only o e insigh in o basic biological ques ions bu also acili a e he de elopmen
Chap e 1. Nano Neu o
19
o diagnos ic and he apeu ic s a egies, o ming a c i ical b idge be ween expe imen al
neu oscience and clinical applica ion (Figu e 1.4).57,64
One way o s udy he ne ous sys em is o isola e i s building blocks – he cells – and examine
hem in con olled labo a o y condi ions (in i o). This anges om immo al cell lines o ac ual
neu ons kep ali e ou side he body. These models le esea che s obse e molecula and
cellula p ocesses up close, ee om he complexi y o a whole o ganism.70,71
Immo alized cell lines a e cells ha can g ow inde ini ely in he lab. Though hey a e no
neu ons, cell lines like HEK293 (human emb yonic kidney cells) o HeLa (human cell line de i ed
om cance cells) a e in aluable o neu obiological esea ch.72 Scien is s o en use hese cells
as hos sys ems o s udy speci ic neu al p o eins. Fo example, HEK293 cells a e widely used o
p oduce and analyze neu onal ion channels o ecep o s by inse ing he ele an genes.73
While hese cell lines do no i e impulses o o m ne wo ks like eal neu ons, hey o e a high-
h oughpu , easy- o-main ain pla o m o dissec molecula de ails. By con as , p ima y
neu ons a e ne e cells aken di ec ly om an animal’s b ain o spinal co d and g own in a dish.
A common example is oden hippocampal neu ons cul u ed om newbo n a s o mice. These
p ima y neu on cul u es do o m synapses and elec ical ac i i y, essen ially c ea ing a minia u e
neu al ne wo k in he dish. Resea che s use hem o s udy synap ic communica ion,
de elopmen o neu al connec ions, and he e ec s o d ugs o gene ic changes on neu on
beha io . Because hey a e eal neu ons, p ima y cul u es mo e closely mimic b ain unc ion
han cell lines. The downside is ha p ima y neu ons a e delica e and ha e a ini e li espan in
i o. None heless, hey ha e been a wo kho se o s udying mechanisms o neu ode elopmen
and neu oplas ici y a he cellula le el ( o example, unco e ing how synapses s eng hen o
weaken du ing lea ning-like p ocesses).70
Ex ending he elemen al powe o p ima y cul u es o he ne wo k scale, b ain-slice
p epa a ions main ain au hen ic synap ic a chi ec u e in a ully manipulable ex- i o se ing.
In oduced by Hen y McIlwain in he 1950s,74 his p epa a ion – main ained in p ecisely de ined
a i icial ce eb ospinal luid – p o ed i s wo h ea ly on by demons a ing no mal es ing
po en ials, in ac synap ic ansmission, and s imula ion-e oked me abolic shi s. Today,
hippocampal slices emain he benchma k o dissec ing long- e m po en ia ion and o he o ms
o synap ic plas ici y, whe eas co ical slices suppo eal- ime imaging o ne wo k oscilla ions
and high- h oughpu pha macological sc eening.75 Thei ela i e “quie ude,” combined wi h
unobs uc ed op ical and elec ode access o dend i es and axons, enables mechanis ic analyses
– om ion-channel kine ics o ci cui -le el disease models – ha a e imp ac ical in i o. B ain-
Chap e 1. Nano Neu o
20
slice wo k hus occupies a pi o al middle g ound be ween cell cul u es and whole-animal
s udies, o e ing a cos -e ec i e, e hically a o able pla o m ha con inues o d i e mode n
neu obiology.
A e olu iona y ad ance in he las decade has been he use o s em cells o model he human
b ain. S em cells (especially induced plu ipo en s em cells, iPSCs, made om adul human cells)
can be guided o de elop in o neu al cells. F om hese, scien is s can g ow 3D b ain o ganoids –
iny, pea-sized blobs o issue ha sel -o ganize in o laye s and cell ypes eminiscen o a
de eloping b ain. These o ganoids a e no ull b ains, bu hey can mimic speci ic aspec s o he
human ne ous sys em, such as o ming co ical-like s uc u es o e en p imi i e eye cups in a
dish. This app oach allows us o s udy human neu al de elopmen and disease in i o. Fo
ins ance, esea che s ha e c ea ed pa ien -speci ic b ain o ganoids o in es iga e mic ocephaly
(a de elopmen al b ain diso de ) and ound ha ce ain gene ic mu a ions o i uses (like Zika
i us) can s un o ganoid g ow h, e lec ing he pa ien ’s condi ion. O ganoids hus se e as
expe imen al a a a s o a human b ain, enabling es s o d ug ea men s o gene ic co ec ions
on pa ien -de i ed issue. The e a e also s uc u es called assembloids, whe e mul iple
o ganoids (e.g., co ical and spinal co d, o b ain and muscle) a e used o s udy in e ac ions
be ween egions, and e en expe imen s ansplan ing human o ganoid issue in o animal b ains
( o obse e how human neu ons in eg a e in i o). While s ill a de eloping a ea, hese s em
cell–de i ed models a e a majo s ep owa d b idging he gap be ween animal models and he
human b ain i sel , especially o unde s anding human-speci ic neu ode elopmen al diso de s
and e olu ion.76,77
Chap e 1. Nano Neu o
21
Figu e 1.4 Spec um o expe imen al models used in neu oscience. This cha o ganizes model sys ems by
biological complexi y ( e ical axis) and esea ch ocus, anging om basic esea ch o clinical applica ions
(ho izon al axis). Models a e ca ego ized as in i o o in i o. In i o sys ems include immo alized cell
lines, p ima y neu ons, s em-cell–de i ed p epa a ions, and b ain slices ob ained om animals. In i o
models span om simple in e eb a es (e.g., Hyd a, ui ly, cu le ish) o aqua ic e eb a es
(e.g., zeb a ish), oden s, non-human p ima es, and humans. These a e selec ed examples om a b oade
a ay o expe imen al models used in neu oscience.
C
C
C
C
C
C
C C C
C
Chap e 1. Nano Neu o
22
2. Nano echnology
Since he de il is said o dwell in he de ails, mode n ma e ials science looks downwa d – owa d
a oms, molecules, and nanoscale s uc u es. Nano echnology is he in e disciplina y ield ha
ocuses on he design, syn hesis, and cha ac e iza ion o s uc u es ypically anging om 1 o
100 nanome e s. A his scale, ma e ials o en display p ope ies ha di e signi ican ly om
hei bulk coun e pa s. These di e ences a ise p ima ily om wo ac o s:
(1) Su ace e ec s, due o a high su ace a ea- o- olume a io, whe e a la ge p opo ion o
a oms eside on he su ace wi h ewe neighbo ing a oms. This can educe cohesi e ene gy,
lowe mel ing poin s (e.g., 2.5 nm gold pa icles mel a much lowe empe a u es han bulk
gold), and enhance chemical eac i i y. Ca aly ic ac i i y, o ins ance, o en inc eases a he
nanoscale due o he abundance o ac i e su ace si es.78,79
(2) Quan um e ec s, which become ele an when pa icle dimensions app oach he
cha ac e is ic leng h scales o elec ons (e.g., he exci on Boh adius). In such cases, ene gy
le els become quan ized, leading o new op ical, elec onic, and magne ic beha io s. Fo
example, while bulk pla inum is non-magne ic, 2 nm clus e s can exhibi magne ism. Simila ly,
semiconduc o nanopa icles show size-dependen bandgaps, esul ing in a blue shi in op ical
abso p ion.78,79
Toge he , hese su ace and quan um e ec s gi e ise o he unique mechanical, he mal,
elec onic, op ical, and ca aly ic p ope ies.80 Nanoma e ials a e commonly classi ied by hei
dimensionali y, acco ding o he numbe o deg ees o eedom in he pa icle momen um.
While nanopa icles – s uc u es wi h all h ee dimensions in he 1–100 nm ange – a e
conside ed ze o-dimensional (0D) due o hei oughly iso opic shape, se e al o he o ms o
nanoma e ials exis .78,81
One-dimensional (1D) ca ego y o nanoma e ials includes nano ubes (e.g., ca bon nano ubes),
nano ods, nanowi es, nano ibe s, and nanoho ns. Elec ons in hese s uc u es a e ypically
con ined in wo dimensions, allowing ee mo emen along he leng h. Two-dimensional (2D)
nanoma e ials ha e nanoscale hickness bu ex ended la e al dimensions. Examples include
nanoshee s, nano ilms, nanolaye s, and g aphene-de i ed ma e ials.23 These s uc u es con ine
elec ons in one dimension. Th ee-dimensional (3D) nanoma e ials e e o bulk assemblies o
s uc u es ha a e no con ined o he nanoscale in any single dimension bu a e composed o
nanoscale building blocks (e.g., a ays o nanopa icles, nanowi es, o nano ubes).78,82
Chap e 1. Nano Neu o
23
Nanoma e ials can be syn hesized using wo gene al s a egies: op-down and bo om-up
app oaches.79,80 Top-down me hods s a wi h bulk ma e ials ha a e physically o mechanically
educed o he nanoscale, using echniques such as high-ene gy ball milling, li hog aphy, lase
abla ion, o a c discha ge.82,83 These me hods a e well-sui ed o la ge-scale p oduc ion bu
o en esul in b oade size dis ibu ions and po en ial s uc u al de ec s. In con as , bo om-
up app oaches assemble nanos uc u es a om-by-a om o molecule-by-molecule h ough
chemical o biological p ocesses.79 Examples include chemical apo deposi ion (CVD), sol-gel
syn hesis, co-p ecipi a ion, and colloidal chemical educ ion. Bo om-up echniques gene ally
o e be e con ol o e pa icle size, shape, and c ys allini y. In many cases, a combina ion o
bo h app oaches is employed o op imize s uc u e and unc ionali y, depending on he ma e ial
sys em and applica ion.84,85
As his hesis cen e s on colloidal nanopa icles, he subsequen sec ions will explo e his class
o nanoma e ials in mo e de ail. Nanopa icles can be classi ied in a ious ways; one use ul
app oach is o g oup hem based on hei chemical composi ion and he dominan physical
phenomena ha go e n hei beha io a he nanoscale:
Semiconduc o quan um do s (QDs) a e semiconduc o nanoc ys als, ypically below 10 nm in
diame e , o en composed om CdSe, CdTe, o InP. QDs a e used in biological imaging, ligh -
emi ing de ices, sola cells, and quan um elec onics.79,86,87
Plasmonic me al nanopa icles, commonly made o gold (Au) o sil e (Ag), bu also non-noble
me als such as coppe (Cu), aluminum (Al) o nickel (Ni), exhibi localized su ace plasmon
esonance (LSPR) – he collec i e oscilla ion o conduc ion elec ons induced by inciden ligh .
This leads o s ong, size- and shape-dependen op ical abso p ion and sca e ing. Common
applica ions a e biosensing, diagnos ics, ca alysis, and su ace-enhanced spec oscopy.84,88,89
Magne ic nanopa icles, such as i on oxides (Fe3O4, Fe2O3) o FeP alloys, can exhibi
supe pa amagne ism a sizes below he single-domain limi . These pa icles espond s ongly o
ex e nal magne ic ields bu exhibi no emanen magne iza ion, making hem ideal o use in
MRI con as agen s, magne ic hype he mia, d ug deli e y, and magne ic sepa a ion
echnologies.90,91
Ca bon-based nanoma e ials include se e al s uc u ally dis inc sys ems. Fulle enes (e.g., C60)
a e sphe ical ca bon cages wi h high elec on a ini y. Ca bon nano ubes (CNTs) a e olled
g aphene shee s wi h excep ional mechanical s eng h and elec ical conduc i i y, a ailable in
single- o mul i-walled o ms. Ca bon quan um do s (CQDs) and ca bon do s (CDs) a e quasi-
sphe ical pa icles below 10 nm, wi h size- o de ec -dependen pho oluminescence. These
Chap e 1. Nano Neu o
24
ma e ials a e applied in bioimaging, sensing, ene gy s o age, and en i onmen al
echnologies.78,82,92
Ce amic nanopa icles, such as i anium dioxide (TiO₂) and zinc oxide (ZnO), a e ino ganic, non-
me allic ma e ials wi h high he mal s abili y and chemical esis ance. Depending on syn hesis
condi ions, hey may be amo phous o c ys alline, dense o po ous. They a e widely used in
ca alysis, biomedical coa ings, pho onics, and ene gy con e sion de ices.93,94
Lipid-based nanopa icles – including liposomes and solid lipid nanopa icles (SLNs) – a e
ypically sphe ical and ange om 10 o 1000 nm. Thei amphiphilic composi ion enables
encapsula ion and con olled elease o he apeu ic agen s, making hem cen al o d ug
deli e y, imaging, and diagnos ic sys ems.90,95,96
Polyme ic nanopa icles a e o med om syn he ic o na u al polyme s and ange om 1 o
1000 nm. They can ca y ac i e subs ances ei he on hei su ace o wi hin a polyme ma ix. A
no able subclass, conjuga ed polyme nanopa icles (CPNs) o polyme do s (Pdo s), displays
high b igh ness and unable pho ophysical p ope ies, making hem sui able o imaging and
biosensing.90,97
Nanopa icles exhibi a b oad di e si y in shape, size, and in e nal s uc u e – anging om
sphe ical, od-like, cylind ical, ubula , and hollow-co e geome ies o chi al o ms; hey may be
c ys alline (single- o mul i-domain), amo phous, uni o m, o composed o mul iple laye s –
e lec ing a apidly expanding lib a y o ma e ials ha con inues o g ow as syn hesis me hods
ad ance.78,98
2.1. Semiconduc o Nanoc ys als
O e he pas decade, quan um do (QD) esea ch has e ol ed om undamen al s udies in
nanochemis y in o a b oad echnology pla o m suppo ing ad ances in displays, ligh ing,
pho o ol aics, quan um in o ma ion sys ems, and bioanaly ics. The ield began in he ea ly
1980s, when Ekimo and B us independen ly obse ed ha semiconduc o nanoc ys als exhibi
size-dependen op ical p ope ies due o quan um con inemen . In 1993, Bawendi and
co-wo ke s in oduced a eliable syn he ic ou e p oducing highly uni o m colloidal QDs wi h
unable emission, which ca alyzed apid p og ess in he ield. Con inued inno a ions in
composi ion, shape, and su ace chemis y ha e since enabled QDs wi h high b igh ness,
s abili y, and spec al con ol. In ecogni ion o hese con ibu ions, Ekimo , B us, and Bawendi
Chap e 1. Nano Neu o
31
he syn hesis o di e se aniso opic mo phologies, including high-aspec - a io ods, bipy amids,
and nanos a s wi h sha p p o usions, whose s uc u al ea u es gi e ise o na ow spec al
linewid hs and unable op ical p ope ies.124 Mo e complex a chi ec u es, including nanoshells
and nanocages, a e ypically syn hesized ia empla ing app oaches, such as gold deposi ion on
dielec ic co es (e.g., silica) o gal anic eplacemen eac ions using sil e empla es.127,128
A key ad an age o gold nanoc ys als lies in hei amenabili y o su ace modi ica ion.121,141 Thei
s ong a ini y o hiol g oups allows s aigh o wa d unc ionaliza ion wi h polyme s,
biomolecules, o o he a ge ing ligands.127,129,142,143 Fo biological applica ions, polye hylene
glycol (PEG) is commonly g a ed o enhance colloidal s abili y and educe non-speci ic
in e ac ions wi h biological componen s.127,131 Speci ici y can be u he in oduced h ough
conjuga ion wi h an ibodies, pep ides, o nucleic acids138,144. In many cases, he emo al o
exchange o oxic su ac an s (such as CTAB) is necessa y p io o in i o use.145,146 Ligand
exchange p o ocols, combined wi h su ace cha ac e iza ion echniques like ζ-po en ial
measu emen s, FTIR (Fou ie ‐T ans o m In a ed Spec oscopy), o XPS (X- ay Pho oelec on
Spec oscopy), a e essen ial o con i m he chemical composi ion and s abili y o he inal
nanos uc u e.
These ea u es make gold nanoc ys als highly e sa ile o biomedical and sensing
applica ions.130,131,138 In pho o he mal he apy, NIR- esonan pa icles – such as nano ods, shells,
o b anched s uc u es – e icien ly con e ligh in o localized hea o abla e a ge ed issue
wi h minimal in asi eness.146,147 In pho oacous ic imaging, pulsed lase exci a ion o AuNPs
gene a es s ong ul asonic signals o deep- issue imaging.121,122 Su ace-enhanced Raman
sca e ing (SERS) exploi s local ield enhancemen s o ul a-sensi i e molecula de ec ion, while
simple agg ega ion-based assays enable apid, colo ime ic biosensing.124,128,138 Fu he mo e,
hei in eg a ion in o lab-on-chip pla o ms allows eal- ime, label- ee de ec ion o analy es
h ough LSPR shi s induced by biomolecula in e ac ions a he nanopa icle su ace.129,138
Looking ahead, con inued p og ess in shape-con olled syn hesis, hyb id design (e.g., mul ilaye
co e-shell s uc u es o Janus s uc u es , wi h wo chemically dis inc su aces), and ailo ed
ligh -ma e in e ac ions is expanding he unc ional landscape o gold nanoc ys als.127,135,146
While challenges such as scalabili y, ba ch- o-ba ch ep oducibili y, and long- e m
biocompa ibili y pe sis , he syne gis ic de elopmen o syn he ic s a egies, su ace
enginee ing, and applica ion-speci ic pe o mance con inues o d i e inno a ion in a eas
spanning nanomedicine,148 diagnos ics,128,149 ca alysis,130,143 and pho onic de ices150,151.
Chap e 1. Nano Neu o
32
Pho o he mal E ec
The pho o he mal e ec is a di ec nanoscale con e sion o abso bed ligh in o hea . When a
plasmonic nanos uc u e is illumina ed a LSPR, he inciden elec omagne ic ene gy is
abso bed, causing he collec i e oscilla ion o ee conduc ion elec ons.147 Acco ding o he
p inciple o ene gy conse a ion, his abso bed op ical ene gy mus subsequen ly be con e ed
o o he o ms.152 The exci ed plasmon decays non- adia i ely wi hin em oseconds ( ypically
~100 s), gene a ing ene ge ic cha ge ca ie s called "ho elec ons" and "ho holes" h ough a
p ocess known as Landau damping.129,153,154 These ho elec ons possess kine ic ene gies highe
han hei equilib ium he mal alues and ini ially do no ollow a he malized Fe mi-Di ac
dis ibu ion. Wi hin app oxima ely 10 s, ho elec ons lose ene gy h ough elec on-elec on
sca e ing, edis ibu ing hei ene gy ac oss he b oade elec on gas and he eby he malizing
in o a Fe mi-Di ac dis ibu ion.123,147 Subsequen ly, his ho elec on gas elaxes by ans e ing
i s ene gy o he ionic la ice o he plasmonic nanopa icle h ough elec on-phonon
in e ac ions. Fo gold, his elec on-phonon elaxa ion occu s o e a cha ac e is ic imescale o
a ound 1.7 ps, esul ing in an inc eased la ice empe a u e o he nanopa icle.121 F om ano he
pe spec i e, his hea gene a ion can be iewed as analogous o Joule hea ing, in ol ing ene gy
dissipa ion wi hin he me allic s uc u e. Finally, hea di uses ou wa d om he nanopa icle
in o he su ounding medium (e.g., liquid o glass), leading o an ele a ed empe a u e in i s
immedia e en i onmen . This ex e nal hea di usion occu s o e ela i ely longe imescales,
ypically anging om app oxima ely 100 ps o a ew nanoseconds, depending on ac o s such
as nanopa icle size and he he mal conduc i i y o he su ounding medium.155
Figu e 1.6 Pho o he mal con e sion in plasmonic nanopa icles: (1) Ligh abso p ion a he localized
su ace plasmon esonance. (2) Non- adia i e plasmon decay (~100 s) gene a es ho elec ons and holes.
(3) Elec on-elec on sca e ing (~10 s) he malizes he ca ie dis ibu ion. (4) Elec on-phonon coupling
(~1.7 ps o Au) ans e s ene gy o he la ice, aising nanopa icle empe a u e. (5) Phonon-phonon hea
dissipa ion in o he su ounding medium occu s o e ≈100 ps o a ew ns.
Chap e 1. Nano Neu o
33
The e iciency o he pho o he mal e ec in plasmonic nanoma e ials depends on nanopa icle
size and mo phology de e mining he balance be ween abso p ion and sca e ing.121 Small
nanoc ys als abso b inciden adia ion, con e ing i e icien ly in o localized hea . Fu he mo e,
he geome y o nanopa icles dic a es he in e nal spa ial dis ibu ion o gene a ed hea :
elonga ed shapes acili a e mo e uni o m hea ing ac oss hei olume, whe eas sphe es
p ima ily hea a he pa icle su ace. 156 Addi ionally, he illumina ion condi ions, pa icula ly
wa eleng h, i adia ion in ensi y, and illumina ion mode, a e decisi e. Tailo ing he exci a ion
wa eleng h o coincide wi h he nanopa icle plasmon esonance maximizes abso p ion-d i en
hea gene a ion and can be adjus ed, o example, o ma ch he biologically anspa en NIR-I
(650–950 nm) o NIR-II (1000–1350 nm) windows.141,157,158 The na u e o illumina ion,
con inuous-wa e e sus pulsed, also shapes he mal dynamics. Unde CW illumina ion, he
sys em eaches a s eady-s a e empe a u e dis ibu ion, wi h he hea dissipa ing in o he
su ounding medium acco ding o a adial p o ile ha decays as 1 𝑟
⁄, whe e 𝑟 is he dis ance
om he hea sou ce. In con as , pulsed illumina ion, pa icula ly wi h ul asho ( em osecond)
pulses, con ines hea mo e igh ly in space and ime. The esul ing empe a u e decays mo e
s eeply wi h dis ance, scaling as 1 𝑟3
⁄, and can gi e ise o addi ional e ec s such as acous ic
wa e gene a ion o s uc u al modi ica ions o he nanopa icle.121,159 In addi ion, hea
dissipa ion a es depend on he su ounding medium’s he mal conduc i i y, in luencing
nanopa icle in e nal empe a u e uni o mi y and en i onmen al hea ing dynamics.129,147,160
Collec i e pho o he mal e ec s eme ge om in e pa icle in e ac ions, ampli ying and
homogenizing empe a u e inc eases ac oss nanopa icle ensembles.120,159 Finally, modi ying
he nanopa icle su ace chemis y ia coa ings o unc ional g oups no only enhances colloidal
s abili y and biocompa ibili y bu also can signi ican ly boos op ical abso p ion and o e all
pho o he mal con e sion e iciency. This enhancemen a ises h ough se e al mechanisms:
inc easing he local e ac i e index, in oducing b oadband-abso bing laye s, and o ming
junc ions ha in ensi y he local elec omagne ic ield. Su ace coa ings such as i anium oxide,
melanin, polydopamine, and g aphene oxide ha e been shown o inc ease lase abso p ion in
plasmonic nanoma e ials.122
In s eady-s a e CW illumina ion, he empe a u e ise expe ienced by a plasmonic nanopa icle
is ul ima ely se by he balance be ween he op ical powe i abso bs and he abili y o he
su ounding medium o conduc ha hea . Exp essing he abso bed powe as 𝑄 = σ𝑎𝑏𝑠 ∙I and
sol ing he s a iona y hea -di usion equa ion gi es he canonical ela ion:
δ𝑇𝑁𝑃 = 𝑄
4𝜋𝜅𝑠𝑅𝑒𝑞 (1.2)
Chap e 1. Nano Neu o
34
whe e 𝑅𝑒𝑞 is a he mal adius (Laplace adius) ha cha ac e izes how he pa icle geome y
in luences hea di usion and 𝜅𝑠 he he mal conduc i i y o he hos medium.147 To simpli y
compa ison ac oss di e en ma e ials and shapes, he op ical e m 𝜎𝑎𝑏𝑠 can be ac o ed ou in o
he dimensionless Joule numbe :
𝐽𝑜 = 𝜎𝑎𝑏𝑠 λ𝑟𝑒𝑓
2𝜋𝑉 (1.3)
igu e o me i ha epo s he ligh - o-hea con e sion capabili y pe uni olume a an a bi a y
e e ence wa eleng h λ𝑟𝑒𝑓 (commonly 1240 nm). Because abso p ion c oss-sec ion 𝜎𝑎𝑏𝑠 scales
wi h he local plasmonic ield enhancemen , 𝐽𝑜 is inc easing as he aniso opy o a ma e ial
inc eases ( he bigge aspec a io, he highe 𝐽𝑜 ). Fo example, in gold nano ods, inc easing he
aspec a io om 1 o abou 8 can inc ease 𝐽𝑜 by nea ly wo o de s o magni ude due o he
s onge and ed-shi ed longi udinal su ace plasmon esonance. By subs i u ing 𝐽𝑜 in o he
exp ession o δ𝑇𝑁𝑃 makes explici how geome y ( h ough aspec a io), ma e ial pe mi i i y
and he ambien he mal conduc ance collec i ely dic a e he empe a u e jump ha d i es he
“nanoscale hea -engine” desc ibed abo e.
