UNIVERSITA’ DEGLI STUDI DI PARMA
DOTTORATO DI RICERCA IN
Fisica
CICLO XXXVI
A co ela i e and mul imodal mic oscopy app oach o s udy
he in e ac ion o pho osensi ize s wi h biological samples
Coo dina o e:
Chia .mo P o . S e ano Ca e a
Tu o i:
Chia .mo P o . C is iano Viappiani
Chia .mo Do . Paolo Bianchini
Do o ando: Ma eo Ma iangeli
Anni Accademici 2020/2021 – 2022/2023
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“…L’amo e è l’unica cosa che iusciamo a pe cepi e che ascenda le
dimensioni di empo e spazio.”
A mia nonna I ana
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Con en s
Con en s ...........................................................................................................................................
In oduc ion .................................................................................................................................... 1
PART 1: Theo y and p inciples ........................................................................................................ 4
Pho odynamic he apy ....................................................................................................... 5
1.1 Applica ion o Bac e ia and Vi uses: Pho odynamic Inac i a ion ........................ 10
1.2 The Mos Used Pho osensi ize s in An i i al PDI .................................................. 12
1.3 Hype icin ............................................................................................................... 13
En eloped i us ................................................................................................................. 15
2.1 SARS-CoV-2 ........................................................................................................... 16
2.1.1 S uc u e ........................................................................................................... 16
2.1.2 In e ac ion wi h human cells: he ole o Spike and i al en elope ................. 17
2.2 Suppo ed lipid bilaye s ........................................................................................ 19
Fluo escence mic oscopy .................................................................................................. 22
3.1 Con ocal Lase Scanning Mic oscopy .................................................................... 25
3.2 Supe - esolu ion and STED mic oscopy ................................................................ 26
3.3 Fluo escence co ela ion spec oscopy ................................................................ 29
A omic Fo ce Mic oscopy ................................................................................................. 32
AFM-Fluo escence co ela i e mic oscopy ...................................................................... 36
PART 2 : Resul s ............................................................................................................................. 37
SARS-CoV-2 ....................................................................................................................... 38
1.1 Hype icin binding o SARS-CoV-2 .......................................................................... 38
1.2 Hype icin A ini y o he i al en elope ............................................................... 41
1.3 Hype icin Dis ibu ion on SARS-CoV-2 Pa icles ................................................... 43
1.4 E ec i eness o Hype icin Agains SARS-CoV-2 .................................................... 44
1.5 Conclusions ........................................................................................................... 46
1.6 Ma e ial and me hods .......................................................................................... 47
1.6.1 Fluo escence mic oscopy. ................................................................................. 47

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1.6.2 Spec oscopy ..................................................................................................... 48
1.6.3 Fluo escence Co ela ion Spec oscopy. .......................................................... 48
1.6.4 Single-Pa icle In ensi y Analysis ...................................................................... 49
1.6.5 Vi al In ec i i y Assays and Vi us Fixa ion ........................................................ 49
Suppo ed Lipid Bilaye s ................................................................................................... 52
2.1 AFM measu emen s o Lipid Bilaye s ................................................................... 53
2.2 AFM imaging ......................................................................................................... 53
2.2.1 AFM imaging wi h Hyp+ligh ............................................................................. 56
2.2.2 AFM imaging wi h Hyp in he da k ................................................................... 58
2.3 AFM mechanical p ope ies .................................................................................. 60
2.3.1 Measu emen s o he Young Modulus ............................................................. 60
2.3.2 AFM mechanical p ope ies: b eak h ough measu emen s ............................ 64
2.4 High speed AFM imaging ...................................................................................... 67
2.4.1 AFM imaging wi h Hyp+ligh ............................................................................. 67
Fo ma ion o po es in he bilaye ......................................................................... 71
Fo ma ion o he dense Lβ phase ......................................................................... 74
2.4.2 AFM imaging o Hyp + da k ............................................................................... 79
2.5 Co ela i e AFM- luo escence .............................................................................. 83
2.5.1 Combined Con ocal and AFM imaging in lipid bilaye s .................................... 83
2.5.2 Two colo Con ocal imaging .............................................................................. 86
2.5.3 Fluo escence co ela ion spec oscopy - p elimina y measu emen s ............. 89
2.6 Conclusions ........................................................................................................... 91