Wi h he physical p inciples and echnical capabili ies o plasmonic hea ing es ablished, we now
explo e how hese can be adap ed o he challenges o neu al modula ion, whe e p ecision,
compa ibili y, and esponsi eness a e pa amoun .
3. Nano Neu o
Since he beginning o he 21s cen u y, nano echnology has s eadily ad anced om a
specula i e on ie in o a p ac ical and e sa ile oolki o neu oscience.161 In 2000, Ma son
and colleagues demons a ed ha unc ionalized mul i-walled ca bon nano ubes (MWCNTs)
could suppo neu onal adhesion and neu i e ou g ow h, es ablishing he i s clea e idence
ha enginee ed nanoma e ials could in e ace di ec ly wi h neu al issue.162 Wi hin a ew yea s,
esea che s began modi ying he su ace chemis y o ca bon nano ubes o selec i ely p omo e
neu al di e en ia ion and synap ogenesis, while also imp o ing conduc i i y and
biocompa ibili y. By 2005, s udies showed ha pu i ied MWCNTs enhanced he e iciency o
elec ical signal p opaga ion in cul u ed ne wo ks, laying he g oundwo k o hei in eg a ion
in o low-impedance neu al p obes.163 In 2007, aligned ca bon nano ibe a ays we e success ully
used o s able ex acellula eco dings and a ge ed synap ic s imula ion, illus a ing he
po en ial o nanoscale elec odes o ch onic neu al in e acing.164 Since hen ca bon nano ubes
Chap e 1. Nano Neu o
35
a e in ensi ely s udied as a ool ha could boos he pe o mance o mic oelec onic in e aces
wi h b ain issue.163
Beyond ca bon allo opes, wo-dimensional g aphene and i s de i a i es ha e become
ounda ional ma e ials o anspa en , lexible neu al in e aces.163 Monolaye and nanopo ous
g aphene shee s a e capable o modula ing ion-channel ac i i y, deli e ing pho o he mal o
pho oelec ical s imula ion, and – when pa e ned in o mic o- ansis o s – eco ding co ical
dynamics ac oss a b oad equency spec um, om in aslow oscilla ions o high-gamma bu s s.
Thei op ical anspa ency p ese es he imaging window, enabling mul imodal in es iga ions.
No ably, la ge-a ea educed g aphene oxide ( GO) elec oco icog aphy oils a e now
unde going i s -in-human ials o in aope a i e b ain mapping, unde sco ing hei
ansla ional po en ial.163
To add ess he mechanical misma ch be ween igid implan s and he so neu al issue, ha may
lead o immunological esponse and cell damage, ecen esea ch has u ned o conduc i e
polyme hyd ogels. These a e semi-in e pene a ing ne wo ks composed o conjuga ed
polyme s (like PEDOT:PSS, polypy ole, o poly hiophene) combined wi h PEG o
gela in-me hac yloyl (GelMA) ma ices. Such composi es exhibi b ain-like elas ic moduli
(≤10 kPa) while main aining high cha ge-injec ion capaci ies (>1 mA·cm-2) and excellen
long- e m elec ochemical s abili y. These hyd ogels a e no only obus unde cyclic s ess and
sonica ion bu a e also compa ible wi h ad anced ab ica ion echniques such as 3D p in ing and
wo-pho on polyme iza ion, enabling mic oscale pa e ning o bioac i e cues o guided axon
g ow h and educed issue in lamma ion.163
Me allic nanos uc u es also con ibu e signi ican ly o he neu o echnology landscape. E en
hough we will explo e plasmonic nanos uc u es – especially gold nanopa icles o
neu omodula ion – in mo e de ail sho ly, i is wo h no ing now ha o he me al-based
nanoma e ials ha e al eady played a signi ican ole in ad ancing neu o echnology. Fo
example, sil e nanowi e ne wo ks, ei he ee-s anding o embedded in ul a hin, lexible
Pa ylene-C polyme ilms, ha e been de eloped as low-impedance elec oco icog aphy a ays.
These a ays a e mechanically complian , can be olded o injec ed, and con o m closely o he
b ain’s su ace, allowing s able signal acquisi ion wi h minimal issue dis u bance. In pa allel,
supe pa amagne ic i on-oxide nanopa icles (SPIONs) ha e enabled wi eless neu al s imula ion.
When exposed o al e na ing magne ic ields, hese pa icles can ei he gene a e localized
hea ing (magne o he mal e ec ) o p oduce mechanical o que. Unde magne ic s imula ion,
SPIONs oscilla e o o a e and apply mechanical o ces o he nea by cell memb ane; his
memb ane s ain opens Piezo- amily mechanosensi i e ion channels, allowing sub-millisecond
Chap e 1. Nano Neu o
36
con ol o neu onal i ing en i ely wi elessly – wi hou he need o implan ed elec odes o
op ical ibe s. P oo -o -concep s udies ha e demons a ed ha such magne ically con olled
pa icles can modula e neu onal ac i i y and beha io in eely mo ing animal models,
highligh ing hei po en ial as minimally in asi e, emo ely con olled neu omodula ion ools.163
Be o e di ing in o he co e ocus o his disse a ion – plasmonic nanopa icles (wi h pa icula
emphasis on gold) and semiconduc o quan um do s – i is impo an o i s conside se e al
c i ical ac o s ha mus be add essed when in eg a ing nano echnology in o biological
esea ch.
Fi s aspec o conside is he blood-b ain ba ie (BBB) ha se e ely es ic s nanopa icle
deli e y o he cen al ne ous sys em. BBB is a highly selec i e memb ane ba ie loca ed in he
cen al ne ous sys em ha plays a c ucial ole in main aining b ain balance and p o ec ing i
om ha m ul subs ances and pa hogens.165 Deli e y o nanoma e ials equi es employing
s a egies as: passi e di usion wi h long-ci cula ing o ul asmall pa icles (a ound 2 nm
diame e ); empo a y BBB dis up ion h ough ocused ul asound (o en combined wi h
mic obubbles), osmo ic agen s such as manni ol, o ni ic oxide- eleasing nanopa icles;
a ge ed deli e y using su ace-modi ied lipid ca ie s, ecep o - a ge ed ligands, ca ionic
esicles, apolipop o ein adso p ion, o ecep o -media ed anscy osis; and magne ic guidance
o i on-oxide o magne oelec ic nanopa icles using ex e nal magne ic ields o enhance b ain
up ake in a minimally in asi e manne .165
Second, once in biological luids, nanopa icles apidly acqui e a “p o ein co ona”.166 In his
spon aneous p ocess laye o biomolecules, p ima ily p o eins, a e adso bed on he su ace o
nanoma e ial d i en by a ious physicochemical in e ac ions be ween p o eins and he
nanoma e ial su ace. P o ein co ona al e s agg ega ion and biodis ibu ion, cellula up ake, o
po en ial oxici y o nanopa icles.163 Unde s anding and con olling co ona composi ion is
essen ial o p edic able in i o beha io . S a egies o minimize undesi ed co ona o ma ion
include su ace modi ica ion wi h polyme s such as PEG o zwi e ionic ligands, coa ing
nanopa icles wi h na u al cell memb anes o mimic biological iden i y, and emo ing
con aminan s ha p omo e non-speci ic in e ac ions.163 Al e na i ely, he p o ein co ona can
be delibe a ely enginee ed – o example, h ough ligand unc ionaliza ion o con olled p o ein
adso p ion – o enhance selec i e up ake, acili a e ecep o -media ed anspo , and educe
o - a ge e ec s.166
Thi d, cellula endocy osis, he p ocess by which cells imme se ex acellula ma e ial in o
memb ane-bound esicles, is a double-edged swo d o nanopa icle applica ions.167–169 On one
Chap e 1. Nano Neu o
37
hand, i enables in acellula deli e y o ca gos such as biosenso s, d ugs, o imaging agen s, and
unde lies he design o nanomedicines ha exploi ecep o -media ed o adso p i e up ake
pa hways o each speci ic o ganelles o cell ypes. Typical endocy ic up ake begins wi hin
minu es – cla h in-media ed pi o ma ion and esicle scission occu wi hin 15-60 seconds, and
ca eolae-media ed p ocesses ake abou 5-10 minu es – while ull in e naliza ion, so ing wi hin
endosomes, and anspo o lysosomes can ake up o one o wo hou s.170 When op imizing
nanopa icles o desi ed applica ion i is c ucial o ake in o conside a ion he up ake kine ics
and p ope ly une he size, cha ge, and su ace o nanoma e ial.171 Fo neu omodula ion
s a egies ha ac i a e he mosensi i e ion channels h ough pho o he mal hea ing, endocy ic
up ake mus be supp essed so ha nanopa icles emain a he memb ane in close p oximi y o
he channels.
Fou h, he unique eac i i y o nanoma e ials aises neu o oxic isks – oxida i e s ess,
in lamma ion, ba ie dis up ion, and di ec memb ane damage – ha depend sensi i ely on
composi ion, coa ing, and dose. This highligh s he need o igo ous and physiologically ele an
oxici y assessmen s, pa icula ly when conside ing in i o use and u u e clinical ansla ion.170
Finally, slow clea ance and long- e m e en ion o non-deg adable pa icles h ea en ch onic
accumula ion and in lamma ion. PEG-coa ed QDs ha e been shown o pe sis in bone ma ow
and lymph nodes o se e al mon hs ollowing in a enous injec ion in animal models.172 While
his p olonged isibili y makes hem aluable as long- e m imaging p obes, i also aises
impo an ques ions abou he po en ial long- e m e ec s and sa e y o nanoma e ial
accumula ion in i o. On he o he hand, magne oelec ic nanopa icles we e de ec able in
co ical issue o abou 24 hou s, wi h hei s imula o y e ec s dissipa ing wi hin h ee days –
sugges ing ha hey a e likely clea ed om he b ain wi hin ha pe iod. These indings sugges
ha nanopa icle e en ion is highly dependen on bo h he ype o nanoma e ial and he
speci ic issue o which i is deli e ed, unde sco ing he impo ance o ho ough in i o s udies
and he de elopmen o biodeg adable ma e ials o ensu e long- e m sa e y and clinical
iabili y. The de elopmen o biodeg adable co es and immunologically ine coa ings is
impe a i e, and add essing hese challenges is key o ha nessing nano echnology sa ely and
e ec i ely in neu oscience.170,173
Chap e 1. Nano Neu o
38
Semiconduc o Nanopa icles in Neu obiology – S a e o he A
Semiconduc o nanoc ys als quickly cap u ed he in e es o biologis s, no only o hei
excep ional b igh ness and pho os abili y as luo escen labels, bu also o hei po en ial as
ools o neu omodula ion. While quan um do -media ed neu onal s imula ion has been
explo ed expe imen ally,174,175 his hesis will no add ess ha di ec ion. Ins ead, he ocus he e
is on hei op ical de ec ion p ope ies. This sec ion i s conside s hei ole in isualiza ion, wi h
a la e discussion add essing hei neu omodula o y po en ial. A key ad an age ha
dis inguishes QDs om con en ional dyes is no only hei highe quan um yield – o en eaching
65-95% compa ed o 30-50% in s anda d luo opho es – bu also hei signi ican ly la ge one-,
wo-, and h ee-pho on abso p ion c oss-sec ions. These p ope ies help o e come
he long-s anding ade-o be ween imaging dep h and spa ial esolu ion.176
When discussing he use o semiconduc o nanoc ys als in deep b ain imaging, i is impo an o
dis inguish be ween wo main modali ies: s uc u al imaging and unc ional imaging. S uc u al
imaging aims o e eal ana omical ea u es such as ascula u e, ibe ac s, and cellula
o ganiza ion. In con as , unc ional imaging cap u es dynamic physiological p ocesses like
neu al ac i i y, memb ane po en ial luc ua ions, o neu o ansmi e elease. The applica ion
o QDs in deep b ain imaging began in he ea ly 2000s, wi h pionee ing s udies ha
demons a ed hei e ec i eness in pene a ing deep issue and p o iding s able,
high- esolu ion signals.
In 2002, Dube e e al. achie ed he i s in i o isualiza ion o QDs by encapsula ing hem in
phospholipid micelles.177 Ea ly b eak h oughs began in 2003, when Dahan e al. demons a ed
eal- ime acking o glycine ecep o s in li ing neu ons using single QDs, ollowed by
La son e al. ha success ully used wa e -soluble quan um do s o in i o mul ipho on
luo escence imaging o labeled blood escels.178–180 The explo a ion o QDs o deep b ain
imaging gained signi ican momen um in he 2010s, wi h main ocus on ha nessing QDs as ools
o ol age sensing.176 The ollowing discussion examines key de elopmen s ha ha e
es ablished QDs as a p ominen ool in bo h s uc u al and unc ional neu oimaging.
In s uc u al imaging applica ions, he high quan um yield and na ow, unable emission spec a
o QDs signi ican ly enhance signal- o-backg ound a ios, pa icula ly a long exci a ion
wa eleng hs (1.3-2.2 µm), whe e issue sca e ing is minimized. Comme cially a ailable p obes
such as Q acke 655, when exci ed a 1700 nm, ha e enabled h ee-pho on imaging o dep hs
o up o 2100 µm in he mouse b ain – ou pe o ming con en ional luo opho es like Texas Red
dex an, which is ypically limi ed o a ound 1340 µm. Cus om-enginee ed he e os uc u es,
Chap e 1. Nano Neu o
39
including CdSe/CdS/ZnS QDs, ha e demons a ed imaging dep hs o 850 µm h ough an in ac
skull and 1550 µm ollowing c anio omy. QDs ha e been employed ac oss mul ipho on imaging
modali ies, suppo ing wo-, h ee-, and e en ou -pho on luo escence de ec ion.181 Among
hese, h ee-pho on exci a ion o e s he g ea es pene a ion dep h wi h educed isk o
pho odamage. Addi ionally, he b oad spec al unabili y o QDs acili a es mul icolo
isualiza ion o ascula u e, neu ons, and mic oglia.176 Mos s uc u al applica ions ha e
ocused on he mouse b ain ascula u e, wi h pa icula ly de ailed imaging o deep subco ical
egions such as he hippocampus. Ad ances in ligand design a e u he expanding he po en ial
o QDs, enabling mo e a ge ed labeling s a egies and imp o ed a e sal o he blood-b ain
ba ie .176
Func ionally, QDs ex end hese op ical ad an ages o eal- ime physiology. Thei esis ance o
pho obleaching allows sus ained exci a ion, suppo ing hemodynamic measu emen s a
signi ican dep hs. Fo example, comme cially a ailable Q acke 800 has been used o quan i y
blood- low eloci ies up o 600 µm deep, while Q acke 655 enables de ailed ascula imaging
a dep hs eaching 1600 µm. Mo e impo an ly, he quan um-con ined S a k e ec and
enginee ed ene gy- o cha ge- ans e (FRET and ET) cons uc s equip QDs wi h millisecond-
scale ol age sensi i i y.176,179
In 2013, Ma shall and Schni ze p oposed he use o QDs as luo escen ol age indica o s,
le e aging he quan um-con ined S a k e ec (QCSE) o de ec changes in memb ane
po en ial.179 This ounda ional concep was subsequen ly ad anced by he esea ch g oups o
Shimon Weiss and James B. Delehan y h ough he de elopmen o memb ane-inse ing QD
nanosenso s.163 Bo h simula ions and in i o expe imen s demons a ed ha single ac ion
po en ials could be esol ed using signi ican la ge op ical esponse han wi h con en ional
ol age-sensi i e dyes o gene ically encoded ol age indica o s (GEVIs). No ably, Delehan y’s
g oup u he in oduced bioconjuga ed QD- ulle ene pla o ms ha enabled he de ec ion o
elec ically e oked co ical esponses h ough cha ge ans e mechanism (Figu e 1.7 A).182
In pa allel, he g oups o Ra ael Yus e and Jona han Owen183 de eloped qua z nanopipe es
wi h ul a-na ow ip diame e s (15–30 nm), coa ed wi h QDs o acili a e wo-pho on
isualiza ion.183 These nanopipe es unc ioned as minimally in asi e nanoelec odes o
in acellula ol age eco dings and we e success ully a ge ed o dend i ic spines o
hippocampal neu ons in bo h cul u ed cells and acu e co ical slices. This app oach was u he
e ined in a ollow-up s udy184 whe e a lexible e sion o he nanopipe e was in oduced and
deployed in i o.184 Using c anial mic op isms, he au ho s we e able o access and eco d om
Chap e 1. Nano Neu o
40
isually iden i ied py amidal neu ons and in e neu ons in bo h anes he ized and awake,
head- ixed mice.
Complemen ing hese ad ances, S. Weiss, K. Pa k, and colleagues (2018) demons a ed ha
od-shaped QDs unc ionalized wi h speci ic pep ides could spon aneously inse in o lipid
memb anes and de ec single-pa icle ol age esponses in model sys ems (Figu e 1.7 C).185
These esul s p o ided expe imen al con i ma ion o p io heo e ical wo k by he same
g oup.112,186 Building on his wo k, S. Weiss oge he wi h A. Ludwig and collabo a o s epo ed
in 2020 ha ype-II ZnSe/CdS seeded nano ods (NRs), when unc ionalized wi h a lipid mix u e
de i ed om b ain issue, could spon aneously inco po a e in o neu onal plasma
memb anes.187 This inse ion was d i en by he in insic compa ibili y o he nano od coa ing
wi h he na i e lipid composi ion o he memb ane. Once embedded, he nano ods, ope a ing
h ough QCSE, we e able o sense local changes in memb ane po en ial. This unc ionali y was
demons a ed ac oss mul iple sys ems, including sel -spiking and pa ched HEK293 cells, as well
as p ima y co ical neu ons – all wi hou he need o gene ic modi ica ion.187,188 Ano he ecen
s udy by Qiangbin Wang’s g oup189 demons a ed ha glu a hione-capped CdSe/ZnS quan um
do s can localize o neu onal memb anes and unc ion as ol age senso s (Figu e 1.7 B). The
sensing mechanism elies on Fö s e esonance ene gy ans e (FRET), wi h dipic ylamine (DPA)
ac ing as an accep o wi hin he lipid bilaye . Changes in memb ane po en ial al e he posi ion
o DPA ela i e o he QD su ace: du ing depola iza ion o hype pola iza ion, DPA mig a es
wi hin he memb ane, he eby modula ing he dono –accep o dis ance and, consequen ly, he
luo escence in ensi y o QDs.189 Cagla e al. (2019) demons a ed ha InP/ZnS QDs could
de ec sub h eshold ol age luc ua ions in li e cells wi h g ea e sensi i i y han calcium-based
indica o s, which is a signi ican p og ess o o e imp o ed biocompa ibili y.190
Chap e 1. Nano Neu o
47
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Chap e 2. Sel -spiking HEK Cells
63
p opo ion o he cul u e su ace co e ed by cells. T ans e ing cells o a new essel a his s age
p e en s o e g ow h and main ains a o able condi ions o con inued eplica ion. Be ween
passages, he cul u e medium may be pa ially o ully eplaced o es o e nu ien s and emo e
was e p oduc s.6 These s anda d p ac ices help ensu e s able cell physiology and ep oducible
expe imen al ou comes. As an immo alized line, HEK293 cells can be p opaga ed inde ini ely,
hough o minimize genomic d i -such as ch omosomal ea angemen s and gene copy numbe
changes-cul u es a e commonly main ained o no mo e han wen y passages.3,5
Se e al gene ically modi ied de i a i es o he o iginal HEK293 cell line ha e been de eloped
o e he yea s o mee speci ic expe imen al and indus ial demands. A pa icula ly inno a i e
adap a ion o he HEK293 cell line was de eloped by Adam E. Cohen’s g oup a Ha a d
Uni e si y o c ea e a eliable and accessible model o elec ical exci abili y h ough he s able
exp ession o de ined ion channels, known as spiking HEK cells (Table 2.2). This enginee ed
sys em enables high- h oughpu sc eening and de ailed cha ac e iza ion o luo escen ol age
indica o s, while also suppo ing a b oad ange o applica ions including ion channel esea ch,
all-op ical elec ophysiology, compound sc eening, oxici y e alua ion, and s udies o
elec opo a ion dynamics. In ou esea ch, i p o ides a cen al pla o m o e alua ing ol age-
sensi i e nanoma e ials wi hin a simpli ied, scalable, and well-con olled expe imen al
con ex .7,8
Spiking HEK cells a e gene ically modi ied o s ably exp ess one o wo key ion channels: NaV1.3
o NaV1.5 - ol age-ga ed sodium channels ha ini ia e ac ion po en ials, and Ki 2.1, an inwa d
ec i ie po assium channel ha s abilizes he es ing memb ane po en ial nea physiological
alues. The inclusion o Ki 2.1 educes he es ing po en ial o app oxima ely -66 mV, allowing
he cells o exhibi epe i i e ac ion po en ial i ing upon depola iza ion. In some e sions o he
sys em, Ki 2.1 exp ession is placed unde he con ol o a doxycycline-inducible p omo e ,
enabling he gene a ion o bo h spiking and non-spiking a ian s om he same line o
compa a i e s udies. Ki 2.1 channel migh be addi ionally agged wi h cyan luo escen p o ein
(CFP) as a luo escen ag, which enables op ical assessmen which cells exp ess he channel.9
When cul u ed as a monolaye a high con luency (90-95%), spiking HEK cells o m a unc ionally
in eg a ed ne wo k h ough in e cellula coupling media ed by endogenous gap junc ions, so
called connexin-45. This elec ical connec i i y suppo s spon aneous, hy hmic ac ion
po en ials ha p opaga e uni o mly ac oss he cul u e in a coo dina ed, me onome-like
pa e n.7 In addi ion o hei in insic ac i i y, ac ion po en ials can be ex e nally igge ed using
elec ical s imula ion. In ce ain a ian s o he cell line, he op ogene ic p o ein CheRi is
addi ionally exp essed, enabling p ecise, ligh -induced ac i a ion o ac ion po en ials h ough
Chap e 2. Sel -spiking HEK Cells
64
blue ligh s imula ion. These elec ophysiological p ope ies make spiking HEK cells pa icula ly
well sui ed o op ical elec ophysiology, including he e alua ion o gene ically encoded ol age
indica o s (GEVIs) and ol age-sensi i e dyes (VSDs). The sys em also enables high- h oughpu
sc eening o pha macological agen s a ge ing ion channels, as well as pheno ypic assays
ele an o neu ological and ca diac diso de s. I is especially aluable o iden i ying modula o s
o exci abili y, assessing po en ial neu o oxici y o ca dio oxici y, and in es iga ing disease
mechanisms h ough all-op ical con ol and moni o ing o memb ane ol age. While spiking HEK
cells do no ully eplica e he biophysical p ope ies o neu ons-due o longe ac ion po en ials
and a iable epola iza ion kine ics- hey o e a obus , scalable, and expe imen ally accessible
model o s udying exci able cell beha io in a con olled se ing.7–9
Table 2.2 Spiking HEK293 cell line a ian s wi h hei ion channel payloads, inducible sys ems, sou ces, and
key p ac ical no es. Comme cially a ailable lines we e de eloped in he Adam E. Cohen labo a o y and a e
dis ibu ed by ATCC (h ps://www.a cc.o g/).