2.7 Ma e ials and Me hods ......................................................................................... 92
2.7.1 SLBs sample p epa a ion .................................................................................. 92
2.7.2 S anda d AFM imaging ...................................................................................... 95
2.7.3 Mechanical p ope ies by AFM: Young Modulus ............................................. 96
2.7.4 Mechanical p ope ies by AFM: b eak h ough measu emen s ....................... 98
2.7.5 High-speed AFM ................................................................................................ 99
2.7.6 Co ela i e mic oscopy ................................................................................... 102
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2.7.7 Con ocal mic oscopy ....................................................................................... 103
2.7.8 Hype icin dilu ion and injec ion ...................................................................... 103
AFM measu emen s o SARS-CoV-2 ............................................................................... 104
3.1 AFM imaging ....................................................................................................... 106
3.2 AFM nanomechanical p ope ies ........................................................................ 108
3.3 Conclusions ......................................................................................................... 111
3.4 Ma e ials and me hods ....................................................................................... 111
3.4.1 Imaging ............................................................................................................ 112
3.4.2 Nanomechanics ............................................................................................... 112
Conclusions and u u e pe spec i es .............................................................................. 113
Aknowledgemen s .......................................................................................................... 115
Bibliog aphy .................................................................................................................... 117
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1 | P h D h e s i s - M a e o M a i a n g e l i
In oduc ion
O e he pas cen u y, signi ican medical ad ancemen s ha e g ea ly enhanced people's quali y
o li e. Key de elopmen s include Alexande Fleming's disco e y o an ibio ics in 1928, pi o al in
comba ing bac e ial in ec ions; he ad en o chemo he apeu ic agen s as i al ools agains
cance ; and he ongoing e ec i eness o accines in p e en ing o e adica ing deadly i al
diseases. Howe e , hese echniques ha e hei limi a ions. Fo example, he o e use o
an ibio ics in ecen decades has led o a su ge in an ibio ic- esis an bac e ia, a majo heal h
conce n oday. Chemo he apy's lack o selec i i y, ha ming bo h cance ous and heal hy cells,
emains a challenge o i s e icacy and ole ance. Despi e hei e iciency agains deadly i uses,
including eme ging ones like SARS-CoV-2, accines ace challenges in p oduc ion and don' always
p o ide comple e p o ec ion agains pa hogens.
In he las 30 yea s, new medical s a egies ha e been de eloped o comba pa hogens and
cance cells, ei he as s andalone ea men s o as adjunc s o es ablished he apies. One such
me hod is Pho odynamic The apy (PDT), which uses ligh and pho osensi i e molecules o a ge
speci ic a eas.
An imic obial PDT is ad an ageous wi h espec o adi ional an imic obials because i doesn’
ely on a singula ac ion mechanism, educing he isk o pa hogen esis ance. Common
an imic obials, wi h hei single ac ion mechanism and un egula ed use, o en con ibu e o
esis an s ain p oli e a ion.
PDT's indisc imina e a ge ing also simpli ies applica ion, as i doesn' equi e an in-dep h
unde s anding o he mic oo ganism's biology. This makes i a quicke , mo e e sa ile op ion
agains a ious pa hogens, including eme ging ones. Despi e hese bene i s, he medical
communi y emains cau ious abou using PDT agains pa hogenic mic oo ganisms, e en hough
i 's a clinically app o ed cance ea men . Consequen ly, unding and basic esea ch in his a ea
a e limi ed, aising ques ions abou hesi ancy owa ds his app oach [1].
The wo k o his hesis is amed in his con ex : many ad anced mic oscopic and spec oscopic
echniques we e applied o unde s and how a pho osensi ize wo ks in physiological
en i onmen s. We ocused on pho odynamic inac i a ion o i uses, a opic o heigh ened
ele ance in ecen yea s, as seen in he igh agains HIV, MERS, and SARS i uses. These i uses
belong o he class o En eloped i uses; hence hey sha e a common ea u e: a phospholipidic
en elope c ucial o hei p o ec ion and eplica ion. Ou esea ch ocused on cha ac e izing he
e ec s o Hype icin, a p omising and e sa ile pho osensi ize known o be e ec i e agains
some en eloped i uses [2], on SARS-CoV-2 and on specially designed model memb anes ha
simula e he i us's en elope.
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As a ma e o ac , in PDI and PDT he pho osensi ize (PS) plays a c ucial ole. A good
pho osensi ize should ha e se e al key cha ac e is ics o be e ec i e, he mos impo an ones
a e:
• High Pho os abili y: The PS should be s able unde ligh exposu e and no deg ade
quickly, losing i s e icacy.
• S ong Abso p ion o Visible Ligh : A PS ha s ongly abso bs isible ligh is p e e able,
especially in he ed and nea -in a ed spec al ange (600-900 nm), whe e issues a e
mo e pene able; his makes he ea men mo e e ec i e o a a ie y o applica ions.
• High quan um yield o he iple s a e: he PS should ha e an exci ed iple s a e wi h
a long li e ime and su icien ene gy (g ea e han 96 kJ/mol); his is di ec ly e lec ing he
abili y o p oduce ROS.
• Low Da k Toxici y: The PS should be non (o minimally)- oxic in he absence o ligh
ac i a ion. This cha ac e is ic is impo an o ensu e ha i doesn' cause ha m o he hos
o ganism o en i onmen unde no mal condi ions.
• Chemical and Biological Compa ibili y: The PS should be chemically and biologically
compa ible wi h he hos o ganism and he en i onmen in which i is used. I shouldn'
in e ac nega i ely wi h o he componen s o he ea men o he biological sys em.