Cell line
Payload
Inducible
sys em
Sou ce
P ac ical no es
Spiking HEK
(O iginal,
No
ca alogued)
Cons i u i e:
•NaV1.3
•Ki 2.1
No luo o ags
None
(cons i u i e
exp ession)
A. Cohen lab;
no comme cially
a ailable
Less s able; Ki 2.1
exp ession los a e
a ew passages.
e -on
Spiking HEK
(ATCC
CRL-3479)
Cons i u i e:
•NaV1.5
Inducible:
• Ki 2.1-CFP
TA-Te -On;
induce wi h
1-2 µg mL⁻¹
doxycycline
o 24-36 h
Comme cial
(ATCC)
HEK cells do no
ole a e sus ained
Ki 2.1-main ain a
mas e pla e wi hou
doxycycline and
induce only pla es
des ined o assays.
CheRi -eGFP
e -on
Spiking HEK
(ATCC
CRL-3480)
Cons i u i e:
•NaV1.5
•CheRi -eGFP
Inducible:
•Ki 2.1-CFP
TA-Te -On;
induce wi h
1-2 µg mL⁻¹
doxycycline
o 24-36 h
Comme cial
(ATCC)
Adds op ical
s imula ion
capabili y ia
CheRi .
Chap e 2. Sel -spiking HEK Cells
65
1.1. Cell Cul u e
S anda d cul u e condi ions o adhe en HEK293 cells in ol e main aining he cells a 37 °C in a
humidi ied a mosphe e wi h 5% ca bon dioxide (CO2).6 Cells a e ypically g own in Dulbecco’s
Modi ied Eagle Medium (DMEM), usually combined wi h nu ien mix u e, and supplemen ed
wi h FBS, L-glu amine, and an ibio ics.6 An ibio ics in cell cul u e se e wo main pu poses: hey
p e en bac e ial con amina ion, commonly h ough he use o agen s such as penicillin and
s ep omycin, and hey unc ion as selec i e agen s o isola e o main ain gene ically modi ied
cells ha ca y an ibio ic esis ance genes. Fo example, compounds such as e acycline,
gene icin, pu omycin, and blas icidin a e ou inely used o selec cells ha ha e been
success ully ans ec ed wi h genes o in e es .4,7
Cul u es a e usually passaged wo o h ee imes pe week. As cells g ow and me abolize, hey
g adually acidi y he su ounding medium. This shi is easily moni o ed by he colo change o
DMEM con aining phenol ed, which ansi ions om ed o o ange o yellow as he pH d ops-
o en se ing as a isual cue ha he cul u e is due o passaging o medium eplacemen
(wo k low illus a ed on Figu e 2.9).
Passaging begins by emo ing he old cul u e medium om he lask. I he cell monolaye is
well a ached and s able, he cells can be gen ly insed wi h phospha e-bu e ed saline (PBS) o
wash away dead cells and was e. To de ach cells om he su ace, a mild enzyma ic ea men
wi h ypsin is used.5 A e a sho incuba ion ( ypically 1-3 minu es a 37 °C), he cells begin o
ound up and li om he su ace. A his poin , he enzyme is neu alized by adding esh
DMEM10. The suspension is pipe ed gen ly o b eak up any clumps, hen ans e ed o a s e ile
cen i uge ube. The cell suspension is cen i uged o pelle he cells, o ming a isible puck a
he bo om o he ube. The supe na an is ca e ully disca ded, and he cells a e esuspended in
esh medium, again pipe ing up and down o ensu e a uni o m, single-cell suspension.
Con olling he seeding densi y is impo an o expe imen s like spiking monolaye cul u es o
imaging, and his equi es a p ecise cell coun using a hemocy ome e . Op ionally, ypan blue
dye can be added o disc imina e be ween iable and non- iable cells, as he dye pene a es
only he memb anes o dead cells. Based on he calcula ed cell concen a ion, he app op ia e
numbe o cells is seeded in o new cul u e lasks and expe imen al sample pla es, using esh
DMEM10 supplemen ed wi h an ibio ics o main ain selec i eness and ensu e consis en gene
exp ession whe e ele an . In some cases, cul u e essels o sample dishes may be coa ed wi h
poly-D-lysine-a syn he ic, posi i ely cha ged polypep ide ha enhances cell adhesion and
suppo s g ow h on he cul u e su ace.3,7 Once seeded, all essels a e clea ly labeled and placed
Chap e 2. Sel -spiking HEK Cells
66
back in o he incuba o o allow he cells o ea ach and con inue g owing unde s anda d
condi ions. A de ailed p o ocol, including he comple e media ecipe, exac eagen olumes,
and he s ep-by-s ep cell coun ing p ocedu e, is p o ided in he Appendix (Sec ion 2.2.1 Cell
Cul u e P o ocols).
Figu e 2.9 Schema ic o e iew o he HEK293 cell passaging wo k low. S eps include eagen p epa a ion,
cell de achmen wi h ypsin, cen i uga ion and esuspension in esh medium, cell coun ing wi h ypan
blue exclusion, and eseeding in o new cul u e essels o con inued incuba ion.
Con uency
80
T ypsin
Disca d
media and
wash cells
wi h PBS
Dead cell
Viable cell
DMEM
Incuba on
37°C
5 CO2
Disca d
supe na an Redispe se
in DMEM
1:1
T ypan
blue
Cell
solu on
37°C
15 min
Hemocy ome e
Chap e 2. Sel -spiking HEK Cells
67
2. Calcium Imaging o Spiking HEK Cells
Fluo escence se es as a undamen al indica o o neu al s uc u e and dynamics, o ming he
basis o nume ous imaging echniques employed by neu obiologis s (see Chap e 1.1.2).
By le e aging luo escen signals, i is possible o achie e high- esolu ion imaging o neu ons
and hei unc ional ac i i ies, he eby enabling explo a ion o cellula mechanisms unde lying
neu al ac i i y.10 The choice o luo escence mic oscope plays a pi o al ole in de e mining he
spa ial and empo al esolu ion achie able, and neu obiological s udies equen ly employ
wide- ield, con ocal, wo-pho on, o mul ipho on mic oscopes, each sui ed o pa icula
expe imen al equi emen s and imaging dep hs. While con ocal mic oscopy o e s excellen
op ical sec ioning o mode a e-dep h samples, wo-pho on mic oscopy has become a mains ay
in imaging neu al ac i i y deep wi hin sca e ing issues, such as in ac b ain p epa a ions.11,12
Wi h hese conside a ions in mind, his chap e desc ibes he use o luo escence mic oscopy o
s udy calcium dynamics in sel -spiking HEK cells. The imaging se up employs s anda d
epi- luo escence illumina ion and came a-based de ec ion, which allows moni o ing o
in acellula calcium changes in esponse o elec ical ac i i y. This s aigh o wa d con igu a ion
was selec ed o suppo he de elopmen o an app oach ha can be easily adop ed in ypical
biological labo a o y en i onmen s wi hou equi ing specialized ins umen a ion.
2.1. Fluo escence Mic oscope Se up
Fluo escence mic oscopy isualizes biological samples by de ec ing emi ed luo escence ligh
om molecules called luo opho es. Fluo opho es abso b exci a ion ligh a a sho e
wa eleng h and emi luo escence a a longe wa eleng h; his shi , known as he S okes shi ,
allows clea sepa a ion o emi ed luo escence om exci a ion illumina ion.13 A ypical
luo escence mic oscope employs an epi- luo escence se up, whe e he same objec i e lens
se es bo h o deli e exci a ion ligh and o collec emi ed luo escence (Figu e 2.10 A).14
Objec i es wi h a high nume ical ape u e (NA) a e p e e ed, as hey inc ease ligh -ga he ing
e iciency and imp o e spa ial esolu ion. Imme sion objec i es-used wi h a medium such as
wa e , oil, o glyce ol be ween he lens and he specimen-can u he enhance image quali y by
minimizing e ac i e index misma ches and educing op ical dis o ions.13
Cen al elemen o such a luo escen se up is he il e ing cube, con aining h ee essen ial
componen s: an exci a ion il e ha ansmi s only speci ic wa eleng hs equi ed o exci e he
luo opho es, a dich oic mi o placed a a 45-deg ee angle ha e lec s exci a ion wa eleng hs
on o he sample and ansmi s emi ed luo escence owa ds he de ec o , and an emission
(ba ie ) il e ha selec i ely allows only he longe -wa eleng h luo escence signal o each
Chap e 2. Sel -spiking HEK Cells
68
he de ec o , blocking esidual exci a ion ligh .11,13 These il e s mus be ca e ully ma ched o
he spec al p ope ies o he luo opho e used. Illumina ion is ypically p o ided by a me cu y
lamp, which emi s in ense, b oad-spec um ligh wi h s ong peaks s ong peaks in he UV and
isible spec um. To egula e illumina ion in ensi y and minimize pho obleaching o
pho o oxici y, neu al densi y (ND) il e s can be in oduced in o he op ical pa h o educe he
amoun o exci a ion ligh eaching he sample.11
Fluo escence signals a e ypically eco ded using sensi i e digi al de ices such as CCDs (Cha ge-
Coupled De ice), EMCCDs (Elec on-Mul iplying CCD), o sCMOS (Scien i ic Complemen a y
Me al-Oxide-Semiconduc o ) came as.10,11,13,14 Came a selec ion should be guided by
expe imen al needs, weighing ac o s such as signal sensi i i y, empo al and spa ial esolu ion,
as well as cos and accessibili y. P io o da a collec ion, key eco ding pa ame e s such as
came a exposu e ime, ame a e ( ames pe second, ps), pixel binning, and he size o he
eco ded a ea mus be ca e ully conside ed. Sho e exposu es enable highe ame a es and
imp o ed empo al esolu ion bu may equi e inc eased exci a ion in ensi y o highe de ec o
sensi i i y o p ese e signal quali y. Binning, which combines adjacen pixels in o la ge uni s,
enhances he signal- o-noise a io a he cos o spa ial esolu ion, and can suppo as e
acquisi ion by educing da a olume. Simila ly, educing he eco ded ield o iew (FOV) allows
as e eadou and highe ame a es. These pa ame e s mus be uned o achie e he
app op ia e balance be ween empo al and spa ial esolu ion, signal quali y, da a size, and
sample iabili y, based on he speci ic goals and cons ain s o he expe imen .13,14
Chap e 2. Sel -spiking HEK Cells
69
Figu e 2.10 Epi- luo escence mic oscopy se up used o calcium imaging expe imen s. (A) Op ical diag am
o he luo escence ligh pa h, showing key componen s including he me cu y a c lamp, exci a ion and
emission il e s, dich oic mi o , imme si e objec i e, and de ec o . The sys em is equipped wi h a
mo o ized il e cube u e , allowing apid swi ching be ween mul iple il e cubes du ing expe imen s.
(B) Pho og aph o he luo escence mic oscope sys em loca ed a he Neu o echnology Cen e , Columbia
Uni e si y.
The mic oscope used o he majo i y o measu emen s p esen ed in his disse a ion was an
up igh ixed s age mic oscope sys em loca ed in he Yus e Labo a o y a he Neu o echnology
Cen e , Columbia Uni e si y. This se up, in e nally e e ed o as he “No h Rig”, was con igu ed
o single-channel calcium imaging using Calcium G een-1, wi h FITC il e cube ma ched o he
dye’s exci a ion and emission cha ac e is ics (Figu e 2.10 B). Exci a ion ligh om a me cu y
lamp was spec ally il e ed h ough a 475/85 nm band-pass exci a ion il e and di ec ed on o
he specimen ia a 500 nm long-pass dich oic mi o and a wa e -imme sion objec i e. The same
objec i e lens collec ed he emi ed luo escence, which hen passed h ough a 535/50 (passing
app oxima ely 510-560 nm wa eleng hs) emission il e be o e being de ec ed by a sCMOS
Hamama su came a moun ed on he mic oscope’s inocula po .11,13 An imme sion objec i e
was used o minimize e ac i e index misma ch be ween he objec i e lens, he co e slip, and
he aqueous medium co e ing he sample, he eby imp o ing ligh collec ion e iciency and
p ese ing spa ial esolu ion. Acquisi ion pa ame e s-including exposu e ime, pixel binning,
and ame a e-we e op imized unde con inuous illumina ion o cap u e apid calcium
ansien s wi h minimal signal loss. Reco dings we e pe o med a 50, 100, and 200 ms exposu e
imes; an exposu e o 100 ms (co esponding o 10 ames pe second) p o ided a sui able
Chap e 2. Sel -spiking HEK Cells
70
balance be ween empo al esolu ion and luo escence signal b igh ness o de ec ing spiking
e en s.
In addi ion o luo escence illumina ion, a seconda y whi e ligh sou ce was posi ioned benea h
he specimen, enabling he acquisi ion o b igh - ield images aligned wi h he co esponding
luo escence ields o iew. The mic oscope is equipped wi h a il e cube u e , allowing apid
swi ching be ween il e se s du ing expe imen s o suppo imaging o di e en luo opho es.
2.2. Resul s and Discussion
The gene ically modi ied HEK cells, desc ibed by Pa k and Cohen (2013), spon aneously gene a e
elec ical spikes when cul u ed as a monolaye a high con luence (80-95%). This ac i i y
eme ges as coo dina ed depola iza ion e en s, wi h epo ed spike equencies a ound 3 Hz and
p opaga ion eloci ies o app oxima ely 2 cm/s. These spikes a e cha ac e ized by a es ing
memb ane po en ial nea -66 mV, peak depola iza ions eaching +34 mV, and ise imes o
app oxima ely 3 ms, e lec ing s able and epea able ac ion po en ial dynamics ac oss he
cul u e.7
Fo imaging, monolaye s we e s ained wi h he chemical calcium indica o Calcium G een-1 and
eco ded in Ty ode’s solu ion, a physiological ex acellula bu e wi h de ined ionic
composi ion; de ailed p o ocols o bo h a e p o ided in he Appendix (Sec ion 2,
Supplemen a y Ma e ial o Chap e #2).
To de e mine he op imal ime window o eliable eco dings, we moni o ed cul u es a days 3,
4, and 5 pos -seeding (Figu e 2.11). Examples shown o each day illus a e ep esen a i e
eco dings om quali a i e analysis o h ee cul u e pla es pe ime poin , wi h se e al egions
examined in each dish. On day 3, he monolaye was o en incomple e, and spon aneous spiking
was no de ec ed. E en in mo e con luen egions, cells appea ed insu icien ly ma u e o
suppo he ion channel exp ession necessa y o obus spiking. In hese a eas, we obse ed
slow calcium ansien s, bu no he as , pe iodic signals cha ac e is ic o elec ical spikes. By
day 4, he cul u es consis en ly o med dense and uni o m monolaye s, and spiking ac i i y
became clea , hy hmic, and ep oducible ac oss samples. Es ima ing dominan equency o he
calcium signals in p esen ed sample yielded peak alue o 0.26 Hz, as de e mined using he
Welch me hod. On day 5, signs o o e g ow h we e e iden , wi h cells g owing in mul iple laye s.
Unde hese condi ions, spiking ac i i y was educed o absen , and when p esen , i was o en
un eliable and no synch onized. The co esponding Welch spec um o he day 5 ace iden i y
a dominan equency a 0.26 Hz, bu he powe spec al densi y o he peak was app oxima ely
Chap e 2. Sel -spiking HEK Cells
71
h ee old lowe compa ed o day 4. These obse a ions de ine a na ow expe imen al window,
wi h day 4 ep esen ing he mos a o able condi ions o cap u ing spon aneous, synch onized
spiking e en s in his sys em.
We nex examined whe he he obse ed ac i i y e lec ed coo dina ed beha io ac oss he
monolaye a he han independen cellula esponses (Figu e 2.12). Fluo escence was eco ded
om a 166.4 × 166.4 µm ield o iew a 10 ames pe second, using 100 ms exposu es. Wi h a
epo ed wa e p opaga ion speed o app oxima ely 2 cm/s, a spike would a e se he ield in
abou 10 ms, well below he empo al esolu ion o ou acquisi ion. Unde hese condi ions,
p opaga ing spikes a e expec ed o appea as simul aneous e en s ac oss he ield, enabling
synch onized ac i i y o be cap u ed as a uni o m signal. The a e aged luo escence ace o
whole FOV (Figu e 2.12 B) shows egula calcium ansien s wi h consis en shape and iming. A
hea map o 500 indi idual cell aces (Figu e 2.12C) e eals a uni o m pa e n: nea ly all cells
wi hin he ield exhibi aligned spiking, no signi ican a ia ion in iming. This synch ony is u he
illus a ed in selec ed single-cell aces (Figu e 2.12 D), which ollow he same hy hm and phase.
These esul s con i m ha he cul u e beha es as a synch onized sys em, wi h cells spiking
collec i ely a egula in e als.
Chap e 2. Sel -spiking HEK Cells
72
Figu e 2.11 Spon aneous spiking ac i i y in Calcium G een-1-labeled cells monolaye s a di e en days
pos -seeding. Top: Fluo escence images om days 3, 4, and 5. Bo om: A e age luo escence aces o e
60 seconds o whole ield o iew ( ull ame a e age). Day 3 monolaye s a e incomple e and show no
spiking. On day 4, uni o m monolaye s s a o exhibi pe iodic spiking. By day 5, cul u es a e o e g own
and ac i i y is diminished o absen . On he igh , powe spec al densi y plo s om Welch analysis
illus a e he equency componen s co esponding o each ace.
Day 3 Day 4 Day 5
50 m50 m50 m
Chap e 2. Sel -spiking HEK Cells
79
(16) Même, W.; Ezan, P.; Venance, L.; Glowinski, J.; e al. ATP-Induced Inhibi ion o Gap
Junc ional Communica ion Is Enhanced by In e leukin-1 β T ea men in Cul u ed
As ocy es. Neu oscience 2004, 126 (1), 95–104.
h ps://doi.o g/10.1016/j.neu oscience.2004.03.031.
Chap e 2. Sel -spiking HEK Cells
80
81
Chap e 3
Quan um Do s o De ec ing Neu onal
Ac i i y
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
82
1. Sphe ical Quan um Wells
Sphe ical quan um wells (SQWs) ep esen an ad anced ca ego y o colloidal semiconduc o
nanoc ys als ha ea u e a mul ilaye ed a chi ec u e, commonly e e ed o as co e/well/shell
he e os uc u es. These s uc u es consis o a cen al semiconduc o co e su ounded by wo
dis inc shell laye s, each con ibu ing o he o e all quan um con inemen and op ical beha io
o he ma e ial.1,2 One o he mos ex ensi ely in es iga ed con igu a ions is he CdS/CdSe/CdS
s uc u e, composed o a CdS co e (2.7 eV bulk bandgap), a middle CdSe laye unc ioning as he
quan um well (1.75 eV bulk bandgap), and an ou e CdS shell.3,4 In his a chi ec u e, he
in e media e CdSe laye ac s as a h ee-dimensionally con ined egion o hole wa e unc ions,
while elec on wa e unc ions a e delocalized o e he en i e olume o he nanoc ys al, which
leads o a quasi- ype-II band alignmen . This unique spa ial dis ibu ion o cha ge ca ie s is
cen al o he dis inc i e op ical and elec onic p ope ies o SQWs, dis inguishing hem om
bo h 2D plana quan um wells and 0D sys ems such as adi ional quan um do s (QDs) o
con en ional co e/shell quan um do s (CSQDs).
S ong con inemen wi hin he sphe ical CdSe laye con ibu es o high pho ochemical s abili y
and quan um yields app oaching uni y.1 Mo eo e , he s ong con inemen leads o well-
sepa a ed elec onic s a es and al e ed ecombina ion dynamics, including supp essed Auge
ecombina ion and ex ended ca ie li e imes.5–7 In pa icula , hick-shell CdS/CdSe/CdS SQWs
ha e achie ed nea -uni y pho oluminescence quan um yield (PLQY) a oom empe a u e
(Figu e 3.16 A). This high e iciency esul s om educed la ice misma ch be ween he CdSe
well and CdS laye s, whe e cohe en s ain supp esses mis i de ec o ma ion. These s uc u es
also exhibi s ongly educed blinking, spending up o 90 o he ime in he emissi e “on”
s a e.1,3,8 Ano he no ewo hy p ope y o SQWs is hei enhanced mul iexci on emission,
especially in la ge-co e s uc u es. SQWs wi h la ge co e diame e s ha e shown high biexci on
quan um yields, some imes app oaching 100%. This e iciency a ises om educed Auge
ecombina ion a es and inc eased e ec i e exci on olumes (Figu e 3.16 B). Fu he mo e, he
well-de ined sphe ical quan um con inemen allows highe biexci on binding ene gies
compa ed o pu ely sphe ical co e nanoc ys als.4,7 Al hough he CdS/CdSe/CdS sys em is
ex ensi ely s udied – bene i ing om he well-es ablished unde s anding o CdSe and CdS
quan um do s and co e/shell s uc u es – o he ma e ial combina ions, including ZnS/CdS/ZnS,9
InP/ZnSe/ZnS (cadmium ee al e na i e)10, and CdSe/CdS/ZnS11 ha e also been in es iga ed.
These al e na i e a chi ec u es aim o ailo exci on dynamics, ca ie con inemen , and band
alignmen s o mee he demands o speci ic echnological applica ions.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
83
Figu e 3.16 Op ical pe o mance enhancemen in semiconduc o nanoc ys al he e os uc u es h ough
sphe ical-well geome y and in e acial alloying. (A) Compa ison o pho oluminescence quan um yield
(PLQY) as a unc ion o CdS shell hickness o CdS/CdSe/CdS sphe ical quan um well (SQW) nanoc ys als
and con en ional CdSe/CdS co e/shell s uc u es. SQW nanoc ys als achie e nea -uni y PLQY and
supp essed blinking a shell hicknesses g ea e han 5 nm, a ibu ed o cohe en s ain in he CdSe laye ,
de ec - ee ou e shell g ow h, and educed hole apping a he su ace. (B) Pho oluminescence decay
dynamics o con en ional CdSe/CdS co e/shell quan um do s wi h an ab up in e ace and co e/alloy/shell
s uc u es inco po a ing a CdSeₓS₁₋ₓ alloy in e laye . The p esence o he alloyed in e ace slows
mul iexci on decay, indica ing supp essed Auge ecombina ion due o a smoo he hole con inemen
po en ial.
Panel A ep in ed wi h pe mission om B. G. Jeong, Y.-S. Pa k, J. H. Chang, e al., ACS Nano, 2016, 10, 10,
9297–9305. Copy igh © 2016 Ame ican Chemical Socie y. Panel B ep in ed wi h pe mission om
W. K. Bae, L. A. Padilha, Y.-S. Pa k, e al., ACS Nano, 2013, 7, 4, 3411–3419. Copy igh © 2013 Ame ican
Chemical Socie y.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
84
Due o hese supe io pho ophysical p ope ies, SQWs ha e been iden i ied as highly p omising
candida es o a wide ange o ad anced op oelec onic applica ions. In quan um ligh sou ces,
hei supp essed blinking and nea -uni y pho oluminescence quan um yield (PLQY) make hem
excellen single-pho on emi e s o quan um c yp og aphy, wi h an ibunching sus ained e en
a elecom wa eleng hs when in eg a ed in o pho onic ca i ies.3,8,12 In ligh -emi ing de ices,
SQWs ha e imp o ed mic oca i y lase pe o mance and enabled lexible displays wi h b oad
colo gamu co e age and ex ended li e imes.4,7 They also show p omise in adia ion de ec ion,
wi h as X- ay esponse imes ha ou pe o m adi ional scin illa o s, opening he possibili y
o new gene a ion o adia ion de ec ion.8,13
Among hese di e se applica ions, bioimaging s ands ou as a pa icula ly p omising a ea.
Quan um do s, including SQWs, a e ac i ely esea ched as luo escen p obes o biosensing
applica ions. Due o hei b igh ness, pho os abili y, and he abili y o be unc ionalized o
a ge ing speci ic cellula s uc u es hey a e widely used o bio-labeling and cellula imaging.14
Thei b igh and unable emission can be ailo ed as p e e ed o pa icula applica ion. I
needed, i can be enginee ed in o he nea -in a ed egion, acili a ing deep- issue imaging wi h
minimal pho odamage and educed backg ound au o luo escence. Addi ionally, hei enhanced
wo-pho on abso p ion c oss-sec ions enable high- esolu ion imaging using nea -in a ed
exci a ion, which is ad an ageous o in i o applica ions.4,7,15
In summa y, SQWs cons i u e a majo ad ancemen in colloidal nanoc ys al echnology,
e ec i ely combining bene icial aspec s om plana quan um wells and classical nanoc ys als.
By s a egically managing exci on dynamics and band alignmen h ough ma e ial and s uc u al
op imiza ion, SQWs signi ican ly ou pe o m con en ional semiconduc o nanos uc u es (Table
3.3), p o iding new oppo uni ies ac oss mul iple scien i ic and indus ial domains.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
85
Table 3.3 Compa ison o con en ional quan um do s and sphe ical quan um well (SQW) nanoc ys als
ac oss s uc u al, op ical, and unc ional p ope ies.
Aspec
Quan um Do s
ingle componen o hin
co e/shell)
Sphe ical Quan um Wells
(co e/well/shell)
Re e ence
Dimensional
con inemen
Con inemen in all h ee
dimensions; band-gap ises
and he DOS becomes disc e e
as he diame e dec eases
Con ined adially, wi h ee
ca ie mo ion a ound he well;
esul s in quasi-2D shell s a es
go e ned mainly by well
hickness, no pa icle diame e
6 Ba aglia e al.
Angew. Chem. 2003
Ene gy-le el
s uc u e,
exci on wa e-
unc ions
1S-1P ene gy spacing se by
co e adius; elec on & hole
wa e- unc ions o e lap
s ongly ( ype-I) unless hick
g aded shells a e added
Quasi- ype-II: hole in CdSe,
elec on in CdS, weak o e lap;
gap se by CdSe hickness – each
added monolaye ed-shi s, size
la gely i ele an
5 Peng e al. APL 2005
6 Ba aglia & Peng
Angew. Chem. 2003
Op ical
p ope ies
(abso p ion& PL)
PL FWHM 30-40 nm (b oade
due o size dis ibu ion and
phonon in e ac ions)
PL FWHM na owe , o en ~20
nm, colo unning by al e ing
well hickness
1 Jeong e al. ACS
Nano 2016
Abso p ion onse is b oade ,
g adual ise, caused mainly by
size dispe sion and a ia ions
in elec on s uc u e
Abso p ion onse is s eep and
well-de ined, om consis en
well hickness and quan ized 2D-
like ene gy le els
S okes shi mode a e (~20
meV), a ies by s uc u e.
PLQY 50-70 a e passi a ion
S okes shi la ge (30-60 meV)
due o quasi- ype-II alignmen ,
PLQY ~ 100%
Ca ie dynamics
( adia i e & non-
adia i e
li e imes)
Radia i e li e imes 5-20 ns;
Auge ecombina ion o
mul iexci ons is as (0.1 ns);
blinking and gain loss
Exci on li e ime simila o QDs,
bu mul iexci on li e ime
ex ended o >1 ns (“gian ”
supp ession); biexci on PLQY 80-
100%
4 Nagamine e al. ACS
Pho onics 2020 ;
7 Ma de e al. ACS
Ma e . Le . 2023
Response o
ex e nal elec ic
ields
Nea ly co-loca ed ca ie s, he
S a k esponse is la gely
quad a ic and ed-shi s s ay
modes (0-2 meV/10 kV cm-1)
Build in adial dipole makes hin-
well SQWs espond linea ly (3-6
meV/10 kV cm-1 o 1-2 ML
wells), bu hick CdS ba ie
sc eens modes ex e nal ields.
1 Jeong e al. ACS
Nano 2016
High-o de
nonlinea op ics
TPA c oss-sec ion σ₂ 103-104
GM; enhanced in aniso opic
s uc u es
SQWs show σ₂ up o 1.8 × 102
GM/nm3 and low ASE h esholds
(~10 µJ/cm2); biexci on binding
unable (-130 o -50 meV)
8 Xiang e al.
Nanoma e ials 2024
Applica ions
demons a ed
QD-LEDs, pho ode ec o s,
LSCs, single-pho on sou ces.
Low- h eshold solu ion-
p ocessed lase s, op ical
ampli ie s, single-pho on
emi e s, down-con e e s o
SSL; quan um-ligh sou ces
16 Ga cía-de-A que e
al. Science 2021;
3 Allemand e al.
Nano echnology 2022
Key challenges
Auge losses & blinking,
Su ace- ap passi a ion
P ecise con ol o well hickness
a ML le el, Scale-up
17 C agg e al. Nano
Le . 2010;
18 Giansan e e al.
JPCL 2017
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
86
The s udy in oduced in his PhD disse a ion ocuses on he CdS/CdSe/CdS con igu a ion as he
p ima y s uc u al model, based on bo h heo e ical p edic ions and expe imen al indings ha
highligh i s high op ical e iciency and s ong sensi i i y o ex e nal elec ic ields.5,8,19 These
a ibu es posi ion SQWs as p omising candida es o sensing neu onal ac i i y, whe e changes
in memb ane ol age could modula e hei op ical emission.20 Such ol age sensi i i y o e s a
po en ial pa hway o non-in asi e neu al imaging wi h high spa ial and empo al esolu ion.21
While expe imen al alida ion is s ill unde way, eme ging s udies sugges ha SQWs could play
a pi o al ole in he de elopmen o ad anced neu oimaging ools and b ain-compu e in e ace
echnologies.
1.1. O e iew o Quan um-do -based Pla o ms
Be o e di ing in o he expe imen al p o ocols and pe o mance analysis, i is impo an o
p o ide a clea o e iew o he quan um do (QD) sys ems explo ed in his s udy. These
nanoc ys al a chi ec u es o m he basis o all subsequen in es iga ions p esen ed in his
chap e . Al hough he a ionale behind he selec ion and de elopmen o each sys em will be
discussed in de ail in la e sec ions, in oducing he ull se o ma e ials he e will help o ien he
eade and p o ide a consis en e e ence poin h oughou he ex .
Figu e 3.17 summa izes he six QDs-based sys ems, iden i ied as Sys ems I-VI, ha we e included
in he cou se o s udy p esen ed in his disse a ion. These sys ems span a ange o co e-shell
geome ies and su ace chemis ies, allowing o a sys ema ic examina ion o how s uc u al
a ia ions in luence bo h op ical pe o mance and biological in e ac ions. Sys em I is a
comme cially a ailable CdSe/ZnS co e-shell nanoc ys al capped wi h i-n-oc ylphosphine oxide
(TOPO), se ing as a widely used e e ence s anda d. Sys ems II, III, and IV sha e a CdS/CdSe/CdS
sphe ical quan um well (SQW) con igu a ion bu di e in hei su ace coo dina ion s a egies:
cadmium olea e (sys em II), zinc olea e (sys em III), and a combina ion o cadmium luo ide and
oc ylamine (sys em IV). Sys em V builds on he s uc u e o III by inco po a ing an addi ional ZnS
ou e shell, enabling e alua ion o he e ec s o ex ended passi a ion. Sys em VI, on he o he
hand, modi ies he ligand en i onmen by eplacing linea olea e wi h a b anched
2-hexyldecanoa e, p o iding insigh s in o how ligand geome y impac s colloidal beha io and
biological in e ac ions.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
87
Figu e 3.17 O e iew o quan um do sys ems in es iga ed h oughou his s udy. Six dis inc nanoc ys al
a chi ec u es we e e alua ed, di e ing in co e-shell s uc u e and su ace ligand composi ion. Sys em I
ep esen s a comme cially a ailable CdSe/ZnS co e-shell quan um do capped wi h i-n-oc ylphosphine
oxide (TOPO), se ing as a e e ence ma e ial. Sys ems II-VI a e cus om-syn hesized CdS/CdSe/CdS o
CdS/CdSe/CdS/ZnS sphe ical quan um wells (SQWs), each modi ied wi h a dis inc su ace coo dina ion
en i onmen : cadmium olea e (II), zinc olea e (III and V), cadmium luo ide and oc ylamine (IV), and a
b anched 2-hexyldecanoa e ligand (VI). This ma e ials lib a y enables a sys ema ic in es iga ion in o how
nanoc ys al a chi ec u e and su ace chemis y in luence op ical beha io , colloidal s abili y, and
memb ane usion e iciency.
All sys ems we e designed and p epa ed wi h ep oducible p o ocols, ensu ing consis en co e
size and quan um well hickness. This uni o mi y enables he e alua ion o he in luence o
su ace chemis y and shell composi ion on he physical o quan um do s. The sec ions ha
ollow will i s de ail he syn he ic s a egies used o each cus om-made nanoc ys al sys em
(3.1.3 Su ace Chemis y on CdS/CdSe/CdS Nanoc ys als), ollowed by in es iga ions o wa e
ans e (3.2 S a egies o Wa e T ans e ), and hei in e ac ion wi h biological ma e ial
(3.3 Cell Memb ane Inse ion). F om his poin o wa d, each sys em will be e e ed o by i s
assigned Roman nume al, wi h Figu e 3.17 se ing as a isual e e ence h oughou he chap e .
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
88
1.2. Syn he ic App oaches
Syn hesis o laye ed CdS/CdSe/CdS nanoc ys als
CdS/CdSe/CdS nanoc ys als wi h laye ed adial composi ion we e syn hesized unde an ine
a gon a mosphe e using a combina ion o single and sequen ial injec ion s a egies a 240 °C.
The me hod elied on he simul aneous injec ion o sul ide and selenide p ecu so s in o a
cadmium olea e and oleic acid solu ion o ini ia e he g ow h o he CdS/CdSe co e/shell
s uc u e, ollowed by he sequen ial g ow h o an ou e CdS shell. The chalcogen p ecu so
mix u e consis ed o hiou ea22 and selenou ea23 de i a i es, which se ed as sul u and
selenium sou ces and enabled p ecise con ol o nuclea ion and g ow h. As epo ed by he
g oup o Jona han Owen, hese p ecu so s elease me al-chalcogen monome s, which se e as
building blocks o nanoc ys als, a unable a es, allowing con ol o e he numbe o nuclei
o med and hus he inal size o he nanoc ys als. Unlike adi ional me hods ha s op he
eac ion ea ly o con ol size, o en a he expense o yield, his app oach allows he eac ion o
p oceed o ull comple ion, p oducing nanoc ys als wi h uni o m size, well-de ined composi ion,
and high e iciency. Fu he mo e, by adjus ing he eac i i y o he p ecu so s, he me hod can
be ailo ed o wo k wi h di e en me al sou ces, enabling he syn hesis o a wide ange o me al
sul ide and selenide nanoc ys als.
La ge-scale syn hesis o ai -s able N,N′-disubs i u ed and N,N,N′- isubs i u ed hiou eas can be
e icien ly achie ed in a single-s ep eac ion, in mos cases pe o med in a oom empe a u e.
This p ocess in ol es he di ec coupling o eadily a ailable subs i u ed iso hiocyana es wi h
p ima y o seconda y amines, esul ing in high eac ion e iciency.22 De ailed p o ocols can be
ound in he Appendix (Sec ion 3.1.1).
Small CdS/CdSe/CdS quan um wells we e syn hesized using a sequen ial injec ion s a egy unde
an ine a gon a mosphe e, inside a glo ebox. Fo he ini ial CdS/CdSe o ma ion, a solu ion o
cadmium olea e (0.244 g, 0.36 mmol), oleic acid (0.204 g, 0.72 mmol), and hexadecane
(28.46 mL, 22 g) was loaded in o a 100 mL h ee-neck ound-bo om lask equipped wi h a
magne ic s i ba , connec ed o he Schlenk line unde A gon and hea ed o 240 °C (Figu e 3.18,
panel A). In pa allel, a chalcogen p ecu so mix u e was p epa ed by dissol ing N-hexyl-N′-
dodecyl hiou ea (0.0496 g, 0.15 mmol) and 1,3-die hy imidazolidine selone (0.0308 g,
0.15 mmol) in e aglyme (0.8 g, 0.848 mL). The chalcogen p ecu so solu ion was swi ly
injec ed in o he ho cadmium mix u e, and he eac ion was main ained a 240 °C o
60 minu es (Figu e 3.18, panel B). The eac ion o his as -con e ing hiou ea (con e sion a e
cons an kcon = 0.03 s-1)22 and slowe -con e ing selenou ea (kcon = 0.001 s-1)23 wi h cadmium
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
95
Ligand exchange o Zn(2-hexyldecanoa e)2
Ligand exchange using Zn(2-hexyldecanoa e)2 was pe o med ollowing he same p ocedu e as
desc ibed o Zn(olea e)2, wi h he only modi ica ion being he subs i u ion o zinc olea e wi h
an equimola amoun o Zn(2-hexyldecanoa e)2. All o he eac ion condi ions and pu i ica ion
s eps emained unchanged.
Ligand exchange – luo ina ion
Cd-olea e-s abilized SQWs (0.100 µmol) we e d ied unde acuum and edispe sed in
oc adecene (15 mL). Oc ylamine (0.0259 g, 0.0331 mL, 0.2 mmol) and benzoyl luo ide (0.0248 g,
0.0205 mL, 0.2 mmol) we e added o he dispe sion, and he eac ion mix u e was s i ed a
oom empe a u e o 1 hou . The esul ing nanoc ys als we e pu i ied by p ecipi a ion wi h
ace one and cen i uga ion.
Shelling wi h addi ional ZnS laye
To ob ain CdS/CdSe/CdS/ZnS mul ilaye s uc u e, Cd-olea e-s abilized CdS/CdSe/CdS SQWs
we e p epa ed as p e iously desc ibed, wi h he CdS shelling s ep modi ied o use hal he
eagen quan i ies and hal he eac ion ime. Following CdS shell g ow h, a ZnS shelling solu ion
was p epa ed by dissol ing zinc olea e (0.3393 g, 0.357 mL, 0.54 mmol), 2-hexyldecanoic acid
(0.1385 g, 0.1556 mL, 0.54 mmol), and N-hexyl-N′,N′-dibu yl hiou ea (0.1472 g, 0.54 mmol) in
oc adecene (14.2 g, 18.0 mL). This mix u e was injec ed in o he eac ion ia sy inge pump o e
2 hou s a a a e o 9 mL/h. The esul ing nanoc ys als we e pu i ied by ou sequen ial
p ecipi a ions using a 5:1 / me hyl ace a e o oluene mix u e.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
96
2. S a egies o Wa e T ans e
In o de o sphe ical quan um wells o be applicable in biological sys ems, hei ansi ion om
o ganic sol en s o wa e o wa e -based bu e s is c i ical. A e he syn hesis, nanoc ys als a e
ypically s abilized wi h o ganic molecules (e.g., ioc ylphosphine oxide, oleic acid), which
makes hem soluble in non-pola o ganic sol en s such as hexane, oluene o chlo o o m and
ensu e colloidal s abili y. This hyd ophobic su ace chemis y also d i es he ini ial associa ion
wi h he cell memb ane: by he classic hyd ophobic e ec , alkyl chains on he QD su ace
minimize un a o able wa e con ac s by pa i ioning in o he lipid bilaye ’s hyd ophobic co e.24
Ba e al. demons a e on simpli ied suppo ed lipid bilaye s ha nega i ely cha ged QDs i s
dock on o he memb ane ia elec os a ic a ac ion and hen pene a e a high‐cu a u e o
de ec si es – whe e hyd ophobic ligand chains inse in o he lipid ails – suppo ing a wo‐s ep,
de ec -media ed mechanism o memb ane embedding.24
Lipophilic-coa ed quan um do s inhe en ly pa i ion in o he hyd ophobic co e o he memb ane
a he han s aying in he su ounding aqueous phase. Ye , o enable e icien cellula deli e y –
and, ul ima ely, eliable ol age sensing – hese nanopa icles mus i s ea u e colloidal
s abili y in physiological media.
To ans e QDs in o aqueous solu ion, se e al s a egies ha e been epo ed in he li e a u e.
Ligand exchange eplaces na i e hyd ophobic ligands wi h hyd ophilic ones (such as hiols o
mul iden a e ligands).25,26 This me hod is ela i ely s aigh o wa d bu can dis u b he QD
su ace and o en esul s in signi ican loss o pho oluminescence quan um yield (QY) and
educed colloidal s abili y. In con as , encapsula ion echniques p ese e he o iginal ligand
shell by w apping he hyd ophobic QD wi h an amphiphilic coa ing. These include polyme o
su ac an micelle encapsula ion and coa ing wi h biomolecules like lipids o p o eins.
Encapsula ion is gene ally mo e complex bu ends o be e e ain he QDs’ op ical p ope ies.
Fo example, amphiphilic polyme coa ings main ain he na i e hyd oca bon ligand
en i onmen , yielding wa e -dispe sible QDs wi h minimal changes in abso p ion o emission
spec a.27,28 Simila ly, p o ein-based coa ings (e.g., embedding QDs in bo ine se um albumin o
polyme -p o ein hyb id shell) can con e wa e solubili y while keeping he o iginal ligand in
place, p ese ing luo escence and p o iding biological in e aces.29–31 Each app oach di e s in
complexi y and e ec i eness. Ligand exchange p oduces smalle hyd odynamic diame e s bu
isks diminished pho os abili y, whe eas polyme o lipid encapsula ion yields la ge
bioconjuga es wi h excellen pho ophysical e en ion.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
97
Among me hods ha main ain he na i e hyd ophobic coa ing on QDs, wo p ominen
s a egies a e encapsula ion in oc yl glucoside (OCG) micelles and lipid esicles (liposomes).
These app oaches a oid di ec chemical modi ica ion o he QD su ace, he eby aiming o
p ese e he o iginal ligand passi a ion and he QD’s pho ophysical p ope ies.
2.1. OCG Micelles
n-Oc yl-β-D-glucoside (OCG) is a small non-ionic de e gen wi h a hyd ophobic oc yl ail and a
hyd ophilic glucose head (Figu e 3.7 A). I is widely used in memb ane biochemis y due o i s
abili y o gen ly solubilize lipid bilaye s while main aining he s uc u al and unc ional in eg i y
o memb ane-associa ed p o eins. OCG is pa icula ly e ec i e o isola ing unc ional
memb ane p o eins, as i dis up s he memb ane en i onmen wi hou dena u a ion and can
be eadily emo ed h ough dialysis o dilu ion.32 Addi ionally, OCG is employed o induce
con olled ansi ions be ween lipid esicles and micelles by modula ing i s concen a ion nea
he c i ical micelle concen a ion.33,34 This p ope y is aluable o s udying memb ane
cu a u e, lipid packing, and he ene ge ics o bilaye assembly.32 In suppo ed lipid bilaye
sys ems, OCG acili a es he p ecise solubiliza ion and e o ma ion o memb anes on solid
subs a es, which is essen ial o echniques such as a omic o ce mic oscopy and o he su ace-
based analyses.
Al hough he encapsula ion o quan um do s wi hin OG micelles has no been p e iously
epo ed, a ela ed s udy by Rasch e al. demons a ed he success ul aqueous phase ans e o
small gold nanopa icles using OCG molecules, esul ing in he o ma ion o nanopa icle- esicle
hyb id s uc u es.35 Since p e ious s udies ha e shown ha TOPO-capped CdSe quan um do s
can be e ec i ely ans e ed o aqueous media h ough encapsula ion wi h su ac an s o ming
micella s uc u es,36 and gi en he widesp ead use o OCG in memb ane biochemis y, we
aimed o in es iga e i s po en ial o memb ane s aining. OCG’s abili y o solubilize hyd ophobic
en i onmen s while p ese ing molecula in eg i y, along wi h i s es ablished ole in modula ing
esicle-micelle ansi ions, makes i a s ong candida e o encapsula ing hyd ophobic quan um
do s wi hou al e ing hei na i e su ace chemis y. In his s udy, we e alua e he use o OCG
micelles o aqueous phase ans e o QDs and assess hei u ili y o ans-memb ane deli e y
in biological sys ems.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
98
Encapsula ion p o ocol
Da a shown in his subchap e , o SQWs encapsula ed wi hin OCG micelles, a e based on he
sample desc ibed in a p e ious sec ion (Figu e 3.19).
P io o conduc ing any expe imen s, i was essen ial o de e mine he concen a ion o
sphe ical quan um do s in solu ion. This was accomplished using he Bee -Lambe law,
exp essed as A = εcl, whe e A is he abso bance, ε is he mola ex inc ion coe icien , c is he
concen a ion, and l is he op ical pa h leng h. In ou calcula ions, we u ilized he abso bance
alue a a wa eleng h o 350 nm and assumed an ex inc ion coe icien o 1.7, which aligns wi h
epo ed alues o CdSe quan um do s o compa able size and op ical p ope ies.37
Figu e 3.22 Encapsula ion o CdS/CdSe/CdS sphe ical quan um wells (SQWs) using he amphiphilic
su ac an β-D-oc yl glucoside (OCG). (A) Chemical s uc u e and schema ic ep esen a ion o OCG,
highligh ing i s amphiphilic na u e wi h a hyd ophilic glucose head (blue) and a lipophilic alkyl ail (yellow).
(B) Schema ic illus a ion o he SQW encapsula ion p ocess. Hyd ophobic SQWs a e phase- ans e ed
om dichlo ome hane (DCM) o wa e ia OCG-assis ed encapsula ion, o ming s able SQWs@OCG
micelles upon DCM e apo a ion a 50 °C.
To op imize he p o ocol, se e al key pa ame e s we e sys ema ically in es iga ed and adjus ed.
These included he choice o o ganic sol en , he wa e - o-sol en a io, he OCG- o-SQW a io,
s i ing speed and empe a u e. Mul iple phase ans e s a egies we e e alua ed, wi h he
scheme p esen ed abo e in Figu e 3.22 ep esen ing he mos ep oducible me hod iden i ied
du ing he s udy. The ollowing sec ion ocuses on he in luence o OCG concen a ion on he
quali y o he micelles ob ained.
In a 4 mL glass ial equipped wi h a magne ic s i e , 500 μL o OCG solu ion in dichlo ome hane
(DCM) a he desi ed concen a ion was mixed wi h 100 μL o SQWs edispe sed in DCM
(0.06 μM). The mix u e was s i ed a oom empe a u e o a leas 5 minu es. Subsequen ly,
250 μL o wa e was added unde igo ous s i ing, and he sys em was s i ed o an addi ional
5 minu es a oom empe a u e. The solu ion was hen hea ed o 50 °C un il he DCM had ully
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
99
e apo a ed. Finally, an addi ional 250 μL o wa e was in oduced, and he esul ing dispe sion
was subjec ed o u he op ical cha ac e iza ion.