• Minimal Side E ec s: The PS should cause minimal o no side e ec s o he hos o ganism
o he su ounding heal hy issues du ing and a e he ea men . High e icacy a low
concen a ions is equi ed o minimize po en ial damage o heal hy cells. Also, as
elimina ion o he molecule om he body is needed o a oid side e ec s.
• Ease o Syn hesis and Modi ica ion: The syn hesis o he PS should be s aigh o wa d,
and i should be amenable o chemical modi ica ions. This lexibili y allows o he
ailo ing o PS p ope ies o sui speci ic applica ions.
• App op ia e Lipophilici y/Hyd ophilici y Balance: The PS should ha e a balanced
lipophilici y and hyd ophilici y o ensu e good solubili y and bioa ailabili y. This balance
also a ec s he PS's abili y o pene a e cell memb anes and each a ge si es wi hin
pa hogens.
The las o hese poin s is c i ical because many pho osensi ize s a e highly hyd ophobic and hus
poo ly soluble in aqueous en i onmen s. A solu ion is o en o in oduce pola subs i uen s o
make hem mo e soluble, as is o en done wi h po phy ins, he mos common pho osensi ize s.
Hyd ophobic molecules in an aqueous en i onmen end o minimize in e ac ions wi h he
sol en and o m agg ega es, which ha e e y di e en pho ophysical p ope ies om dissol ed
molecules. Agg ega ed pho osensi ize s poo ly abso b ligh , and he ew exci ed elec onic s a es
gene a ed quickly quench, p e en ing he p oduc ion o ROS. Howe e , he bes me hod o
o e come his p oblem is o use a ehicle o hei elease ha is biocompa ible wi h he

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he apy's a ge en i onmen [9]. This app oach has addi ional bene i s. Pho osensi ize s a e no
inhe en ly selec i e o hei in ended a ge s, necessi a ing a molecula a ge ing mechanism o
ensu e he he apy’s e ec i eness. Gi en ha 1O2 has a li espan o abou 3-4 s [10][11] in
aqueous en i onmen s and can only di use sho dis ances o 100 o 200 nanome e s, i needs
o be in close p oximi y o he in ended a ge . I no , he e's a isk o unin en ionally causing
oxida i e ha m o heal hy cells, which would coun e ac he he apeu ic goals.
In PDT, i 's c ucial o he pho osensi ize o di e en ia e be ween cance ous and no mal cells.
This is achie ed h ough ca ie s ha deli e he pho osensi ize . These ca ie s ake ad an age
o he umo 's unique physical and chemical p ope ies o passi e a ge ing. Al e na i ely,
ca ie s can employ ac i e a ge ing by capi alizing on he al e ed ecep o densi ies ound in
umo cells[12]. Va ious s udies ha e also shown ha pho odynamic he apy (PDT) can enhance
an i- umo immuni y [13]. Mice whose umo s we e ea ed wi h PDT demons a ed an abili y o
esis subsequen umo s imula ion, indica ing he p esence o immunological memo y. A
cu en challenge is o demons a e he ex en o which PDT applied o umo s such as Non-
Small-Cell Lung Ca cinoma can s imula e he immune sys em o adap and coun e ac cance
ecu ence [14] [15].
This app oach o exploi he immune sys em o he ea men o cance is ela i ely new and is
called cance immuno he apy [16][17]. I is a e olu iona y app oach in he ea men o cance ,
le e aging he body's own immune sys em o comba he disease. This ield has seen signi ican
ad ancemen s and has become a i al pa o he oncologic he apeu ic a senal [18]. Unlike
adi ional ea men s like chemo he apy and adia ion, which di ec ly a ge cance cells,
immuno he apy wo ks by enhancing o modi ying he immune sys em's esponse o cance . I
can ei he s imula e he immune sys em o wo k ha de o sma e in a acking cance cells, o i
can p o ide he immune sys em wi h addi ional componen s, such as man-made immune sys em
p o eins.
We will no dig any deepe in o his aspec o applica ion o PDT, ins ead we will ocus on he PDI
inac i a ion o pa hogens.
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1.1 Applica ion o Bac e ia and Vi uses: Pho odynamic Inac i a ion
Al hough PDT is mo e commonly known o i s applica ion in cance he apy, i s pho odynamic
e ec has ex eme ele ance in he con ex o implemen a ion agains pa hogens.
Mic obial in ec ions (including hose caused by bac e ial, ungal, and i al pa hogens) esul ed in
nea ly 8.5 million dea hs wo ldwide in 2016, wi h 700,000 di ec ly a ibu ed o d ug- esis an
in ec ions. This numbe could apidly inc ease in he coming yea s because, in he wo s -case
scena io, up o 10 million people could die each yea due o an ibio ic- esis an in ec ions i le
unchecked. The p ima y cause o mic obial an ibio ic esis ance is he o e use and misuse o
an ibio ics in bo h human and animal applica ions [19].