Cha ac e iza ion
The concen a ion o β-D-oc yl glucoside (OCG) used du ing encapsula ion had a p onounced
e ec on he physical and op ical quali y o he esul ing SQW o mula ions. Abo e i s c i ical
micelle concen a ion (CMC, ~20-25 mM), OCG molecules spon aneously assemble in o micelles
in aqueous media. When hyd ophobic SQWs a e in oduced in o an aqueous OCG solu ion, he
alkyl chains o OCG in e ac wi h he hyd ophobic ligand shell o he SQWs ia he hyd ophobic
e ec , while he suga headg oups o ien owa d he su ounding wa e , esul ing in he
o ma ion o a micella colloid. The encapsula ion p ocess can be ca ied ou unde mild
condi ions, in ol ing mode a e s i ing and gen le hea ing, which makes i s aigh o wa d o
implemen while minimizing he isk o al e ing he nanoc ys al su ace chemis y. As shown in
Figu e 3.23, samples p epa ed wi h 25 mM (sample 1), 50 mM (sample 2), and 100 mM
(sample 3) OCG display p og essi ely g ea e u bidi y and ligh sca e ing, pa icula ly a he
highes concen a ion. Dynamic ligh sca e ing (DLS) measu emen s suppo his obse a ion:
sample 1 exhibi s small micelles wi h a hyd odynamic diame e o 35.2 ± 7.5 nm and a low
polydispe si y index, indica ing uni o m size. In con as , highe OCG concen a ions yield much
la ge and mo e polydispe se agg ega es, wi h hyd odynamic diame e s o 260.8 ± 126.0 nm
and 347.2 ± 146.4 nm o he samples 2 and 3, espec i ely (Figu e 3.23 B).
Pho ophysical cha ac e iza ion con i med ha micelliza ion p ese es he op ical p ope ies o
he SQWs. The co esponding UV-Vis spec a o sample 1 shows p onounced sca e ing, while
lowe -concen a ion samples show minimal de ia ion om baseline, sugges ing he p esence o
well-dispe sed micelles o pa icles smalle han 100 nm (Figu e 3.23 C). Emission spec a e eal
a concen a ion-dependen blue shi in pho oluminescence maximum om 529 nm (sample 1,
25 mM) o 526 nm (sample 2, 50 mM) and 523 nm (sample 3, 100 mM) (Figu e 3.23 D). All
micella samples a e he e o e sligh ly ed shi ed ela i e o he same SQWs dispe sed in
oluene (520 nm, Figu e 3.19).
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
100
Figu e 3.23 Visual appea ance and op ical cha ac e iza ion o sphe ical quan um wells (SQWs)
encapsula ed wi h inc easing concen a ions o β-D-oc yl glucoside (OCG). (A) Pho og aphs o samples
con aining 25 mM, 50 mM, and 100 mM OCG unde ambien whi e ligh ( op) and UV illumina ion
(bo om). Samples wi h 25 mM and 50 mM OCG (samples 1 and 2) a e op ically clea wi h a sligh yellow
in , while he 100 mM sample (sample 3) appea s inc easingly u bid due o ligh sca e ing. (B) Dynamic
ligh sca e ing (DLS) analysis shows hyd odynamic diame e s o 35.2 ± 7.5 nm, 260.8 ± 126.0 nm, and
347.2 ± 146.4 nm o he 25 mM, 50 mM, and 100 mM samples, espec i ely. (C) UV-Vis ex inc ion spec a
exhibi a concen a ion-dependen inc ease in baseline in ensi y, consis en wi h enhanced sca e ing in
he 100 mM sample. (D) Emission spec a o he same samples display a blue shi in he
pho oluminescence maximum wi h inc easing OCG concen a ion.
Despi e he di e ences in he abso p ion spec a o sample 1 and sample 3, hei
pho oluminescence li e imes a e no ably simila (Figu e 3.24). Bo h samples exhibi
a bi-exponen ial decay p o ile, wi h componen s o 44.0 ± 1.0 ns and 6.0 ± 0.22 ns o sample 1,
and 43.0 ± 1.1 ns and 9.1 ± 0.31 ns o sample 3. These co espond o a e age luo escence
li e imes o 18.09 ns and 23.69 ns, espec i ely. Compa ed o SQWs dispe sed in oluene
(τ1 = 44.0 ± 2.1 ns, τ2 = 13.0 ± 0.79 ns), bo h encapsula ed samples exhibi a sho ened a e age
li e imes, mo e p onounced a he lowes OCG concen a ion. In e es ingly, he sample wi h he
lowes OCG concen a ion and smalles hyd odynamic diame e displays he sho es
luo escence li e ime. In smalle micelles, a highe su ace- o- olume a io places a g ea e
ac ion o SQWs nea he micelle-wa e in e ace, inc easing exposu e o OCG headg oups and
he aqueous phase. This p oximi y may enhance su ace- o ap-media ed non adia i e
elaxa ion, a ec ing he as e τ2 componen while lea ing he long-li ed componen la gely
unchanged. This is a possible con ibu ing ac o , alongside o he micelliza ion- ela ed e ec s
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
101
such as changes in local dielec ic en i onmen , in e acial s ain, o su ace ligand
ea angemen .
Figu e 3.24. Pho oluminescence li e ime analysis o encapsula ed CdS/CdSe/CdS SQWs. (A) PL decay cu e
o sample 1, i ed wi h a biexponen ial model yielding li e imes o τ₁ = 44.0 ± 1.0 ns and τ₂ = 6.0 ± 0.22 ns,
co esponding o an a e age li e ime o 18.4 ns. (B) PL decay o sample 3, showing τ₁ = 43.0 ± 1.1 ns and
τ₂ = 9.1 ± 0.31 ns, esul ing in a longe a e age li e ime o 23.4 ns. The inc eased τ₂ componen in sample
3 sugges s imp o ed passi a ion o al e ed su ace dynamics.
T ansmission elec on mic oscopy analysis o he CdS/CdSe/CdS SQWs encapsula ed in OCG
micelles e eals ha he micelles exhibi a sphe ical mo phology wi h diame e s anging om
app oxima ely 20 o 100 nm (Figu e 3.25 A). Highe -magni ica ion imaging con i ms ha each
micelle con ains mul iple SQWs densely packed wi hin he micella co e (Figu e 3.25 B).
S a is ical analysis o 250 micelles yields an a e age diame e o 44.3 ± 11.6 nm (Figu e 3.25 C),
which is la ge han he a e age hyd odynamic diame e o 35.2 ± 7.5 nm de e mined by
dynamic ligh sca e ing (Figu e 3.23). The shi in dis ibu ion maximum be ween TEM and DLS
measu emen s may esul om popula ion misma ch due o sample polydispe si y o om
d ying-induced de o ma ion o so micelles du ing g id p epa a ion.
O e all, he OCG micelle encapsula ion me hod is an e ec i e s a egy o p ese ing he
pho ophysical p ope ies o nanoc ys als du ing phase ans e , p o iding a apid and p ac ical
ou e o ob ain wa e -dispe sible QDs while main aining he o iginal spec al ea u es.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
102
Figu e 3.25 TEM analysis o CdS/CdSe/CdS sphe ical quan um wells encapsula ed wi hin OCG micelles.
(A) Low-magni ica ion TEM image showing SQWs encapsula ed wi hin micelles, wi h o e all micelle sizes
anging om 20 o 100 nm. (B) Highe magni ica ion image o he selec ed a ea in (A) e eals ha
indi idual micelles enclose mul iple SQWs. (C) His og am o micelle size dis ibu ion ob ained om he
analysis o 250 micelles based on TEM images. The a e age micelle diame e is 44.3 ± 11.6 nm.
2.2. Lipid Vesicles
Ano he widely employed s a egy o ans e ing hyd ophobic nanoma e ials in o aqueous
en i onmen s in ol es hei encapsula ion wi hin lipid bilaye esicles, commonly e e ed o as
liposomes. This me hod enables he anspo o lipophilic, ligand-coa ed nanopa icles h ough
wa e -based media while p ese ing hei na i e su ace chemis y.
Liposome o ma ion e lec s a gene al o ganiza ional p inciple obse ed in biological sys ems,
whe e amphiphilic molecules spon aneously a ange in o bilaye s uc u es o o m cell
memb anes, anspo esicles, and compa men alized eac ion spaces. Amphiphilic lipids
con ain pola headg oups and non-pola ails, and hei assembly is d i en p ima ily by he
hyd ophobic e ec , suppo ed by an de Waals o ces, elec os a ic in e ac ions, and hyd ogen
bonding. Al hough each o hese non-co alen in e ac ions is indi idually weak, hei combined
e ec yields s able bilaye memb anes. Kine ic ac o s, such as mixing a e, can in luence he
assembly pa hway, po en ially leading o s uc u al a ia ions in memb ane hickness o esicle
mo phology.
Owing o hei a chi ec u e and composi ion, liposomes can encapsula e bo h hyd ophilic
species in he aqueous co e and hyd ophobic species in he bilaye . These p ope ies ha e
enabled hei widesp ead use as model sys ems o s udying memb ane-associa ed phenomena
and as ca ie s o a b oad ange o subs ances, including he apeu ic d ugs and
nanoma e ials.38,39 Encapsula ion wi hin liposomes can educe nanopa icle cy o oxici y and
imp o e s abili y, acili a ing biomedical applica ions. 38,40 Examples include gold nanopa icles
o s imuli- esponsi e elease, 41 sil e nanopa icles o an imic obial ea men s,42,43 zinc
50 nm
BA
200 nm
C
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
103
oxide44,45 and ce ium oxide46 nanopa icles o cance he apy, i anium dioxide nano ubes o
sus ained d ug deli e y, and supe pa amagne ic i on oxide nanopa icles in magne oliposomes
o hype he mia, a ge ed chemo he apy, and magne ic esonance imaging (MRI).47–50
Liposomes ha e also been used o encapsula e semiconduc o quan um do s o bioimaging,51,52
and hyb id sys ems combining liposomes wi h silica, polyme s, o g aphene-based ma e ials
ha e been de eloped o enhance encapsula ion e iciency and enable s imuli- esponsi e
elease.53,54
Typically, he nanopa icles a e co-dissol ed wi h phospholipids in an o ganic sol en ; a e
sol en e apo a ion and hyd a ion, he lipids spon aneously sel -assemble in o bilaye esicles,
wi h he hyd ophobic nanoc ys als becoming embedded wi hin he nonpola egion o he
memb ane.
We conduc ed a se ies o expe imen s o op imize he syn hesis o phospholipid esicles
inco po a ing quan um do s wi hin hei lipid bilaye s. The phospholipids used we e selec ed
based on hei s uc u al and unc ional p ope ies, which suppo bo h s able esicle o ma ion
and he po en ial o memb ane usion – an aspec ele an o ou en isioned biomedical
applica ions. A de ailed jus i ica ion o he lipid composi ion is p o ided in Sec ion 3.2,
Fusosomes – Fusogenic Lipid Vesicles (page 127). Howe e , i is wo h no ing he e ha he
molecula cha ac e is ics o hese lipids we e chosen o enhance he in e ac ion o he esicles
wi h biological memb anes, which is cen al o he aim o his s udy.
P epa a ion o lipid esicles
Vesicles can be classi ied as unilamella , bilamella , o mul ilamella based on he numbe o
lipid bilaye s hey con ain. Phospholipids, depending on hei molecula size and h ee-
dimensional a angemen , commonly o m ei he small unilamella esicles o gian unilamella
esicles.55 To mee he cen al objec i es o he p esen wo k, he goal was o p oduce small,
unilamella esicles wi h minimal size a iabili y.
The lipid composi ion used in hese expe imen s was adap ed om he wo k o
G. Gopalk ishnan e al.56 A lipid solu ion was p epa ed in chlo o o m a a o al concen a ion o
1 mM, comp ising 5% o he ca ionic lipid DOTAP, 0.5% DPPE-PEG2000, and 74.5% DMPC
(see molecula s uc u es in Figu e 3.26 A) , and was mixed wi h 3 × 10-7 mmol o quan um do s.
Once he mix u e was ho oughly homogenized, he sol en was e apo a ed unde a s eam o
ni ogen, and he esul ing lipid ilm was u he d ied unde acuum o 2 hou s. Following
dehyd a ion, 1 mL o PBS bu e was added o he ial, and he solu ion was o exed o
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
104
edispe se he quan um do s. A magne ic s i e was in oduced o ensu e ho ough mixing. To
acili a e comple e dispe sion, he ial was in e mi en ly o exed and b ie ly imme sed in a ho
wa e ba h.
A e ob aining a homogeneous mix u e o lipids and quan um do s in PBS, a eeze- haw cycle
was pe o med by al e na ely placing he sample ial in a liquid ni ogen ba h (~-200 °C) and a
wa e ba h main ained a 50 °C (Figu e 3.26 B). This echnique is closely ied o he lipids' phase
ansi ion empe a u e, o en e e ed o as he mel ing empe a u e (Tm) – he poin a which
lipid molecules shi om a solid-like gel phase o a luid-like liquid c ys alline s a e. This
ansi ion plays a c i ical ole in liposome p epa a ion, as i in luences memb ane s abili y and
pe meabili y. Below he Tm, lipid bilaye s a e igh ly packed wi h es ic ed molecula mobili y;
abo e he Tm, hey become mo e luid, allowing la e al di usion o lipid molecules wi hin he
memb ane. Repea ed eeze- haw cycles, commonly employed o ans o m mul ilamella
esicles in o unilamella ones, le e age his phase beha io . Du ing eezing, ice c ys al
o ma ion dis up s he bilaye s, b eaking la ge esicles in o smalle agmen s; upon hawing,
hese agmen s eassemble in o unilamella s uc u es. The e iciency o his p ocess depends
on se e al ac o s, including lipid composi ion, ionic s eng h o he bu e , and he Tm, which is
especially c i ical du ing he hawing phase when memb ane luidi y is equi ed o esicle
usion. P ope ly op imizing hese pa ame e s leads o mo e uni o m and s able esicle
popula ions, he eby imp o ing he eliabili y and ep oducibili y o esicle-based sys ems.57,58
DOTAP exhibi s a ansi ion empe a u e below 5 °C, DMPC has a well-cha ac e ized ansi ion
empe a u e o app oxima ely 24 °C, and DSPE unde goes a phase ansi ion a a ound 74 °C;
while PEGyla ion may sligh ly al e his alue, i is expec ed o emain ela i ely high.59 All alues
a e aken om da a p o ided by A an i Pola Lipids (h ps://a an i esea ch.com/). To calcula e
he ansi ion empe a u e o a mixed phospholipid sys em, we can use a weigh ed a e age
app oach based on he composi ion and indi idual ansi ion empe a u es o he lipids.
Tm(mix u e) = (5 °C × 0.05) + (74 °C × 0.005) + (24°C × 0.745) = 18.5 °C (3.1)
The e o e, he es ima ed ansi ion empe a u e o he mixed phospholipid sys em is
app oxima ely 18.5 °C. I is impo an o no e ha his is a simpli ied calcula ion and he ac ual
ansi ion beha io o mixed lipid sys ems can be mo e complex. Fac o s such as lipid-lipid
in e ac ions, domain o ma ion, and he p esence o PEGyla ed lipids can a ec he o e all
phase beha io . Mo e accu a e de e mina ions would equi e expe imen al measu emen s
using echniques such as di e en ial scanning calo ime y (DSC) o nanoplasmonic sensing (NPS)
as desc ibed in he li e a u e.57,60
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
111
S abili y s udy
The isual inspec ion o he solu ions con aining esicles and QDs e ealed sligh haze unde
whi e ligh Figu e 3.34 and b igh luo escence unde UV illumina ion (λex = 365 nm) indica ing
homogenous dis ibu ion o quan um do s (Figu e 3.19).
Figu e 3.34 Visual compa ison o esicle solu ions a h ee di e en ex usion s ages unde (A) whi e ligh
and (B) UV i adia ion.
To e alua e long- e m s abili y o esicle, we moni o ed he change o hyd odynamic diame e
o samples ex uded h ough 100 nm (Ex usion IV), 400 nm (Ex usion III), and 800 nm
(Ex usion II) memb anes o e a i e-day pe iod. The esul s a e p esen ed in Figu e 3.35 which
includes ex inc ion spec a (A, B, C), emission spec a (D, E, F), and dynamic ligh sca e ing (DLS)
da a (G, H, I) o each esicle size. The ex inc ion spec a (Figu e 3.35 A, B, C) emain la gely
unchanged h oughou he obse a ion pe iod, sugges ing s uc u al in eg i y a e well
p ese ed. In he emission spec a (Figu e 3.35 D, E, F), he luo escence p o ile emains
consis en o e i e days; howe e , a g adual dec ease in in ensi y is obse ed. This decline may
esul om ac o s such as esicle agg ega ion, pa ial deg ada ion o quan um do s in
he aqueous en i onmen , o mino sample dilu ion du ing epea ed handling.
Dynamic ligh sca e ing (DLS) measu emen s o he esicles om Ex usion IV, Ex usion III, and
Ex usion II, which ep esen p og essi ely la ge a e age esicle sizes, we e pe o med o e
a i e-day pe iod o p o ide insigh s in o hei s abili y. Fo he smalles esicles om Ex usion
IV (Figu e 3.35 G) he Z-a e age diame e dec eased om 116.4 nm on day 1 o 109.4 nm on
day 5 (-6%), accompanied by a modes inc ease in he polydispe si y index om 0.129 o 0.158
(Figu e 3.36).
Vesicles ex uded h ough 400 nm memb anes (Ex usion III, Figu e 3.35 H) showed no no able
change a e 5 days o incuba ion, wi h di e ences below 2%: he Z-a e age diame e dec eased
om 203.8 nm o 201.2 nm, while he PdI showed a sligh d op om 0.216 o 0.210 (Figu e
WHITE LIGHT UV‐LIGHT
Ex usion II
(800 nm)
A B
Ex usion III
(400 nm)
Ex usion IV
(100 nm)
Ex usion II
(800 nm)
Ex usion III
(400 nm)
Ex usion IV
(100 nm)
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
112
3.36). The la ges esicles om Ex usion II, ob ained using an 800 nm-po e memb ane (Figu e
3.35 I), exhibi ed an inc ease in Z-a e age diame e om 256.8 nm o 269.6 nm (+5%),
accompanied by a ise in PdI om 0.247 o 0.270 (Figu e 3.36). The DLS p o ile b oadened
signi ican ly by day 5, indica ing inc eased he e ogenei y.
These esul s sugges a endency owa d esicle usion o agg ega ion o e ime, highligh ing
he compa a i ely lowe s uc u al s abili y o la ge esicles du ing p olonged s o age. Vesicles
subjec ed o h ee o ou ex usion cycles h ough smalle -po e memb anes (400 nm and
100 nm) main ained ela i ely s able diame e s o e he i s wo days, wi h changes below 1%
(116.4 o 115.3 nm and 203.8 o 203.7 nm, espec i ely). Howe e , all h ee esicle popula ions
exhibi ed measu able size o PdI a ia ions o e i e days, indica ing ha he dispe sions a e
mos eliable when used immedia ely a e p epa a ion o wi hin a sho ime ame, p e e ably
wi hin 24 hou s.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
113
Figu e 3.35 S abili y assessmen o esicles ex uded h ough memb anes o 100 nm (Ex usion IV; A, D, G),
400 nm (Ex usion III; B, E, H), and 800 nm (Ex usion II; C, F, I) o e a i e-day pe iod. Ex inc ion spec a
(A, B, C) show minimal changes. Emission spec a (D, E, F) e eal a g adual dec ease in luo escence
in ensi y, possibly due o agg ega ion o QD deg ada ion. DLS measu emen s (G, H, I) indica e a sligh
inc ease in esicle size, pa icula ly in he 800 nm samples, sugges ing some deg ee o agg ega ion o
usion o e ime.
A B C
D E F
G H I
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
114
Figu e 3.36 Summa y o DLS measu emen o esicles ex uded h ough 100 nm (Ex usion IV), 400 nm
(Ex usion III), and 800 nm (Ex usion II) memb anes o e a i e-day pe iod. The able summa izes
Z-a e age diame e s and polydispe si y indices ob ained om dynamic ligh sca e ing measu emen s on
days 1, 2, and 5. The g aph illus a es empo al changes in a e age esicle diame e o he h ee samples,
showing mino a ia ions o Ex usion IV and Ex usion III esicles and an inc ease Ex usion II esicles.
Fluo escence li e ime measu emen s we e conduc ed o gain deepe insigh in o he
pho ophysical beha io o he esicle samples o e ime. Measu emen s we e pe o med on
esicles ob ained a e ex usion h ough 100 nm-, 400 nm-, and 800 nm-po e memb anes on
days 1, 2, and 5. The esul ing luo escence decay cu es we e i ed using a iple exponen ial
model, a commonly applied app oach o desc ibing complex, mul i-componen decay p ocesses
(Appendix, Figu e A.2). The unc ional o m o he model is shown below:
𝑓(𝑡)= 𝐻 + 𝐴1𝑒{−𝑡
𝜏1}+ 𝐴2𝑒{−𝑡
𝜏2}+ 𝐴3𝑒{−𝑡
𝜏3} (3.2)
He e, ( ) ep esen s he luo escence in ensi y a ime ; H is he baseline o se ; A1, A2, and A3
a e he ampli udes o he h ee decay componen s; and 𝜏1, 𝜏2, 𝜏3 a e he co esponding li e imes.
This model accoun s o mul iple emissi e species o en i onmen s con ibu ing o he o e all
decay p o ile. Al hough he i quali y showed mino de ia ions beyond 100 ns in some cases,
his had li le e ec on he analysis, as he majo i y o pho on coun s occu ed wi hin he i s
40 ns – he ime window mos c i ical o accu a e li e ime de e mina ion.
A compa a i e analysis o he i ed decay cu es is shown in he ollowing Figu e 3.37,
highligh ing changes in he a e age luo escence li e imes (𝜏ₐᵥ), calcula ed as:
𝜏
a
=𝐴1𝜏1+𝐴2𝜏2+𝐴3𝜏3
𝐴1+𝐴2+𝐴3 (3.3)
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
115
ac oss he di e en days o measu emen . The model inco po a es wo exponen ial decay
componen s, each cha ac e ized by a dis inc li e ime 𝜏1, 𝜏2, and 𝜏3. The p e-exponen ial ac o s,
A1, A2, A3, ep esen he ela i e con ibu ions o hese wo decay p ocesses o he o e all
luo escence in ensi y.
The a e age luo escence li e ime o esicles ex uded h ough 100 nm memb anes d opped
ma kedly o e ime, om 2.1 ± 3.0 ns on day 1 o jus 0.5 ± 0.2 ns by day 5, indica ing a
subs an ial ise in non- adia i e decay p ocesses. This apid dec ease aligns wi h mechanisms
such as ap-assis ed ecombina ion and Auge - ype in e ac ions, bo h o which can become
mo e p ominen when become su ace-cha ged o de elop new ap s a es.61 A likely
con ibu ing ac o is cu a u e-induced s ess in he lipid memb ane: a highe cu a u e, as
seen in smalle esicles, he lipid bilaye may exhibi packing de ec s and ansien pe meabili y
o wa e , oxygen, and ions. This inc eased pe meabili y can acili a e oxida i e damage o ion
o ma ion, bo h o which deg ade pho ophysical pe o mance o e ime.62,63 E en hough
bilaye hickness and lipid composi ion a e iden ical ac oss all ba ches, he smalle adius
ampli ies su ace- o- olume a io and memb ane ension, accele a ing hese deg ada ion
pa hways. By con as , esicles ex uded h ough 400 nm and 800 nm memb anes, whose
hyd odynamic diame e s a e a ound 200 nm, main ained s able li e imes (Figu e 3.37). The
modes ly la ge adius lowe s cu a u e s ess, educes de ec densi y, and limi s sol en /ion
ing ess, he eby supp essing he c ea ion o su ace aps and keeping Auge ecombina ion
a es nea hei ini ial alues. Collec i ely, he esul s e eal a cu a u e-dependen
pho os abili y: smalle esicles expe ience as e li e ime e osion owing o cu a u e-induced
memb ane leakiness, while sligh ly la ge esicles (< 200 nm) p ese e QD op ical in eg i y o e
se e al days. Fo expe imen s equi ing consis en op ical ou pu , esicles should ideally be used
sho ly a e p epa a ion; howe e , ex usion h ough memb anes ≥ 400 nm and keeping he
diame e o a esicle sligh ly la ge p esen s a s aigh o wa d s a egy o imp o ing
o mula ion obus ness.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
116
Figu e 3.37 Time- esol ed luo escence decay analysis o lipid esicles wi h di e en diame e s: 100 nm
(A, B), 400 nm (C, D), and 800 nm (E, F), measu ed on Day 1, Day 2, and Day 5 pos -syn hesis. Panels A, C,
and E p esen he i ed luo escence decay cu es showing how luo escence li e imes e ol e o e ime
o each esicle size. The co esponding a e age li e imes a e summa ized in panels B, D, and F,
espec i ely. No ably, 100 nm esicles exhibi a ma ked dec ease in luo escence li e ime o e ime,
indica ing educed pho ophysical s abili y, while la ge esicles (400 nm and 800 nm) main ain mo e
consis en li e imes o e he i e-day pe iod. This sugges s ha esicle size plays a ole in he empo al
s abili y o encapsula ed luo opho es o quan um do s.
PLQY in he p esence o lipids
The ini ial phase o ou wo k on op imizing lipid esicle p epa a ion was conduc ed using
comme cially a ailable CdSe/ZnS co e/shell quan um do s (QDs) unc ionalized wi h
ioc ylphosphine oxide (TOPO). Gi en hei low cos and wide a ailabili y, hese QDs se ed as
an ideal sys em o p elimina y p o ocol de elopmen and op imiza ion o he expe imen al
wo k low. Th oughou hese ea ly expe imen s, we obse ed ha he luo escence o he
A B
C D
E F
AVERAGE LIFETIME
100 nm esicles:
Day 1: 2.1 ns ± 3.0 ns
Day 2: 0.9 ns ± 1.0 ns
Day 5: 0.5 ns ± 0.2ns
Day 5, 100 nm: 0.5 ns ± 0.2 ns A e age li e me o Day 5, 400 nm: 1.8 ns ± 2.2 ns A e age li e me o Day 5, 800 nm: 1.8 ns ± 2.8 ns
100 nm: 0.5 ns ±0.2 ns A e age li e me o Day 5, 400 nm: 1.8 ns ± 2.2 ns A e age li e me o Day 5, 800 nm: 1.8 ns ± 2.8 ns
AVERAGE LIFETIME
400 nm esicles:
Day 1: 1.5 ns ± 2.3 ns
Day 2: 1.9 ns ± 2.7 ns
Day 5: 1.8 ns ± 2.2 ns
AVERAGE LIFETIME
800 nm esicles:
Day 1: 1.6 ns ± 2.6 ns
Day 2: 1.9 ns ± 3.0 ns
Day 5: 1.8 ns ± 2.8ns
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
117
comme cial QDs emained s able in lipid en i onmen s, wi h any mino dec ease a ibu ed
p ima ily o ma e ial loss du ing he ex usion p ocess. Howe e , achie ing he b oade
objec i es o his esea ch equi ed ansi ioning o ou ma e ial o choice: CdS/CdSe/CdS
sphe ical quan um wells (SQWs), syn hesized in Jona han Owen’s labo a o y. These mo e
sensi i e nanos uc u es a e be e sui ed o u u e applica ions due o hei unique op ical and
elec onic p ope ies.
The inco po a ion o SQWs in o lipid sys ems e ealed displayed a sha p decline in
pho oluminescence almos immedia ely upon mixing wi h lipids in non-pola sol en s. This
e ec was especially p onounced in he p esence o ca ionic lipids like DOTAP, sugges ing ha
su ace pola iza ion migh be igge ing enhanced non- adia i e ecombina ion pa hways. To
p ese e luo escence, su ace passi a ion mus be imp o ed o mi iga e quenching e ec s.
Se e al app oaches o con olling su ace chemis y we e implemen ed. SQWs unc ionalized
wi h Cd-olea e we e e alua ed i s . This sys em, howe e , p o ed o be highly ulne able o
lipid-induced luo escence loss, wi h PLQY alues d opping om 31.6% in DCM o jus 2.2% and
0.25% in he p esence o DOTAP and DSPE-PEG, espec i ely (Figu e 3.38). We hen explo ed
luo ina ed passi a ion using CdF2 combined wi h oc ylamine. Al hough hese nanopa icles
exhibi ed a high ini ial PLQY o 94.0% in DCM, hey oo expe ienced quenching in lipidic
en i onmen s, highligh ing he insu icien pho os abili y o his passi a ion s a egy (Figu e
3.39).
The su ace ea men wi h Zn-olea e ligands showed imp o ed esis ance o quenching, wi h
PLQY alues emaining ela i ely s able a 51% in DCM in he p esence o DMPC and d opping
o 10-12% in DOTAP and DSPE-PEG solu ions. This e ec can be a ibu ed o he s onge
a ini y o Zn-olea e o anionic sul u compa ed o Cd-olea e, which leads o mo e obus binding
o Zn-olea e ligands o he nanoc ys al su ace and mo e e ec i e passi a ion o su ace s a es.64
We u he in oduced a ZnS shell o o m a obus co e/shell s uc u e, which ma kedly
enhanced he op ical s abili y o he SQWs. ZnS is pa icula ly e ec i e because
unde coo dina ed sul u a oms a he nanoc ys al su ace ac as ap si es ha p omo e
non adia i e ecombina ion; o e coa ing wi h ZnS supp esses hese de ec - ela ed pa hways
and passi a es su ace s a es, he eby s abilizing he emission. In addi ion, he wide bandgap
o ZnS ela i e o CdS o CdSe inc eases ca ie con inemen wi hin he CdSe co e and mi iga es
Auge ecombina ion, u he imp o ing bo h pho oluminescence e iciency and s abili y.11,65 As
a esul , hese ZnS-shelled SQWs s abilized wi h Zn-olea e ligands main ained high PLQY alues
(~54%) ac oss all lipid o mula ions es ed, including DMPC, DOTAP, and DSPE-PEG, and e ained
a no able 29% PLQY e en in aqueous PBS esicle suspensions (Figu e 3.40). This inal sys em
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
118
demons a ed ha wi h he igh combina ion o ino ganic shell and su ace ligands, SQWs can
be e ec i ely shielded om en i onmen al pe u ba ions, pa ing he way o hei eliable use
in biological applica ions.
Ex inc ion and emission spec a co esponding o each s udied sys em (Sys em I – IV) a e
p o ided in he Appendix (Sec ion 3.3, Figu e A.3).
Figu e 3.38 Pho oluminescence esponse o CdS/CdSe/CdS sphe ical quan um wells (SQWs) unc ionalized
wi h Cd-olea e upon exposu e o lipid en i onmen s. (A) Schema ic o SQW su ace unc ionaliza ion wi h
cadmium olea e ligands. (B) Pho oluminescence quan um yield (PLQY) measu emen s o SQWs in a ious
media, demons a ing signi ican luo escence quenching upon mixing wi h lipid componen s, pa icula ly
DOTAP and DSPE-PEG. (C) Summa y o PLQY alues co esponding o he samples shown in panel B.
(D) Digi al images o SQW samples unde UV-ligh illumina ion, isually con i ming he luo escence loss
in lipid-con aining en i onmen s.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
119
Figu e 3.39 Pho oluminescence o CdS/CdSe/CdS unc ionalized wi h CdF2 and oc ylamine in lipid
en i onmen s. (A) Illus a ion o SQW su ace passi a ion (B) PLQY measu emen s showing a sha p decline
in emission upon exposu e o lipid componen s, pa icula ly DOTAP and DSPE-PEG, and nea - o al
quenching in PBS esicles. (C) PLQY alues om B. (D) Digi al images o SQW samples unde UV i adia ion.
Figu e 3.40 Pho oluminescence s abili y o passi a ed CdS/CdSe/CdS/ZnS s abilized wi h Zn-olea e in lipid
en i onmen s. (A) Schema ic ep esen a ion o he nanoc ys al ea u ing a ZnS ou e shell. (B) PLQY
measu emen s showing consis en emission in lipids, wi h only a modes dec ease obse ed in PBS.
(C) PLQY alues o each condi ion as shown in B. (D) Digi al images o he SQW samples unde UV-ligh
i adia ion, con i m luo escence e en ion in all lipid media es ed, and unde sco ing he e ec i eness o
he ZnS/Zn-olea e su ace passi a ion s a egy.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
120
3. Cell Memb ane Inse ion
The nex s ep was o assess hei abili y o SQWs o in e ace wi h biological memb anes – an
essen ial equi emen o hei in ended use in de ec ing neu onal ac i i y. This sec ion ocuses
on he memb ane inse ion capabili ies o SQWs deli e ed ia he wo s a egies p e iously
de eloped: OCG micelles and lipid esicles. The p ima y objec i e was o e alua e whe he hese
deli e y sys ems could success ully embed SQWs in o cell memb anes in a manne compa ible
wi h hei pho oluminescen unc ion and biological s abili y.
To alida e memb ane inse ion in a biologically ele an con ex , we employed cul u ed HEK
cells as a model sys em. Human emb yonic kidney (HEK293-T) cells p o ide a obus and well-
cha ac e ized pla o m o ini ial biological alida ion. In some expe imen s, unmodi ied
HEK293-T cells we e used o s udy undamen al memb ane in e ac ions. In o he s, we u ilized
sel -spiking HEK cells ans ec ed wi h ol age-ga ed sodium and po assium channels, p o iding
a dynamic model o es ing ac i i y-dependen luo escence esponses. De ails o he cell line
used a e p o ided in he desc ip ion o each expe imen al esul .
The expe imen s p esen ed in he ollowing subsec ions aim o assess bo h he memb ane
in eg a ion o SQWs and hei unc ional esponsi eness unde condi ions o eal- ime neu onal
ac i i y, es ablishing a ounda ion o hei u u e use in all-op ical elec ophysiology, as de ailed
la e in Subchap e 4, Reco dings o Spiking HEK Cells wi h Sphe ical Quan um Do s (page 153).
3.1. OCG Micelles
We began by e alua ing he memb ane inse ion capabili ies o SQWs encapsula ed wi hin OCG
micelles. The cen al ques ion was whe he hese micelles could b ing SQWs in o close
apposi ion wi h he plasma memb ane o li e cells wi hou comp omising cell iabili y – c i ical
p e equisi es o op ical sensing applica ions.
Ini ial expe imen s we e conduc ed using a specialized HEK cell line ob ained om ATCC
(CRL‑3479), o iginally de eloped by Adam Cohen’s labo a o y.66 This Te -On “spiking” HEK cell
line is gene ically enginee ed o cons i u i ely exp ess he ol age-ga ed sodium channel
NaV1.5, while he inwa d- ec i ie po assium channel Ki 2.1 is placed unde doxycycline-
inducible con ol. The Ki 2.1 channel is also used o cyan luo escen p o ein (CFP), which emi s
luo escence wi h an exci a ion peak a ound 456 nm and an emission peak nea 480 nm. Upon
addi ion o doxycycline ( ypically 1-2 µg/mL), Ki 2.1‑CFP is obus ly exp essed, leading o
memb ane hype pola iza ion. In combina ion wi h NaV1.5, his enables he cells o gene a e
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
127
The unusually b igh cell in he lowe - igh co ne is mos likely a ibu able o spec al bleed-
h ough om CFP, as p e iously no ed.
Ac oss mul iple eplica es and expe imen al condi ions, his punc a e, ex acellula
accumula ion pa e n was consis en ly obse ed, e en when micelle o mula ions we e
supplemen ed wi h addi i es such as Lipo ec amine o Plu onic F-127 – nei he o which al e ed
he dis ibu ion o p omo ed memb ane in eg a ion. These esul s sugges ha al hough he
pu i ied OCG micelles a e biocompa ible and capable o app oaching he cell su ace, hei
s uc u al and chemical p ope ies a e insu icien o suppo e icien inse ion o SQWs in o
he lipid bilaye . This limi a ion p omp ed a s a egic shi owa d al e na i e deli e y pla o ms,
speci ically lipid esicles, which o e a mo e memb ane-compa ible in e ace o SQW
inco po a ion, as desc ibed in he ollowing sec ions.
3.2. Fusosomes – Fusogenic Lipid Vesicles
In an ea lie chap e (2.2 Lipid Vesicles, page 102), when in oducing he p epa a ion o lipid
esicles and he encapsula ion o quan um do s, he speci ic choice o lipids was delibe a ely le
wi hou de ailed jus i ica ion. The esicles desc ibed so a we e no designed me ely as ca ie s
o solubilizing hyd ophobic quan um do s in aqueous media. Thei o mula ion was d i en by
enabling hem a usion wi h biological memb anes. Th ough ca e ul selec ion o lipid
componen s, a con en ional liposome can be ans o med in o a usosome – a esicle wi h he
in insic abili y o me ge wi h cellula memb anes and deli e i s con en s di ec ly in o he
memb ane o he cy osol.
Fusogenic liposomes, o usosomes, a e lipid-based nanoca ie s enginee ed o me ge di ec ly
wi h cellula memb anes, enabling e icien cy osolic deli e y o he apeu ic o diagnos ic ca go.
These sys ems o en inco po a e i al usion pep ides o syn he ic ligands o imp o e a ge
speci ici y and enhance memb ane usion e iciency.
Memb ane usion i sel is a undamen al biological p ocess in which wo lipid bilaye s me ge o
o m a con inuous memb ane, allowing he con en s o p e iously dis inc compa men s o mix.
This mechanism unde lies essen ial cellula ac i i ies, including endocy osis, exocy osis, synap ic
esicle elease, and in acellula esicle a icking. Fusion p oceeds h ough a se ies o
o ches a ed s eps: memb ane ecogni ion, docking, local bilaye des abiliza ion, hemi usion
(ou e lea le me ging), and inally ull usion, cha ac e ized by po e o ma ion and con en
mixing.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
128
Gene al amewo ks o designing usosomes should conside ew c ucial elemen s:
1. Building-Block Lipids
• S uc u al sca old: A zwi e ionic phospha idylcholine (PC) lipid (e.g. POPC)
p o ides a s able bilaye “skele on” ha main ains esicle in eg i y and luidi y
unde physiological condi ions.67
• Cu a u e modula o s: Cone-shaped lipids such as phospha idyle hanolamine
(PE) o high-cu a u e POPC en ich he bilaye wi h he necessa y nega i e
cu a u e, helping o s abilize he s uc u e o esicle and main ain memb ane
in eg i y.67
• Memb ane s i ene s: Choles e ol in e cala es be ween phospholipids,
inc easing bilaye o de and igidi y o p e en p ema u e ca go leakage
wi hou abolishing usogenici y (memb ane- usion capaci y).67
• Cha ge ancho s: Ca ionic lipids (e.g. DOTAP) enhance elec os a ic a ac ion o
he ypically anionic cell su ace, p omo ing esicle docking and usion
ini ia ion.68,69
2. Fusogenic T igge s
• A oma ic lipids: Lipids bea ing a oma ic headg oups (e.g. luo escen BODIPY-
o hodamine- agged PE) in e cala e in o memb anes, locally pe u bing lipid
packing and educing he ac i a ion ene gy o s alk o ma ion; hey also se e
as buil -in ace s o imaging. 68,70,71
• Conical lipids: In addi ion o hei ole as key cu a u e-inducing building blocks
in he bilaye , hei p onounced cone shape ac i ely d i es usion by c ea ing
local memb ane s ain ha s abilizes hemi usion in e media es and p omo es
po e opening.
• Fusogenic pep ides: Sho pep ides de i ed om i al usion p o eins inse in o
and des abilize a ge bilaye s; s udies show hey induce liposome sh inkage
and local lipid ea angemen s ha p ime memb anes o usion.72,73
3. Applica ion Speci ic Su ace Modi ica ions:
• An i ouling zwi e ions: Inclusion o addi ional zwi e ionic lipids (e.g. DOPC)
esis s se um-p o ein adso p ion, p ese ing usogenic ac i i y in complex
media.74
• PEGyla ion: G a ing PEG chains ( ia DSPE-PEG) u he educes nonspeci ic
in e ac ions, enhances colloidal s abili y in se um, and ex ends ci cula ion ime
in i o, wi h minimal impac on usion when op imized a low mola a ios.56,74
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
129
• Ta ge ing and epo ing: In oduce eac i e headg oups o an ibody o ligand
conjuga ion,69,70,72,75, luo escen lipids o imaging,70,71 o s imuli- esponsi e
lipids o con olled ca go elease52.
• Inco po a e lipids bea ing hyd ophobic ancho s (e.g., choles e ol o ocophe ol)
o secu e usosomes on o a ge memb anes o solid suppo s.76
By balancing hese elemen s – s uc u al lipids, ac i e usogens, and adap i e su ace
modi ica ions – usosomes can be a ionally uned o achie e apid, e icien , and speci ic
memb ane usion o cy osolic deli e y o d ugs, nucleic acids, p o eins, o imaging agen s.77
The usosome composi ion in his s udy consis ed o DMPC (1,2-dimy is oyl-sn-glyce o-3-
phosphocholine), DOTAP (1,2-dioleoyl-3- ime hylammonium-p opane), and DPPE-PEG2000
(1,2-dis ea oyl-sn-glyce o-3-phosphoe hanolamine-N-me hoxy(polye hylene glycol)-2000).56 A
physiological empe a u e (37 °C), DMPC exis s in a luid, liquid-c ys alline phase (Tm ≈ 24 °C),
p o iding a lexible and s able bilaye s uc u e ha suppo s he dynamic memb ane
ea angemen s equi ed o usion. The addi ion o 5 mol% DOTAP, a ca ionic lipid, in oduces
a posi i e su ace cha ge ha acili a es s ong elec os a ic in e ac ions wi h nega i ely cha ged
cellula memb anes. This enhances memb ane binding and lowe s he ene ge ic ba ie o ini ial
bilaye des abiliza ion and hemi usion.56,70,77 An impo an componen is DPPE-PEG2000 ha
plays a mul i ace ed ole. While PEGyla ion is adi ionally associa ed wi h p o iding s e ic
s abiliza ion and educing agg ega ion in se um-con aining en i onmen s, a low concen a ions
i also suppo s usion.56,76 The PEG chains help exclude in e acial wa e , educing hyd a ion
epulsion be ween bilaye s – a phenomenon o en e e ed o as “PEG-induced
dehyd a ion”.78,79 In addi ion, he bulky PEG headg oups in oduce la e al p essu e a he
memb ane in e ace, gene a ing packing de ec s and localized cu a u e ha can se e as
nuclea ion si es o lipid mixing.80 This e ec is pa icula ly bene icial when used alongside
usogenic o ca ionic lipids, as i p omo es close memb ane apposi ion wi hou comple ely
shielding he su ace cha ge. Toge he , his ca e ully balanced combina ion o lipids ensu es
bo h colloidal s abili y and high usogenic po en ial, enabling he esicles o unc ion e ec i ely
as usosomes o di ec memb ane in e ac ion and in acellula deli e y.56
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
130
Figu e 3.45 Schema ic ep esen a ion o usosome s uc u e and usion mechanism. (A) C oss-sec ional
iew o a usosome composed o 74.5 mol% DMPC, 5 mol% DOTAP, and 0.5 mol% DPPE-PEG2000, wi h
nanoc ys als embedded be ween he lipid bilaye s. The bilaye a chi ec u e is s abilized by DMPC, which
exis s in a luid, liquid-c ys alline phase a physiological empe a u e (37 °C). Su ace-exposed DOTAP
in oduces a ne posi i e cha ge, enhancing elec os a ic in e ac ions wi h nega i ely cha ged cellula
memb anes. PEGyla ed lipids (depic ed wi h ex ended blue chains) con ibu e s e ic s abiliza ion and
p omo e memb ane usion by acili a ing in e acial dehyd a ion and inducing packing de ec s. (B) Upon
con ac wi h a nega i ely cha ged cellula memb ane, he posi i ely cha ged usosome engages in
elec os a ic in e ac ions, esul ing in spon aneous memb ane usion. This p ocess is d i en by educed
hyd a ion epulsion and memb ane cu a u e s ess induced by he PEGyla ed lipids, p omo ing lipid
mixing and di ec bilaye me ge . The embedded nanoc ys als a e ans e ed di ec ly in o he a ge
memb ane, whe e hey emain localized wi hin he ene ge ically a o able hyd ophobic en i onmen o
he lipid ails.
In o de o con i m he usogenic capabili y o he liposome o mula ion, we pe o med a se ies
o in- i o expe imen s using liposomes labeled wi h he lipophilic luo escen dye DiI
(1,1'-Dioc adecyl-3,3,3',3'-Te ame hylindoca bocyanine Pe chlo a e). DiI is a highly
hyd ophobic, ca ionic indoca bocyanine dye ha ancho s in o lipid bilaye s ia i s wo long C18
alkyl chains and se es as a highly sensi i e p obe o memb ane usion. No ably, DiI exhibi s
minimal luo escence in aqueous en i onmen s due o agg ega ion and in amolecula
lexibili y, which a o non- adia i e decay. Upon inse ion in o a lipid bilaye , he dye becomes
b igh ly luo escen as i s ch omopho e is s abilized wi hin he o de ed, low-pola i y memb ane
en i onmen . This ansi ion dis up s agg ega e o ma ion, es ic s o sional mo ion o he
polyme hine chain, and signi ican ly enhances i s quan um yield – making luo escence
e ec i ely ga ed by memb ane embedding. The chemical s uc u e o he dye is shown in Figu e
3.46 A, and i s spec oscopic p o ile a e inco po a ion in o esicle memb anes (Figu e 3.46 B)
e eals a dis inc abso p ion peak nea 550 nm and an emission cen e ed a 570 nm. This
en i onmen -induced luo escence makes DiI an ideal ace o de ec ing lipid mixing and
esicle-cell usion e en s.
In his se o expe imen s, we a ied amoun s o PEGyla ed lipid (DSPE-PEG2000) and kep
cons an DMPC and DOTAP. To assess how PEG densi y in luences memb ane usion e iciency,
we sys ema ically adjus ed he PEG con en o 0.5 mol%, 1.0 mol%, 2.5 mol%, and 5.0 mol%.
This concen a ion ange was es ablished based on p e ious indings by Gopalak ishnan e al.56
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
131
To isualize usion e en s, we added 10 mol% o he luo escen lipid DiI o each o mula ion.
The emaining 90 mol% was used o p ese e he o iginal DMPC/DOTAP a io (app oxima ely
74.5:25), wi h he speci ied amoun o PEG-lipid inco po a ed in o his ac ion. While he
addi ion o DiI sligh ly educed he absolu e pe cen ages o he o he componen s, hei ela i e
p opo ions emained consis en ac oss samples. This design ensu ed ha any di e ences in
usion beha io could be a ibu ed o he PEG con en , wi hou in oducing a iabili y om
changes in he co e lipid composi ion.
Panels C and D (Figu e 3.46) display cu e es con aining he DiI-labeled esicle suspensions
unde dayligh and UV illumina ion, espec i ely. In whi e ligh , all samples look nea ly iden ical,
showing only a ain pink in om he inco po a ed DiI, whe eas UV exci a ion e eals he
expec ed uni o m o ange- ed luo escence ac oss e e y o mula ion.
Figu e 3.46 Cha ac e iza ion o DiI-labeled con ol liposomes used o e alua e esicle-cell usion.
(A) Chemical s uc u e o DiI [1,1ʹ-dioc adecyl-3,3,3ʹ,3ʹ- e ame hylindoca bocyanine pe chlo a e], a
lipophilic ca ionic ca bocyanine bea ing wo C18 alkyl ails ha ancho s ably in o lipid bilaye s.
(B) Abso p ion (dashed blue) and pho oluminescence (solid pu ple) spec a o DiI-labeleld liposomes. The
dye, embedded wi hin he esicle memb ane, exhibi s a s ong, na ow abso p ion peak cen e ed a
550 nm and a luo escence emission maximum a 570 nm, esul ing in in ense ed-o ange luo escence.
(C, D) Pho og aphs o liposome suspensions con aining 10 mol% DiI and inc easing ac ions o
DSPE-PEG2000 (0.5, 1.0, 2.5, and 5.0 mol%). Images we e aken unde ambien whi e ligh (C) and UV
illumina ion (D). Uni o m b igh - ed luo escence con i ms success ul inco po a ion o DiI in all
o mula ions, enabling isual acking o esicle usion in subsequen cell assays.