These ala ming s a is ics ha e ekindled in e es in pho odynamic inac i a ion (PDI), which has
ecen ly gained conside able a en ion. Reac i e oxygen species p oduced can simul aneously
a ack a ious biomolecula si es in he a ge pa hogen (p o eins, lipids, nucleic acids...), o e ing
mul iple and a iable ac ion si es. This lack o speci ici y owa ds he a ge bypasses
con en ional esis ance mechanisms and inhibi s he de elopmen o esis ance o he agen s
hemsel es [6] [20].
An ibac e ial PDI can be used in he con ex o localized in ec ions accessible o ligh , such as skin
in ec ions, ch onic wounds, diabe ic oo and leg ulce s, bu he use o op ical ibe s also makes
a eas such as he uppe espi a o y ac , gas oin es inal ac , and ea s accessible. Howe e ,
only a ew clinical ials ha e been conduc ed in his di ec ion [1], and he me hod is cu en ly
clinically app o ed o he ea men o pe iodon al o endodon ic den al in ec ions [6].
PDI applied o i uses has his o ically been explo ed o a lesse ex en , al hough an i i al e ec s
ha e been known o some dyes/pho osensi ize s such as me hylene blue and o he
pheno hiazine de i a i es since he 1960s, like ac idine o ange among o he s [20]. In he con ex
o an i i al PDI, a ecen s udy explo ed he e ec i eness o me hylene blue-based
pho odynamic inac i a ion agains in acellula B-CoV and SARS-CoV-2 i uses. The s udy
assessed he e icacy o PDI unde a ious ligh sou ces in i o, ocusing on i s po en ial as a
ea men op ion o in ec ions caused by hese i uses [21].
Clinical applica ions o an i i al PDI ha e been con o e sial o some ime, and mo e han hal
o he publica ions on his opic ha e been dissemina ed in he las 15 yea s, sugges ing ha
despi e a long his o y o pho odynamic in es iga ions, he use o an i i al PDI is s ill a young and
apidly g owing ield.
In he medical ield, he main a eas ha ha e seen wide applica ion o an i i al PDI a e he
pu i ica ion o blood de i a i es [22] and he ea men o a ew i uses [23]. PDI has been used
on a ious o ms o He pes Simplex Vi us (HSV) in ec ions, including o al, geni al, ocula , and
he pes zos e in ec ions; on ex e nal Human Papilloma i us (HPV) mani es a ions [24], and he e
is also eme ging e idence sugges ing he e icacy o PDT in ea ing eac i a ed Va icella Zos e
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Vi us VZV, wi h case epo s indica ing signi ican clinical imp o emen and educed ecu ence
a e ea men [25].
Mo eo e , Recu en Respi a o y Papilloma osis (RRP) caused by HPV, is one o he ew in e nal
diseases cu en ly being ea ed wi h PDI. The ea men is ypically combined wi h s anda d
emo al o dis up ion me hods, and s udies ha e shown p omising esul s wi h signi ican
educ ion in ecu ence a es and manageable side e ec s [26].
Fu he mo e, an i i al PDI has also ga ne ed signi ican in e es in a eas such as wa e [27], ood
[28] and su ace decon amina ion [29].
Recen ly, PDI is being p oposed as an al e na i e ea men in classical an i i al he apy based on
d ugs. Jus like an ibio ic esis ance, esis ance o an i i al d ugs is cu en ly a cause o conce n
[30] [31]. The e o e, pho odynamic inac i a ion (PDI) o i uses is o in e es as an al e na i e
ool in an i i al ea men s.
Addi ionally, ecen s udies ha e e ealed sys emic e ec s o pho odynamic ea men , including
he igge ing o immune esponses, jus as we men ioned happening in PDT agains umo s [23]
[32][33]. This disco e y enhances he appeal o PDI as no jus an al e na i e, bu also a
complemen a y app oach o con en ional he apies.
Anyway, despi e being an exci ing and p omising me hod, PDI is s ill la gely in he esea ch and
de elopmen phase, especially conce ning i s applica ion o a ious ypes o pa hogens and in
di e en se ings. The implemen a ion in clinical and o he p ac ical se ings a e dependen on
he ongoing esea ch, and will bene i om echnological ad ancemen s, and be subjec o
egula o y app o als.
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1.2 The Mos Used Pho osensi ize s in An i i al PDI
In he ad ancemen o bo h an i umo and an imic obial he apies, a a ie y o compounds
known o hei abili y o gene a e eac i e oxygen species ha e been explo ed. Among hese,
speci ic classes ha e shown ema kable e ec i eness in inac i a ing nume ous i al species,
pa icula ly in in i o s udies. The mos no able classes include:
• Polyphenols such as cu cumin;
• Ribo la in and i s de i a i es;
• Pheno hiazinium de i a i es, like me hylene blue and oluidine blue;
• Fulle enes and ca bon nanoma e ials;
• Phenalenone de i a i es;
• Te apy oles, he mos di e se class o pho osensi ize s, including po phy ins,
ph halocyanines, chlo ins and bac e iochlo ins. These molecules ha e long played a
decisi e ole in an i umo PDT;
• Xan hene de i a i es: like Eosin Y, E y h osine and Rose Bengal ha a e all de i ed om
luo escein;
• Quinones, such as Hype icin.
Figu e 1.4: a ew examples o pho osensi ize s used in PDI, image om [6]. a) Me hylene Blue; b) Toluidine Blue; c)
TMPyP (a modi ied po phy in); d) chlo in-e6; e) ulle ene C60, ) SAPYR (a wa e -soluble phenalenone de i a i e).
Adap ed om [6].
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1.3 Hype icin
Figu e 1.5: S uc u e o Hype icin.
Hype icum pe o a um, commonly known as S . John's wo , is an he b his o ically used in
adi ional medicine o healing wounds, ea ing bu ns, and alle ia ing mild o mode a e
dep ession. I s use in con empo a y ea men s o mild dep ession pe sis s, al hough he exac
mechanisms o ac ion a e no ully elucida ed. I 's hough o wo k by inhibi ing he eup ake o
exci a o y neu o ansmi e s like se o onin, no epineph ine, and dopamine.
Hype icin (Hyp) (Figu e 1.5) is he p ima y ac i e compound ound in Hype icum species and is
conside ed key o he he apeu ic e ec s o S . John's wo . I is a polycyclic a oma ic
hyd oca bon, speci ically a naph hodian h one, known o i s hyd ophobic na u e and insolubili y
in wa e . Hype icin possess se e al pho ophysical cha ac e is ics when i is solubilized in an
apola en i onmen .
Figu e 1.6: a) abso p ion and exci a ion spec um o Hyp in a lipophilic en i onmen [34]. b) Hyp is poo ly soluble in
aqueous en i onmen s ( op), while i eco e s all i s pho ophysical p ope ies, luo escence comp ised (bo om). a) is
adap ed om [34].
b

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I is dis inguished by i s b igh ed colo and s ong abso p ion in bo h he isible and UV
spec ums (Figu e 1.6a). Owing o hese in iguing p ope ies, Hype icin has been he subjec o
inc eased medicinal and he apeu ic in e es in ecen yea s. Pho ophysical s udies e eal ha
Hype icin is an e ec i e luo opho e, wi h a quan um yield (ϕF=0.35) in DMSO, making i
sui able o luo escence-based in es iga ions.
No ably, Hype icin's mos signi ican a ibu e is i s po en abili y o gene a e single oxygen,
e idenced by a ema kable quan um yield o (ϕΔ=0.33) [35]. This makes i a po en
pho osensi izing agen , opening a enues o i s applica ion in pho odynamic he apy.
In ecen yea s, esea ch has demons a ed ha Hype icin displays a p e e en ial in e ac ion wi h
cance cells o e heal hy ones, showing minimal oxici y o he la e [36]. I s pho odynamic
ac i i y can induce he dea h o cance cells. Fu he mo e, Hype icin has shown signi ican
cy o oxic e ec s on umo issues e en wi hou ligh exposu e. Va ious in i o and in i o cance
models ha e epo ed subs an ial inhibi o y e ec s by Hype icin. Howe e , he e ha e been
ela i ely ew clinical ials conduc ed o e alua e i s sa e y and ac ual clinical e ec i eness in
pa ien s [37].
The e ec i eness o Hype icin in pho odynamic inac i a ion (PDI) has been s udied in a ious
con ex s, agains bo h bac e ia and i uses.
Hyp was ound e ec i e agains a a ie y o bac e ia, in e es ingly o bo h G am-nega i e such
as Esche ichia Coli [38] , o G am-posi i e like S aphylococcus Au eus and En e ococcus aecalis
[39] wi h a consis en e ec also on bio ilm agg ega es o he la e [40].
Hyp has also demons a ed signi ican an i i al ac i i y in a ious s udies. The e ec i eness o
Hype icin in comba ing i uses is la gely de e mined by i s a ini y owa ds i al componen s and
he numbe o ac i e molecules ha a ach o indi idual i uses. S udies ha e demons a ed ha
Hype icin can signi ican ly inac i a e se e al i uses when exposed o isible ligh and, in some
cases, e en in he absence o ligh [41][42] . This e icacy is pa icula ly no able agains a a ie y
o lipid con aining en eloped i uses [2], such as in luenza A i us, he pes simplex, mu ine
cy omegalo i us, Sindbis, hepa i is B and C, equine anemia, and HIV [43]; howe e , Hype icin is
inac i e agains non-en eloped i uses, ha don’ con ain any phospholipid en elope [44].
Hyp, as we al eady said, is a hyd ophobic ch omopho e, and he e o e possesses a p e e en ial
localiza ion in lipid memb anes [45], media ed by i s lipid solubili y [46]. Hyp’s inc easing
e idence on he e ec i eness agains memb ane-en elope i uses sugges ed i s po en ial as a
ea men o SARS-CoV-2 in ec ions, an en eloped i us i sel .
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En eloped i us
Co ona i uses a e a di e se g oup o i uses ha can in ec many animals, including humans.