Fusion expe imen s we e pe o med on spiking HEK293 monolaye s, and he ex en o
memb ane s aining was e alua ed ia luo escence mic oscopy and quan i a i e in ensi y
p o iling (Figu e 3.47). A low PEG concen a ions (0.5 mol% and 1.0 mol%), DiI luo escence
appea ed spa se and punc a e, wi h in ensi y p o iles cha ac e ized by na ow, isola ed peaks
1 2 1 2
hi e ligh i ill mina on
n i ogen il ain
1,1 ‐Dioc adecyl‐3,3,3 ,3 ‐
Te ame hylindoca bocyanine Pe chlo a e
AB
C D
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
132
and o e all lowe signal le els. This con as s wi h he highe PEG condi ions, e lec ing mo e
obus and con inuous memb ane labeling. This pa e n is indica i e o limi ed lipid mixing,
sugges ing incomple e hemi usion wi hou subs an ial memb ane inco po a ion. In con as , a
highe PEG concen a ions (2.5 mol% and 5.0 mol%), he in ensi y p o iles showed a con inuous
and uni o m dis ibu ion o DiI along he cell pe iphe y. The signal emained ele a ed ac oss he
scan, wi h minimal luc ua ions owa d lowe in ensi y alues. These ends pa alleled he
quali a i e di e ences obse ed in he luo escence images, con i ming ha esicle usion
e iciency inc eases in a PEG-dependen manne . Ac oss all condi ions, b igh - ield images
con i med in ac cell mo phology, indica ing no cy o oxic e ec s. Toge he , hese da a alida e
he unc ional in eg i y and usogenic design o he liposomes p oposed he e.
Figu e 3.47 PEG-dependen usion o DiI-labeled con ol liposomes wi h HEK293 cells. B igh - ield ( op ow)
and co esponding luo escence (middle ow) mic oscopy images acqui ed a e 30 minu es o incuba ion
wi h liposomes con aining 0.5, 1.0, 2.5, o 5.0 mol% DSPE-PEG2000. All liposomes we e labeled wi h
10 mol% DiI o ack lipid mixing wi h he plasma memb ane. In ensi y p o iles (bo om ow) ac oss he
dashed pu ple lines e eal spa se, low-in ensi y, punc a e signal a 0.5% and 1.0% PEG, and p og essi ely
s onge , mo e con inuous luo escence a 2.5% and 5.0% PEG, consis en wi h e icien memb ane
labeling.
1.0 PEG0.5 PEG 2.5 PEG 5.0 PEG
100 m
100 m
100 m
100 m
100 m
100 m
100 m
100 m
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
133
Gi en he high e iciency o memb ane usion obse ed wi h DiI-labeled esicles con aining
5 mol% PEG-lipid, we nex examined he minimum incuba ion ime equi ed o achie e
de ec able lipid mixing a he cell su ace. This is an impo an expe imen al pa ame e since he
usion ime cons ains he ime window needed o ob ain ep esen a i e da a. Tha is, one needs
o ob ain as usogenesis ha p olonga e as long as possible o assu e cons an condi ions o
analysis o spikes dynamics. We es ed du a ions ange om 5 o 30 min o assess he kine ics
o usion unde op imized condi ions. As shown in Figu e 3.48, memb ane-associa ed DiI
luo escence was al eady de ec able a e jus 5 minu es, wi h a con inuous ou line ma king he
cell pe iphe y. Howe e , he signal a his ea ly ime poin appea ed no iceably dimme
compa ed o longe incuba ions. By 10 and 15 minu es, he luo escence in ensi y inc eased
compa ed o he 5-minu e ime poin and closely esembled he pa e n obse ed a 30 minu es,
al hough wi h sligh ly lowe o e all b igh ness. This sugges s ha memb ane usion ini ia es
apidly and p og essi ely in ensi ies o e ime. B igh - ield images con i med p ese ed cell
mo phology h oughou he ime cou se, indica ing ha apid usion does no comp omise
memb ane in eg i y. These esul s demons a e ha usion occu s wi hin minu es unde op imal
condi ions, and con i m ha 30-minu e ime window is su icien o ensu e comple e and
uni o m memb ane labeling.
In he ollowing sec ions o his wo k, he e ms esicle, liposome, and usosome will be used
in e changeably, each e e ing speci ically o he DMPC/DOTAP/DPPE-PEG2000-based
esicula s uc u es desc ibed abo e (unless speci ied o he wise).
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
134
Figu e 3.48 Time-cou se analysis o memb ane usion using DiI-labeled liposomes (5 mol% PEG). B igh -
ield ( op ow) and co esponding luo escence (middle ow) images o HEK293 cells incuba ed wi h
DiI-labeled esicles o 5, 10, 15, 30 minu es. A con inuous luo escen ou line is isible as ea ly as
5 minu es, indica ing apid ini ia ion o lipid mixing. In ensi y p o iles (bo om ow) show p og essi e
inc eases in signal ampli ude and uni o mi y o e ime, wi h he 30-minu e ace eaching he highes and
mos consis en alues, e lec ing e icien and widesp ead memb ane labeling. Cell mo phology emains
p ese ed a all ime poin s, con i ming ha usion p oceeds wi hou comp omising memb ane in eg i y.
Memb ane s aining
Building on he six nanoc ys al sys ems in oduced in 3.1.1 O e iew o Quan um-do -based
Pla o ms and illus a ed in Figu e 3.17 (page 87), his sec ion examines hei in e ac ion wi h
cell memb anes. The se ies includes one comme cially a ailable CdSe/ZnS e e ence (Sys em I)
and ou cus om-designed sphe ical quan um wells (Sys ems III-VI), each de eloped i e a i ely
in esponse o expe imen al challenges encoun e ed h oughou he s udy. Wi h aqueous
ans e p ocedu es in place, he nex s ep is o e alua e how e ec i ely hese quan um do s
associa e wi h usogenic esicles. The memb ane-s aining esul s assessmen o labeling
pe o mance and se e as a unc ional p esen ed he e p o ide a compa a i e, quali a i e
p elude o he usion beha io explo ed.
5 min
100 m
100 m
30 min
100 m
100 m
15 min
100 m
100 m
10 min
100 m
100 m
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
135
Sys em I
To e alua e he usion capaci y o he o mula ed usosomes, we i s examined esicles
inco po a ing comme cially a ailable CdSe/ZnS quan um do s capped wi h TOPO – e e ed o
as Sys em I (Figu e 3.49, Panels A and B) – on HEK293 cell monolaye s. Con ocal luo escence
mic oscopy was used o moni o esicle-cell in e ac ions in eal ime, wi h he goal o
de e mining whe he usion wi h he plasma memb ane occu ed and whe he he quan um
do s became inco po a ed in o he memb ane s uc u e. The emission spec um o he quan um
do s showed a peak a 542 nm (Figu e 3.49 B), enabling clea isualiza ion o memb ane-
associa ed luo escence. These expe imen s se ed as an ini ial alida ion o esicle-cell usion
and p o ided a basis o e alua ing he memb ane in eg a ion o he quan um do s.
To sys ema ically explo e he a iables in luencing usion e iciency, we in es iga ed a ange o
pa ame e s: cell seeding densi y (which a ec s monolaye con luency a he ime o imaging),
cell age (numbe o days pos -seeding), he medium used du ing usion (g ow h medium
s. PBS), usion du a ion, ime spen ou side he incuba o , and he olume o usosomes
suspension applied.
A c i ical challenge du ing imaging was he p esence o cellula au o luo escence, which can
obscu e weak signals om memb ane-bound quan um do s. This endogenous backg ound
signal a ises om na u ally luo escen biomolecules such as NADH, la ins, and a oma ic amino
acids, and ends o in ensi y as cells age o unde go s ess. The cellula au o luo escence can
con ibu e backg ound signals ha may be mis akenly in e p e ed as p obe-de i ed emission.
The e o e, we ca e ully assessed imaging pa ame e s o minimize such in e e ence and ensu e
accu a e e alua ion o usosome-media ed memb ane s aining.
Unde he op imized condi ions es ablished h ough pa ame e sc eening, he mos consis en
and obus memb ane labelling was achie ed app oxima ely 25-30 minu es a e esicle
applica ion. As shown in Figu e 3.49 (Panels C-E), con ocal luo escence imaging o HEK293 cell
monolaye s (80% con luen , 24 hou s pos -seeding) incuba ed wi h quan um do -loaded
liposomes e ealed a dis inc g een luo escence ou lining he cell pe iphe y, accompanied by a
limi ed numbe o b igh in acellula punc a.
Fo hese expe imen s, 500 µL o liposome suspension – DMPC, DOTAP, and DSPE-PEG2000 a
a 74.5:25:0.5 mola a io and ex uded h ough a 100 nm memb ane – was added di ec ly o
2 mL o comple e cul u e medium (DMEM supplemen ed wi h 10 FBS) in each well. This gen le
deli e y s a egy was chosen o p ese e cell iabili y and minimize s ess by a oiding ab up
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
136
changes o he en i onmen . Cells we e incuba ed wi h he liposomes o 10-30 minu es, wi h
op imal memb ane-associa ed signal obse ed consis en ly a he 25-30-minu e ime poin .
Imaging was pe o med using a Leica TCS SP5 con ocal mic oscope wi h 458 nm lase exci a ion
se o 30% o maximum powe . Emission was collec ed using s anda d se ings op imized o
memb ane-associa ed luo escence. Full acquisi ion pa ame e s a e p o ided in he Appendix
(Sec ion 3.2.3).
The esul ing images, p esen ed on Figu e 3.49, exhibi a la gely con inuous memb ane-localized
luo escence signal, s ongly sugges i e o quan um do in eg a ion in o o associa ion wi h he
plasma memb ane. The occasional p esence o b igh in acellula spo s may a ise om used
esicle agg ega es o local QD accumula ion. Howe e , he lack o ex ensi e cy oplasmic o
pe inuclea luo escence a gues agains la ge-scale in e naliza ion as he dominan mechanism.
The in ensi y plo p o iles co esponding o he luo escence mic og aphs wi hin yellow
ec angles (Fig. 3.31, panels C and D) p o ide u he suppo o he memb ane-associa ed
localiza ion o quan um do s. These p o ile scans show dis inc peaks in luo escence in ensi y
a posi ions aligning wi h he cell pe iphe y, indica ing a highe concen a ion o luo escen
signal a he plasma memb ane compa ed o he cell in e io . The dual-peak pa e n obse ed
ac oss indi idual cell p o iles is consis en wi h luo escence a ising om le and igh side o
he memb ane. The dis ance be ween he peaks co esponds o he diame e o HEK-293 cells,
ypically 10–15 µm, u he con i ming hei assignmen o cell bounda ies. Backg ound
luo escence emained s able a app oxima ely 10 a.u., whe eas memb ane-associa ed egions
ep oducibly eached alues wo- o h ee- old highe . Quan i ica ion ac oss a popula ion o
app oxima ely 30 cells con i med his obse a ion: s a is ics o luo escence in ensi ies
measu ed a he cell ou line and in he cy oplasm showed clea ly sepa a ed dis ibu ions, wi h
a calcula ed memb ane- o-cy oplasm a io (MCR) o 2.79 (Fig. 3.31 E). O e all, hese spa ial
in ensi y p o iles align wi h he in e p e a ion ha quan um do -labeled esicles p e e en ially
associa e wi h, o in eg a e in o, he plasma memb ane a he han being in e nalized.
While de ini i e dis inc ion be ween usion, adso p ion, and endocy osis would equi e
colocaliza ion wi h memb ane o endosomal ma ke s, he o e all luo escence pa e n
demons a es e ec i e memb ane s aining and con i ms ha he liposome o mula ion eached
and in e ac ed ex ensi ely wi h he plasma memb ane unde he es ed condi ions.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
143
Figu e 3.52 Rep esen a i e esul s o Sys em III (CdS/CdSe/CdS SQWs) deli e ed using esicles con aining
5 mol% PEG-lipid. (A) Schema ic o SQW s uc u e wi h Zn-olea e ligands; lipid mix: DMPC 75%, DOTAP
25%, DSPE-PEG2000 5%. (B) UV-Vis (dashed blue) and PL (solid pu ple) spec a; λem = 530 nm.
(C-D) Fluo escence emains punc a e and non-memb ane-associa ed despi e inc eased PEG concen a ion.
(E-F) In ensi y p o iles om A eas #1 and #2 (C and D espec i ely) show spa se, na ow peaks nea con ol
le els, con i ming lack o memb ane labeling.
C C e C n lea e
Zn(COO C17H33)2
(COO C17H33)2
C
A B
C
D
100 m
100 m
100 m
100 m
100 m
100 m
B igh Field BF Em Emission
B igh Field BF Em Emission
C 2
E F
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
144
Replacing he olea e shell wi h a mixed cadmium- luo ide/oc ylamine passi a ion laye
(Sys em IV) p oduced colloidal SQWs ha e ained he a che ypal CdS/CdSe/CdS a chi ec u e
(Figu e 3.53 A) while emi ing a na ow g een band cen e ed a 541 nm (Figu e 3.53 B). The new
ligand se imp o ed he in e ac ion be ween he nanoc ys al-loaded esicles and he plasma
memb ane. In bo h p esen ed egions (Figu e 3.53, panels C and D), he luo escence channel
e ealed pa e n coinciden wi h he cell bounda ies seen in b igh - ield mode. Only a ew
disc e e in acy oplasmic spo s we e de ec ed, and ex acellula agg ega es we e la gely absen .
The me ged BF + Em o e lays u he con i m ha he pho oluminescen signal is con ined o
he cell pe ime e , indica ing ha he liposome ca go eached he ou e lea le o he HEK293
memb ane. The absence o di use cy osolic glow a gues agains esicle endocy osis, whe eas
he uni o m im sugges s lipid- o-lipid usion a he han adso p ion.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
145
Figu e 3.53 Rep esen a i e memb ane s aining wi h Sys em IV liposomes loaded wi h CdS/CdSe/CdS SQWs
capped by CdF2/oc ylamine. (A) Schema ic o SQW s uc u e and ligand composi ion. Lipid mix:
DMPC 74.5%, DOTAP 25%, DSPE-PEG2000 0.5%. (B) Op ical spec a showing UV-Vis (dashed blue) and PL
(solid pu ple), λem = 541 nm. (C-D) Fluo escence mic oscopy o HEK293 cells a e 30 min incuba ion shows
uni o m memb ane-associa ed signal. (E-F) In ensi y p o iles e eal consis en signal abo e baseline wi h
egula ly spaced peaks, indica ing e ec i e memb ane localiza ion.
CdF2
NH(oc yl)
C
C C e C C 2 c lamine
A B
C
D
100 m
100 m
100 m
100 m
100 m
100 m
B igh Field BF Em Emission
B igh Field BF Em Emission
C 2
E F
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
146
This in e p e a ion is u he suppo ed by he luo escence in ensi y p o iles shown in panels E
and F, co esponding o he emission egions om A eas #1 and #2. In bo h aces, he signal
emains abo e he baseline ob ained om an uns ained con ol (Appendix, Figu e A.4), wi h a
se ies o peaks indica i e o luo escence along he cell pe iphe y. This pa e n s ands in con as
o he p o iles obse ed o Sys em III, which exhibi ed i egula spikes co esponding o punc a
and showed no indica ion o memb ane associa ion. The b oade peaks obse ed o Sys em IV
sugges homogeneous dis ibu ion o he luo opho es and associa ion wi h he plasma
memb ane ac oss di e en egions o he sample. These spa ial p o iles, in ag eemen wi h he
imaging da a, suppo he conclusion ha he CdF2/oc ylamine su ace chemis y acili a es
esicle-memb ane in e ac ion while main aining pho oluminescence o imaging a 100 ms
exposu e. Compa ed o he discon inuous signal seen in Sys em III, Sys em IV achie es
memb ane-speci ic labeling, highligh ing he impo ance o su ace ligand composi ion in
di ec ing esicle beha io a he cellula in e ace. Howe e , while he signal is de ec able unde
cu en condi ions, he o e all luo escence in ensi y emains below he le el equi ed o high-
speed imaging o neu al dynamics. Ou goal is o de elop a labeling sys em ha no only enables
accu a e memb ane a ge ing bu also exceeds he b igh ness o con en ional indica o s,
allowing o sho e exposu e imes and highe ame a es.
To enhance signal in ensi y while main aining esis ance o lipid-induced quenching, we
subsequen ly explo ed Sys em V, which ea u es a CdS/CdSe/CdS co e-well s uc u e u he
passi a ed wi h an ou e ZnS shell. As desc ibed in Sec ion 3.2.2 PLQY in he p esence o lipids,
his addi ional laye signi ican ly imp o es op ical pe o mance, yielding a PLQY o
app oxima ely 50% in non-pola lipid- ich solu ions and e aining a ound 30% in aqueous media
– making i he mos pho os able and e icien o mula ion wi hin ou cu en nanoc ys al
lib a y.
Despi e his a o able pho ophysical p o ile, memb ane s aining using Sys em V was less
e icien han ha achie ed wi h he CdF2/oc ylamine-capped SQWs o Sys em IV. Also, no e ec
o PEG concen a ion was obse ed on he quali y o s aining. Fluo escen mic oscopy o
samples e ealed a punc a e luo escence pa e n dis ibu ed spa sely h oughou he sample,
wi h no con inuous memb ane-associa ed signal, ega dless o PEG amoun . Emissi e spo s
we e equen ly obse ed away om he cell pe iphe y, ei he adjacen o o de ached om he
memb ane, sugges ing ine icien deli e y o nanoc ys als o he plasma memb ane and an
absence o clea e idence o memb ane usion. Se e al ac o s may con ibu e o his educed
usogenic beha io . The addi ion o he ZnS shell inc eases he o e all pa icle size, which may
ele a e he ene ge ic ba ie o lipid mixing and bilaye inco po a ion. Fu he mo e, he long
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
147
and con o ma ionally igid chain o Zn-olea e ligands may c ea e a s e ic ba ie ha limi s close
con ac be ween he SQWs and he cell memb ane, po en ially hinde ing e ec i e mixing wi h
he acyl chains o he lipid bilaye . These esul s unde sco e he delica e balance be ween op ical
op imiza ion and memb ane in e ac ion, highligh ing he need o ailo bo h su ace chemis y
and s uc u al pa ame e s o achie e e icien and b igh memb ane labeling.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
148
Figu e 3.54 Fluo escence mic oscopy o Sys em V: CdS/CdSe/CdS/ZnS SQWs wi h Zn-olea e ligands.
(A) Schema ic o nanoc ys al s uc u e; lipid o mula ion: DMPC 74.5%, DOTAP 25%, DSPE-PEG2000 0.5%.
(B) UV-Vis (dashed blue) and PL (solid pu ple) spec a in non-pola sol en ; λem = 489 nm. (C-D) Imaging
o HEK293 cells shows spa se spo s wi h no clea memb ane-associa ed signal. (E-F) In ensi y p o iles
ac oss blue ec angles in C and D e eal weak, i egula peaks nea baseline, indica ing poo memb ane
localiza ion.
Zn(COO C17H33)2
n (COO C17H33)2
C C e C n n lea e
A B
C
D
100 m
100 m
100 m
100 m
100 m
100 m
B igh Field BF Em Emission
B igh Field BF Em Emission
C 2
E F
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
149
Figu e 3.55 Rep esen a i e esul s o Sys em V (CdS/CdSe/CdS/ZnS SQWs) deli e ed using esicles
con aining 5 mol% PEG-lipid. (A) SQW s uc u e wi h Zn-olea e ligands and ou e ZnS shell; lipid mix:
DMPC 75%, DOTAP 25%, DSPE-PEG2000 5%. (B) UV-Vis (dashed blue) and PL (solid pu ple) spec a;
λem = 489 nm. (C-D) B igh - ield and luo escence images show spa se, non-memb ane-associa ed punc a
despi e inc eased PEG con en . (E-F) In ensi y p o iles om A eas #1 and #2 (C and D espec i ely) emain
nea baseline, con i ming lack o memb ane s aining.
Zn(COO C17H33)2
n (COO C17H33)2
C C e C n n lea e
A B
C
D
100 m
100 m
100 m
100 m
100 m
100 m
B igh Field BF Em Emission
B igh Field BF Em Emission
C 2
E F
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
150
Sys em VI
To u he op imize he su ace chemis y o QD we pe o med a ligand exchange in which
zinc bis(2-hexyldecanoa e) [Zn(2-hexyldecanoa e)2] was used in place o Zn(olea e)2 as he
su ace capping agen . In his con igu a ion, Zn2+ ions coo dina ed by b anched
2-hexyldecanoa e ligands bind o su ace chalcogenide (S2-) si es, p ese ing elec onic
passi a ion while o ming a mo e s e ically bulky and loosely packed ligand shell. Compa ed o
he linea C18 olea e, he seconda y-alkyl b anching o 2-hexyldecanoa e dis up s e icien chain
packing, educes in e -ligand an de Waals in e ac ions, and inc eases con o ma ional en opy
a he nanoc ys al in e ace. These p ope ies ha e been linked o signi ican ly enhanced
solubili y, educed agg ega ion, and imp o ed ilm o ma ion, as demons a ed by Yang e al.,
who desc ibed such ligands as “en opic” in hei beha io .81 Based on his, we hypo hesized
ha he b anched-ligand shell would acili a e lipid pene a ion and imp o e compa ibili y wi h
he plasma memb ane. To es his, SQW-loaded esicles we e p epa ed using 5 mol% DSPE-
PEG2000 – an op imized o mula ion p e iously shown o suppo apid and e icien usion in
DiI-labeled sys em – and e alua ed o hei abili y o achie e uni o m memb ane s aining.
The s aining pe o mance o Sys em VI is p esen ed in Figu e 3.56. Panels A and B show he
nanoc ys al a chi ec u e and op ical cha ac e iza ion, wi h he emission peak cen e ed a
app oxima ely 495 nm. The lowe panels display ep esen a i e luo escence and co esponding
b igh - ield images acqui ed a e a 30-minu e incuba ion wi h HEK293 cells. Each column
depic s a dis inc ield o iew om sepa a e samples, including wo independen liposome
ba ches p epa ed and imaged on di e en days. The consis ency ac oss hese measu emen s
con i ms he ep oducibili y o he obse ed ou come.
In all samples, b igh , con inuous luo escence clea ly delinea es he cell pe ime e , indica ing
e icien deli e y o SQWs o he plasma memb ane ia esicle usion (Figu e 3.56). In each o
he h ee analyzed egions, he luo escence signal emains consis en ly ele a ed abo e
he uns ained con ol baseline (yellow ace; Appendix, Figu e A.4) ac oss nea ly he ull scan
leng h, dipping only a loca ions co esponding o spaces be ween neighbo ing cells. The p o iles
exhibi b oad, g adually ising and alling signal pla eaus, in con as o he na ow, i egula
spikes cha ac e is ic o Sys ems III and V, con i ming a uni o m dis ibu ion o SQWs along he
plasma memb ane. Signal in ensi y and pa e n a e highly consis en ac oss A eas #1, #2, and #3,
unde sco ing he ep oducibili y o he s aining achie ed wi h independen ly p epa ed liposome
ba ches on h ee independen samples. The co esponding b igh - ield images con i m he
in eg i y and con luency o he HEK293 monolaye , indica ing ha bo h he usogenic esicle
o mula ion and he modi ied SQWs a e well- ole a ed by he cells.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
151
Sys em VI yielded isibly s onge luo escence and ma kedly highe memb ane- o-backg ound
con as . No ably, i suppo ed sho e exposu e imes (as low as 30 ms), enabling ame a es
up o ~33 Hz – beyond he capabili y o Sys em IV unde he same condi ions. This ep esen s
an impo an s ep owa d high empo al esolu ion, eal- ime imaging such as neu onal ac i i y
imaging, whe e empo al esolu ion is c i ical. S ill, he achie ed b igh ness emains below he
po en ial o quan um do -based p obes, sugges ing u he op imiza ion o ligand chemis y and
nanoc ys al a chi ec u e is needed o ully ealize hei pe o mance in as , high- esolu ion
cellula imaging.