They a e cha ac e ized by speci ic ea u es such as hei ype o nucleic acid, a lipid en elope,
and a dis inc shape. These i uses a e single-s anded posi i e-sense RNA i uses, a ying in size
om 60 nm o 140 nm. Unde a mic oscope, hei spike p o eins on he memb ane gi e hem a
'c own-like' appea ance, leading o hei name [47]. They belong o he Co ona i idae amily
wi hin he Nido i ales o de (Figu e 2.1). These i uses a e ca ego ized in o α-, β-, γ-, and δ-
co ona i uses, wi h α- and β-co ona i uses p ima ily in ec ing mammals and γ- and δ-
co ona i uses a ec ing bi ds and bo h mammals and bi ds, espec i ely. The mos conce ning
o humans a e he α- and β-co ona i us gene a [48] [49].
Fou human-a ec ing co ona i uses, HKU1, NL63, 229E, and OC43, gene ally cause mild
espi a o y illnesses. Howe e , in he las wen y yea s, he e ha e been signi ican ou b eaks
due o animal co ona i uses jumping o humans. The i s was in 2002-2003 wi h he eme gence
o SARS-CoV, a β-co ona i us om ba s, in China's Guangdong p o ince. I used palm ci e s as an
in e media y and led o 8422 in ec ions and 916 dea hs. A decade la e , MERS-CoV, ano he ba -
o igin β-co ona i us, eme ged in Saudi A abia, whe e d omeda ies we e he in e media e hos s.
This i us in ec ed 2494 people and caused 858 dea hs.
Figu e 2.1: axonomy o Co ona i uses. Analogy wi h human axonomy. The ounde s o i ology and o he eminen
scien is s ep esen indi idual human beings. Adap ed om [49].
16 | P h D h e s i s - M a e o M a i a n g e l i
2.1 SARS-CoV-2
In Decembe 2019, Wuhan, a ci y in China's Hubei p o ince, epo ed 27 unexplained pneumonia
cases. A common link among ea ly pa ien s was hei p oximi y o Wuhan's Huanan Sea ood
Ma ke [50].
Chinese au ho i ies in o med he WHO abou he ou b eak on Decembe 31, 2019, ollowing
in es iga ions in o he pa ien s' espi a o y samples by specialized labs. The Wuhan ma ke was
shu down he nex day. Tes s nea he ma ke de ec ed he i us, sugges ing he ma ke as a
po en ial o igin o he zoono ic i us sp ead. Mo eo e , he i us's abili y o ansmi be ween
humans was con i med by he ising numbe o cases un ela ed o he ma ke .
By Janua y 7, his i us was iden i ied as a no el co ona i us, sha ing o e 95% gene ic simila i y
wi h ba co ona i uses and a ound 80% wi h SARS-CoV. Classi ied as "se e e acu e espi a o y
synd ome- ela ed co ona i us" in he β-co ona i us genus, i was named SARS-CoV-2 [47]. I s
close gene ic ela ion o ba co ona i uses indica ed a possible ba o igin, wi h ansmission o
humans po en ially in ol ing an in e media y species.
2.1.1 S uc u e
SARS-CoV-2 is s uc u ally e y simila o o he co ona i uses, and i ’s composed o a ew
undamen al componen s (Figu e 2.2):
• RNA Genome: he gene ic ma e ial o he i us, single-s anded RNA genome, which is
posi i e-sense. This means he i al RNA can be di ec ly ansla ed in o i al p o eins by
he hos cell's ibosomes.
• Nucleocapsid (N) P o ein: a p o ein ha holds he RNA genome. They mainly play a ole
in i us assembly.
• Memb ane (M) and En elope (E) P o eins: p o eins embedded in he lipid en elope.
• En elope: a phospholipidic bilaye which is de i ed om he hos cell memb ane du ing
i al eplica ion and budding, om he lipids o he Endoplasmic Re iculum (ER) [51][52]
(Table 1). I plays a c ucial ole in he i us's abili y o in ec hos cells.
Mol% o ER memb ane lipids
Phospha idylcholine
58%
Phospha idyle anolamine
22%
Phospha idylse ine
3%
Phospha idylinosi ol
10%
Sphingomyelin
3%
Choles e ol
0.018%
Table 1: lipid cons i uen s o he i al en elope, which a e iden ical o hose ound in he ER.
17 | P h D h e s i s - M a e o M a i a n g e l i
• Spike (S) P o eins: he mos no able ea u e, a p o ein which p o udes om he i us
su ace, p o iding he cha ac e is ic c own-like appea ance. These p o eins a e c i ical o
he i us abili y o in ec hos cells; hei binding o he ACE2 ecep o acili a es he i us
en y in o he hos cell.
Figu e 2.2: SARS-CoV-2 s uc u e (le ) and ep esen a ion o he unc ional subuni s o S, wi h ocus on he binding
be ween S1 and Ace2 ( igh ). C ea ed wi h BioRende .com.