Chap e 3. Quan um Do s o De ec ing Neu onal Ac i i y
152
Figu e 3.56 Rep esen a i e memb ane s aining esul s o Sys em VI: CdS/CdSe/CdS SQWs capped wi h
b anched 2-hexyldecanoa e ligands. (A) Schema ic o SQW s uc u e; esicles con ained DMPC 75%,
DOTAP 25%, DSPE-PEG2000 5%. (B) UV-Vis (dashed blue) and PL (solid pu ple) spec a; λem = 495 nm.
(C-H) Fluo escence and b igh - ield images o HEK293 cells a e 30 min incuba ion show s ong,
con inuous memb ane labeling. (I-K) In ensi y p o iles om A eas #1-3 con i m uni o m luo escence abo e
baseline, suppo ing e icien and ep oducible deli e y o he plasma memb ane.
A B
- n
C C e C 2 e l ecanoa e
C 2
100 m
100 m
100 m
100 m
100 m
100 m
B igh Field
Emission
B igh Field B igh Field
Emission
Emission
C D E
F G H
I J K
APPENDIX
255
Emission was de ec ed in a single channel ia PMT 2, con igu ed a 750 V gain, wi h 8-bi
digi iza ion and a linea LUT (gamma = 1.0). The image acquisi ion was pe o med a a zoom
ac o o 2.00, esul ing in a inal sampling esolu ion o 0.1217 µm pe pixel in he la e al (X-Y)
plane ac oss a 512 × 512 pixel as e , yielding a o al ield o iew o 62.29 µm × 62.29 µm. The
acquisi ion was limi ed o a single op ical plane, wi h Z-s ep size se o ze o and he axial scanne
deac i a ed. These pa ame e s ensu ed Nyquis -su icien la e al sampling and eliable op ical
sec ioning o subsequen in ensi y-based image quan i ica ion.
3.2.4. Fluo escence Mic oscope
The luo escence mic oscope se up used in his s udy was iden ical o he con igu a ion
desc ibed in he supplemen a y ma e ials o Chap e 2. All imaging pa ame e s, op ical
componen s, and acquisi ion se ings emained consis en unless o he wise no ed in he main
ex .
APPENDIX
256
3.3. Addi ional Figu es
S abili y S udy - Fluo escence Li e ime T aces
Shown below a e he o iginal luo escence decay cu es eco ded o esicles ex uded h ough
100 nm, 400 nm, and 800 nm memb anes on days 1, 2, and 5 (Figu e A.102). These aces we e
used o iple exponen ial i ing, wi h a e aged li e ime alues p esen ed in he main ex
(Figu e 3.22, Chap e 3, Sec ion 2.2).
Figu e A.102 Raw luo escence li e ime decay cu es o esicles o di e en sizes—100 nm (A, D, G),
400 nm (B, E, H), and 800 nm (C, F, I) - measu ed on Day 1 (A-C), Day 2 (D-F), and Day 5 (G-I). The o e laid
yellow lines ep esen he i ed iple exponen ial decays.
A B C
D E F
G H I
APPENDIX
257
Op ical cha ac e iza ion o SQWs sys ems used o lipid encapsula ion
As discussed in Chap e 3, Sec ion 2.2, pho oluminescence quan um yield (PLQY) measu emen s
we e pe o med o selec ed quan um well sys ems in he p esence o lipid esicles. Shown
below a e he co esponding abso p ion and emission spec a o all syn hesized sys ems.
Figu e A.103 No malized abso p ion (dashed blue lines) and pho oluminescence (solid pu ple lines) spec a
o he in es iga ed sys ems: (A) Sys em I – CdSe/ZnS@TOPO, (B) Sys em II – SQWs@Cd-olea e, (C) Sys em
III – SQWs@Zn-olea e, (D) Sys em IV – SQWs@Cd-F2 and oc ylamine, (E) Sys em V –
SQWs@ZnS@Zn-olea e, and (F) Sys em VI – SQWs@b anched ligand. Emission maxima (λem) a e indica ed
in each panel.
AB
CD
EF
APPENDIX
258
Memb ane s aining expe imen s – con ol sample
The image below shows he non-s ained HEK293 cell sample used as a baseline e e ence in
memb ane s aining expe imen s discussed in Chap e 3, Sec ion 3.2. This con ol was imaged
unde iden ical condi ions as s ained samples and se ed as a backg ound e e ence o he
in ensi y p o iles shown in Figu es 3.36-3.41.
Figu e A.104 B igh - ield (A) and GFP channel (B) images o non-s ained HEK293 cells used as a con ol.
Panel (C) shows he luo escence in ensi y p o ile ex ac ed along he indica ed egion in he GFP channel
image. The ace ep esen s baseline au o luo escence in he absence o any memb ane dye o SQW
s aining.
Spiking HEK Cells Reco dings – T aces Compa ison
To addi ionally suppo he esul s p esen ed in Chap e 3, Sec ion 3.2, ep esen a i e
luo escence aces a e shown o HEK cells s ained wi h SQWs (Sys em VI), o cells ea ed wi h
TTX, and o a non-s ained con ol (baseline). These aces se e as he basis o e alua ing signal
dynamics. Raw baseline-co ec ed and il e ed aces a e p esen ed bo h in o se iew (Figu e
A.105) and o e laid o di ec compa ison (Figu e A.106).
100 m100 m
B igh Field GFP channel
ABC
APPENDIX
259
Figu e A.105 Rep esen a i e luo escence aces o SQW-labeled HEK cells, TTX- ea ed cells, and non-
s ained con ol. (A) Baseline-co ec ed aw aces.(B) Fil e ed aces (bandpass), shown wi h e ical
o se .
Figu e A.106 Same aces as abo e o e laid o di ec compa ison. (A) Baseline-co ec ed aw aces.
(B) Bandpass- il e ed aces, highligh ing di e ences be ween spiking, TTX- ea ed, and con ol condi ions.
A
B
Compa ison plo s
A
B
APPENDIX
260
T ace a e aging analysis
To assess he p esence o pe iodic ac i i y in he eco ded luo escence signals, a ial-a e aging
app oach was applied. Based on he dominan equency de ec ed in he calcium signal, each
ace was di ided in o equal-leng h segmen s co esponding o he expec ed spike pe iod. In
he absence o an ex e nal synch oniza ion igge , he segmen a ion s a poin was a ied
using a se ies o o se s. Fo each o se alue, he ace was di ided acco dingly, and he
esul ing segmen s we e a e aged. This sliding-window app oach allowed iden i ica ion o he
mos a o able alignmen o pe iodic ea u es. Consis en , well-aligned ansien s we e
obse ed in calcium eco dings. In con as , aces om SQW-labeled and non-s ained con ol
cells lacked pe iodic s uc u e, and hei a e aged signals appea ed andom and
indis inguishable. The analysis was pe o med on bo h aw (Figu e A.107) and il e ed aces
(Figu e A.108).
APPENDIX
261
Figu e A.107 T ace a e aging o di e en o se s applied o aw (baseline-co ec ed, un il e ed)
eco dings. (A) Calcium ace shows consis en spike-aligned ansien s ac oss o se s. (B) SQW-labeled
signal appea s noisy and non-pe iodic. (C) Non-s ained con ol esembles SQW ace, indica ing lack o
s uc u ed signal. Yellow lines indica e a e aged esponse; g ey lines ep esen indi idual segmen s.
A
B
C
APPENDIX
262
Figu e A.108 T ace a e aging o di e en o se s applied o p ocessed (baseline-co ec ed and il e ed)
eco dings. (A) Calcium ace shows obus , well-aligned pe iodic signal ac oss o se s. (B) Non-s ained
con ol and (C) SQW-labeled aces emain inconsis en , wi h a e aged signals lacking clea pe iodic
ea u es. Yellow lines indica e a e aged esponse; g ey lines ep esen indi idual segmen s.
A
B
C
APPENDIX
263
4. Supplemen a y Ma e ial o Chap e #4
4.1. Ma e ials
Gold (III) chlo ide ihyd a e (HAuCl4 ∙ 3H2O), isodium ci a e dihyd a e (C6H5Na3O7 ∙ 2H2O)
we e pu chased om Sigma Ald ich. Hexadecyl ime hylammonium b omide (CTAB) and
hexadecyl ime hylammonium chlo ide (CTAC, 25 w % solu ion), sodium hypochlo i e solu ion
(NaClO), sodium bo ohyd ide (NaBH4), sodium hyd oxide (NaOH), sil e ni a e (AgNO3) ci ic
acid, asco bic acid, hyd ochlo ic acid solu ion (HCl) we e pu chased o m Sigma Ald ich and use
d wi hou u he pu i ica ion. A idin, Neu A idin, Neu A idin-FITC conjuga e and
Neu A idin-DyLigh ™ 633 conjuga e along wi h EZ-Link™ NHS-PEG12-Bio in (No-Weigh™
Fo ma ) we e pu chased om The moFishe Scien i ic (Molecula P obes™).
Cell cul u e eagen s, including DMEM/F-12, Neu obasal Plus, B-27 Plus supplemen s,
Glu aMAX, B-27, Penicillin-S ep omycin (10,000 U/mL), poly-D-lysine, and Gel ex™ we e
pu chased om Gibco. Texas Red™-phalloidin (by In i ogen) and DAPI used o s aining we e
pu chased om The mo Fishe Scien i ic.
4.2. Me hods
4.2.1. P o ocols
Tu ke ich Syn hesis o small Au NPs@ci a e
Glasswa e was eshly cleaned wi h aqua egia (3:1 / HCl : HNO3), insed wi h ul apu e wa e
(18.2 MΩ cm) and d ied a 120°C. An aqueous solu ion o chlo oau ic acid (HAuCl4·3H2O, 500
mL, 0.50 mM Au) was ans e ed o a 1 L lask equipped wi h a magne ic s i ing ba . The
suspension was hea ed o he boiling poin o he solu ion (≈100 °C) while s i ing a 700 pm.
In pa allel, a 1% w/ isodium ci a e dihyd a e solu ion (25 mL, 38.8 mM) was p e-hea ed o
~ 95°C. Once he gold solu ion eached boiling poin , he ho ci a e solu ion was in oduced in
one quick sho . Hea ing was main ained o 15 min; du ing his pe iod he colou e ol ed om
colou less o dim b igh blue/pu ple shade o wine- ed.
A e 15 min he hea ing pla e was u ned o and he lask was allowed o cool o oom
empe a u e unde con inued s i ing. The inal dispe sion olume was 525 mL, gi ing an Au0
concen a ion o 0.48 mM and a ci a e: Au mola a io o 5:1. Samples we e s o ed in ials
a 4°C.
APPENDIX
264
Tailo ed size 30 nm sphe ical Au NPs@CTAB
Gold nanosphe es (30 nm) we e syn hesized ia a h ee-s ep seed-media ed g ow h me hod. In
s ep one, 4.7 mL o 0.1 M CTAB was p ehea ed a 35°C o 5 min, ollowed by addi ion o 25 µL
o 0.05 M HAuCl4. A e ano he 5 min incuba ion a 35°C, 0.3 mL o eshly p epa ed 0.01 M
ice-cold NaBH4 was injec ed in one sho unde igo ous magne ic s i ing. S i ing con inued o
2 min, ollowed by incuba ion o 30 min a 27°C. The seed solu ion (Au0 ≈ 0.25 mM) should ha e
an abso bance o 0.6 a 400 nm. Immedia ely, 72 mL o 0.1 M CTAC was mixed wi h 360 µL o
0.05 M HAuCl4 and s i ed mildly a oom empe a u e o 10 min. Then, 27 mL o 0.1 M asco bic
acid was apidly added, ollowed by immedia e injec ion o 0.6 mL seed solu ion. The eac ion
p oceeded o 60 min unde mild s i ing a oom empe a u e, yielding 12 nm Au NPs
(Au0 ≈ 0.225 mM, A400 ≈ 0.54). The p oduc was cen i uged a 8500 pm o 10 min and
edispe sed in 5 mL wa e . The gold concen a ion was adjus ed o 2.8 mM. Fo he inal g ow h,
0.518 mL o he 12 nm seed solu ion was added o 100 mL o 15 mM BDAC p e-incuba ed a
35°C o 10 min. Then 0.5 mL o 0.1 M asco bic acid was injec ed, ollowed by 0.5 mL o 0.05 M
HAuCl4. The solu ion was s i ed o 30 min a 35°C. The esul ing 30 nm Au NPs had
Au0 ≈ 0.26 mM and A400 ≈ 0.624. Oxida i e shape co ec ion was pe o med by adding 360 µL o
1% / NaClO d opwise and s i ing 10 min a 35 °C. Then, 100 µL o 0.05 M HAuCl4 was added
d opwise, ollowed by a 30 min incuba ion a 35°C. The nanopa icles we e cen i uged wice a
14 500 pm o 10 min each and edispe sed in 10 mM CTAB o each a inal gold concen a ion
o 0.4 mM.
Seed-Media ed Syn hesis o Gold Bipy amids
Gold seeds we e p epa ed by mixing 10.0 mL o 50 mM CTAC, 0.05 mL o 50 mM HAuCl4 and
0.05 mL o 1 M ci ic acid in a 20 mL glass ial and s i ing a 20°C. F eshly p epa ed 0.25 mL o
25 mM NaBH4 was hen injec ed unde igo ous s i ing, esul ing in an immedia e colo change
om yellow o b ownish; he abso bance a 400 nm was eco ded (A400 ≈ 0.6 o a 0.25 mM gold
concen a ion) o ensu e comple e educ ion. The ial was sealed and he seed solu ion aged by
hea ing a 80 °C wi h gen le s i ing o 90 min, du ing which he colo e ol ed om b own o
ed.
Fo bipy amid g ow h, 100 mL o 100 mM CTAB was hea ed o 30°C and sequen ially mixed wi h
5.0 mL o 10 mM HAuCl4, 1.0 mL o 10 mM AgNO3, 2.0 mL o 1 M HCl and 0.8 mL o 100 mM
asco bic acid. Unde igo ous s i ing a 30°C, 1.1 mL o he aged seed solu ion was apidly
injec ed and he mix u e was le undis u bed a 30 °C o 2 h. The esul ing gold bipy amids
APPENDIX
271
Figu e A.112 Con ocal imaging o p ima y co ical (CTX) neu ons incuba ed wi h FITC-labeled gold
bipy amids (BPs@Neu A *FITC) a a ious ime poin s o moni o nanopa icle in e naliza ion. Each ow
co esponds o a di e en incuba ion ime: 0.5 h, 1 h, 6 h, and 24 h, wi h a con ol sample (no BPs) a he
bo om. Columns show: (le ) g een luo escence channel (FITC), (middle) me ged luo escence and b igh -
ield, and ( igh ) b igh - ield alone. o addi ional nuclea o cy oskele al s ains we e applied o hese
obse a ions.
h
1h
2 h
h
Con ol
o
50 m50 m50 m
50 m50 m50 m
50 m50 m50 m
50 m50 m50 m
50 m50 m50 m
APPENDIX
272
5. Da a and Code Reposi o y
All da ase s, analysis sc ip s, and supplemen a y iles associa ed wi h his hesis a e a ailable in
an online eposi o y a h ps://saco.csic.es/s/WoC52KNgGsWw6WC. The eposi o y con ains
aw and p ocessed expe imen al da a, code used o s a is ical analyses and igu e gene a ion,
and addi ional documen a ion ha suppo s he esul s p esen ed in he main ex . The
s uc u e o he eposi o y is o ganized by chap e s, enabling di ec access o he unde lying
da a and analysis pipeline.
QR code o he eposi o y:
6. Documen a ion o Digi al Tools
6.1. Da a Analysis
Da a analysis and isualiza ion we e ca ied ou using Py hon code execu ed in Jupy e Lab,
O iginLab so wa e, and ImageJ/Fiji o mic oscopy image p ocessing. Cus om sc ip s a e
included in he accompanying online eposi o y o ensu e ep oducibili y o he esul s.
6.2. Figu e P epa a ion
Unless o he wise indica ed, all igu es and g aphical elemen s included in his hesis we e
p epa ed by he au ho . Figu e assembly was pe o med using Mic oso Powe Poin . Indi idual
elemen s we e c ea ed o p ocessed using he ollowing so wa e ools (in alphabe ical o de ):
BioRende , ChemD aw, Cha GPT (OpenAI), GIMP, Inkscape, Jmol, and Pexels.
6.3. Assis ed Edi ing and Li e a u e Sea ch
So wa e p o ided by OpenAI (Cha GPT models) was used o suppo ph asing, language
co ec ions, and gene al w i ing assis ance. Pe plexi y and No ebookLM we e used as sea ch
engines o iden i y and o ganize ele an sou ces. Zo e o was used o managing e e ences and
ci a ion o ma ing. All scien i ic con en , da a in e p e a ion, and conclusions we e de eloped
by he au ho .
273
Acknowledgmen s
I would like o acknowledge he oppo uni ies and esou ces p o ided by my supe iso s, Ma ek
G zelczak and Ra ael Yus e, which made his esea ch possible. I am also g a e ul o Jona han
Owen and Mónica Ca il o welcoming me in o hei labo a o ies and o hei in ol emen in
he p ojec . Access o hese esea ch en i onmen s shaped bo h he p og ess o his wo k and
my scien i ic g ow h.
These ou yea s ha e been a demanding jou ney, bu I ha e always conside ed mysel e y
lucky. Whe e e I go, I ind ex ao dina y people who accompany me on my pa h.
The s a ing poin o my PhD was Donos ia. A big shou -ou o my AAA suppo g oup – Adam,
Anish, and Alba. You encou agemen , guidance, and unde s anding mean a g ea deal o me.
You insigh s and ad ice helped shape he esea ch, and e en om a dis ance, you emained
ac i ely in ol ed du ing he w i ing phase, helping me na iga e momen s o unce ain y. Thank
you o e e y hing. Tha g a i ude ex ends o he ou h A – Ane Escoba . You posi i e ene gy
was a eal suppo . Thank you o you biological insigh s, o ecognizing he e o I was pu ing
in, and o you encou aging and mo i a ing eedback.
Adam! Dziunia – Twoja obecność i wspa cie wybiega dużo dalej niż mu y CFM. Dziękuję za każdą
po cję gnocchi po udnym dniu, wspólne wieczo y ilmowe, pływanie, gó skie wycieczki,
wspólne zajawki i memy – dużo memów. You “Make Li e Ha de Easie ”.
Alba – you a e my Ecuado ian win, and I am so glad we me . Thank you o all he help
na iga ing bo h scien i ic and adminis a i e challenges. Thank you o being my ansla o and
my Spanish oice whene e needed. Fo lis ening, o unde s anding, and o b inging colo –
no only o my hai .
Ka e yna, Adam, Ma ia, Paschalis – hank you o making ou apa men o eel like HOME.
The ac is ha we always could ind he e exac ly wha we needed. Ou house was many hings
(a ho el, a cinema, a es au an , a bake y, an a ena o igh insec s) – bu mos impo an ly, i
was a place I always el com o able coming back.
Jo ge – o you g ea , uni e sal ad ice (“SHUMP, Zuzanna, SHUPM!”) and swimming
ad en u es. Augus – o making “Bilbao o Vo e and Mo e” uly “Mo e”. Lo enzo – o
ca bona a lesson and sho , ye meaning ul, p esence. Joze – o he good h ows and you calm
a i ude. Đo đe – o c osswo ds and you Eas e n-Eu opean sa casm.
ACKNOWLEDGMENTS
274
The hesis w i ing p ocess was closely obse ed by my new o ice ma es – Quimey, Asie , Vic o ,
Rubén, Ma in, and Juli. Thank you o he pas ies, he en ing cha s, upli ing s o ies and o
eminding me wha day o he week i was. A big hank-you o Raúl, An o, Sabine, Na haniel,
Ca o, Phuong, Idoia, and e e yone else who made Donos i a good place o be.
Ano he impo an pa o his jou ney ook place in New Yo k. Noah – hank you million o
being he bes ad en u e seeke . Thanks o you I s a ed seeing NY in a di e en ligh and
app ecia ed i s pe ks. Fo all he escapades, ques s, hidden gems, quizzes, laughs, la e nigh s in
he lab bu also scien i ic guidance, discussions and sha ing you scien i ic cu iosi y – hank you.
Te esa – only you can uly unde s and how a eling back and o h could be he ha des , ye
mos meaning ul pa o his PhD expe ience. Thank you o being a g ea companion h ough i
all. You a e he pe son connec ing bo h wo lds – all happy momen s o di icul ies sha ed wi h
you always el well unde s ood.
Hakim – o you iendship. I alue all you expe ise, help wi h he se up and scien i ic inpu
bu also all he discussions ha happened along. Thank you o he museums, he conce s,
balle pe o mances, and o making he lab a be e place. Jus in – o all he un you we e
in oducing e e y day. Fo being posi i e e en du ing ough imes, inding joy ega dless, o
being “a moun ain o a man”. Jojo – o being a mo al compass, and eminding me wha is uly
impo an . Fo swimming, hiking, all he cha s, oo op hangou s and eali y checks. Fo making
me a pa o you e y colo ul wo ld. Bo is – Thank you o being such a g ea scien i ic suppo ,
o all he sugges ions and ips. Thank you o celeb a ing all my wins wi h me, and o pa ien ly
helping me unde s and he easons behind he se backs. Thank you o all he co ees, lunch
b eaks and la e cinema e enings as a ellow A-lis e . You b ough esh ene gy when he lab
began o change. Thank you, Tzi zi and Vic o H., o you pa ien guidance, especially a he
beginning o my biological esea ch. You bo h helped a lo when no hing seemed o make sense.
Da ik and Ellie – hank you o being g ea iends. Toge he wi h Ca he in and Beans, you made
me eel like pa o he local li e and communi y. To ully unde s and Washing on Heigh s, you
ha e o li e i ! We su i ed he Dumps e Fi e, and i looks like we’ e bo h s ill s anding. I was
a pleasu e being a silen wi ness o you p ac ical jokes on Noah.
Ł a z lona i Miszon dzięki za wspaniałe owa zys wo na iniszu w Nowm Jo ku. Dzięki Wam
odk yłam nowe aspek y ego mias a i nowe pasje – bi d wa ching w Cen al Pa ku o os a nia
zecz jakiej się po sobie spodziewałam. Zadbaliście o o żebym nie zaginęła w odmę ach
labo a o ium i dawaliście poczucie no malności, p zypominając co jes ważne. Wspaniale było
mieć Was za sąsiadów. Dziękuję!
ACKNOWLEDGMENTS
275
A big hank-you o Be eke , Dan and Galde o you help wi h chemis y aspec s, and o all
membe s o he Owen Lab o welcoming me in o you space and making me eel a pa o i .
To Bella, Samu, Alex, Wa a u, Jesus, Taba a, Doug, Mei wi h he gang (Bill, Bobby and Ma ie),
and e e yone else who made New Yo k much mo e han “jus a esea ch s ay” – hank you.
Dziękuję całemu mojemu ozbudowanemu sys emowi wspa cia, k ó y pozos aje w mocy mimo
upływu la i dzielących nas kilome ów: San o kowi, Ma cie, G ego owi z D ż ną, Zo ce,
Anecie, z ś o i i Pio owi, Mul ipolkom i B udasom+ (Adamowi, Ze asowi, And zejowi,
Pucha owi i Kozia owi – w az ze wszys kimi „plusami”).
And las bu no leas , I wan o hank my amazing amily, o whom I am always enough. To my
wonde ul and suppo i e pa en s Ka a zyna and Leszek, my siblings Aga a and Wik o . And o
Paschalis, o being my g ea es suppo on day- o-day basis. Wi hou you, w i ing his hesis
would be so much ha de . You helped me o keep my ocus while ensu ing p ope es . Thank
you o g ounding me, o all you ca e, and you lo ing suppo .
DZIĘKUJĘ!