2.1.2 In e ac ion wi h human cells: he ole o Spike and i al en elope
As we said, he Spike (S) is essen ial o in oducing i s gene ic ma e ial in o he cells o he hos
o ganism. I is a glycop o ein abundan ly loca ed on he i us's phospholipid en elope. The S
p o ein exis s in a me as able o m, and i ’s designed o unde go signi ican ea angemen s,
enabling he usion o he i al memb ane wi h ha o he hos cell, hus acili a ing i al en y
[53]. I has a Mw = 440 kDa and i is composed o h ee iden ical subuni s. These subuni s o m a
ime ic s uc u e, which is a common ea u e among co ona i us spike p o eins. Each subuni in
he ime is di ided in o wo main unc ional pa s: he S1 and S2 subuni s (Figu e 2.2).
The S1 subuni o he i us a ge s and a aches o he hos cell's ecep o ia a speci ic a ea
called he ecep o binding domain (RBD). The ecep o in ques ion on he hos cell is he Ace2
p o ein, an angio ensin-con e ing enzyme, which plays a key ole in egula ing he ho mone
angio ensin. Ace2 is p edominan ly ound in cells o he lungs, kidneys, in es ines, and hea .
The spli ing o he S1 and S2 subuni s is key o he subsequen ac i a ion o he S2 subuni ; his
p ocess is acili a ed by he ac ion o a se ine p o ease known as TMPRSS2 [54]. This enzyme
induces i e e sible con o ma ional changes, enabling he ac i a ion p ocess o S2.
S2 subuni ac ually plays a c i ical ole in media ing he i al usion and en y p ocess in o he
hos cell. I exploi s se e al domains, ha a e esponsible o b inging he i al and cell
memb anes close enough o use, and plays a ole in dis up ing and connec ing he lipid bilaye s
o he hos cell memb ane, essen ial o i al usion and en y [55]. In his amewo k, he i al
en elope plays a undamen al ole in he in ec ion, as well. Indeed, he i al en elope plays a
24 | P h D h e s i s - M a e o M a i a n g e l i
The molecule hen loses some ene gy h ough a ious non- adia i e mechanisms (in e nal
con e sion, ib a ional elaxa ion) o he lowes ib a ional le el o he i s exci ed s a e S1. F om
his ene gy le el he adia i e luo escence emission can occu (Kasha’s ule). The loss o ene gy
associa ed wi h non- adia i e elaxa ion leading o he lowes ib a ional le el o S1 means ha
he emission o pho ons occu s a a longe wa eleng h han abso p ion. Such molecules, which
can emi luo escence wi h high e iciency, a e called luo opho es. In luo escence mic oscopy,
he sample is illumina ed wi h a speci ic wa eleng h o ligh (exci a ion) ha he luo opho e
abso bs, i s consequen emission which happens a a di e en wa eleng h (S okes shi ) is
eco ded. This spec al sepa a ion is exploi ed by he ins umen , and by means o some op ical
componen s, he exci a ion and emission can be sepa a ed, so ha only he emission om he
luo opho e is de ec ed. Because o ha he mos common con igu a ion exploi s only one
objec i e lens, o ei he he exci a ion and he emission ligh (epi luo escence).
Figu e 3.3: a) illus a ion o he pho ophysical mechanism o luo escence, b) scheme o an epi luo escence wide ield
mic oscope. b) is adap ed om Wikipedia, [66].
Since he luo opho es can be chemically a ached o a ge molecules h ough se e al di ec o
undi ec me hods, e.g., an ibody labeling o gene ic exp ession o usion p o eins, his
mic oscopy echnique achie es high speci ici y, allowing o sensi i e de ec ion o biological
p ocesses a he cellula and molecula le els.
One o he mos s aigh o wa d implemen a ions o luo escence mic oscopy is ep esen ed by
he wide ield mic oscope. This modali y in ol es he uni o m illumina ion o he en i e specimen
unde obse a ion. The en i e ield o iew is exci ed simul aneously, allowing o he obse a ion
o luo escen ly labeled s uc u es wi hin he sample. The image is o med by he ligh coming
om he sample and is cap u ed ei he h ough an eyepiece o di ec obse a ion o on o a
came a senso . This app oach is ela i ely simple and as , making i sui able o a a ie y o
applica ions whe e apid imaging o la ge a eas is equi ed. The main limi a ion is ep esen ed
by he backg ound luo escence since all planes o he sample a e illumina ed and emi ligh
simul aneously, po en ially educing image esolu ion and con as .

25 | P h D h e s i s - M a e o M a i a n g e l i
3.1 Con ocal Lase Scanning Mic oscopy
Con ocal lase scanning mic oscopy is an ad anced op ical imaging echnique, which majo
ea u e is he op ical sec ioning, i.e. he abili y o image only a speci ic plane (o op ical sec ion)
o he specimen a a ime. In his way i o e comes he limi a ion p e iously desc ibed o he
wide ield luo escence mic oscope.
To achie e sec ioning con ocal mic oscopy uses a poin illumina ion. The ligh is ocused o a
small poin on he specimen and scanned ac oss he specimen in a as e pa e n (bo h la e ally
and axially) o build up a h ee-dimensional (3D) image, one poin a a ime. The ligh emi ed o
e lec ed om ha spo is collec ed and ocused on o a single-poin de ec o (PMT, APD, SPAD,
e c.). The exci a ion sou ce is cons i u ed by a lase which p o ide in ense, monoch oma ic, and
cohe en ligh , ideal o p ecise illumina ion and o exci ing luo escen dyes.
Such a mic oscope in ol es he use o wo pinholes, small and ci cula ape u es; one is placed
a e he exci a ion sou ce and he o he be o e he de ec o . The wo pinholes a e con igu ed
such ha hei ocal planes coincide (o belong o conjuga ed ocal planes), as well as he objec ,
om which he name “con- ocal” [67] [68].
The ligh om he in- ocus plane o he specimen passes h ough he pinhole and is de ec ed,
while hose coming om planes abo e o below he ocal poin is mos ly ou -o - ocus when i
eaches he pinhole and is hus no de ec ed. By excluding he ou -o - ocus ligh i ’s possible o
achie e he op ical sec ioning. A co ec pinhole size should yield a PSF wi h a maximum diame e
co esponding o he cen al b igh spo o he Ai y disk, i.e. ≤ 1 Ai y Uni (AU). In addi ion, by
educing he dimension o he pinhole is also possible o sligh ly imp o e he esolu ion, ei he
la e al [69] o axial [70] [71], bu in expense o he signal- o-noise a io [72].
Figu e 3.4: ypical a chi ec u e o a con ocal mic oscope. Adap ed om Encyclopedia B i annica [73]. Copy igh ©
2012 Encyclopedia B i annica, Inc.
26 | P h D h e s i s - M a e o M a i a n g e l i
3.2 Supe - esolu ion and STED mic oscopy
O e he cou se o he las decades, mic oscopis s ha e made many e o s in de eloping new
echniques o imp o e he esolu ion o luo escence mic oscopes, b eaking he limi imposed
by di ac ion, wi h he aim o explo e biological s uc u es and p ocesses happening a he
nanoscale. Many supe - esolu ion echniques ha e been p oposed o ci cum en his limi . These
me hodologies employ a ious s a egies o go beyond he di ac ion limi , bu hey all ely on
he same p inciple: he selec i e swi ching be ween he “on” and he “o ” s a es o he
luo opho es. This p ocess can be s ochas ic o de e minis ic [74].
S ochas ic echniques like STORM (STochas ic Op ical Recons uc ion Mic oscopy) [75][76] o
PALM ( luo escence Pho oAc i a ion Localiza ion Mic oscopy) [77] pe o m a andom ac i a ion
and subsequen localiza ion o indi idual luo escen molecules. A any momen , only a ew
luo opho es a e in he “on” s a e, allowing hei posi ions o be de e mined wi h high p ecision.
Repea ing his p ocess o e ime and loca ing he posi ions o such molecules allows o he
econs uc ion o a supe - esolu ion image.
On he o he hand, de e minis ic echniques like STED (S imula ed Emission Deple ion) [78] o
SSIM (Sa u a ed S uc u ed Illumina ion Mic oscopy) [79] spa ially selec which a e he
luo opho es ha should be on he “on” o “o ” s a e by means o a con olled illumina ion.
The g oundb eaking wo k on supe - esolu ion mic oscopy echniques was wo h he Nobel P ize
in Chemis y o S. Hell, E. Be zig and W.E. Moe ne in 2014 [80].
The STED mic oscope is a lase scanning mic oscope, wi h an a chi ec u e e y simila o he
con ocal. The main di e ence elies on he p esence o an addi ional lase beam, he STED beam,
which is shaped as a doughnu ea u ing a ze o-in ensi y cen e . This beam is designed o quench
he luo escence emission in he pe iphe y o he illumina ed a ea by s imula ing he exci ed
molecules o e u n o he g ound s a e by s imula ed emission, lea ing only he molecules in he
e y cen e o he doughnu -shaped beam o be de ec ed.
Figu e 3.5: s imula ed emission is a compe i i e p ocess wi h espec o he spon aneous emission ( luo escence)
(le ). Example o an ideal ange o wa eleng h o he STED lase , a gi en spec a is indica ed wi h he ed ba ha
lies on he ail o he emission spec um ( igh ). The image on he igh is adap ed om [81] Copy igh © 2022
Angs om Technologies, Inc. All igh s ese ed.
27 | P h D h e s i s - M a e o M a i a n g e l i
STED imaging equi es a co ec empo al and spa ial alignmen o he STED beam. Indeed, he
STED beam should be synch onized wi h he exci a ion beam o a oid ha luo escence occu s
be o e he STED pulse eaches he molecule. Addi ionally, since any impe ec ions on he ze o-
in ensi y a he cen e o he beam impac s nega i ely on he signal o noise a io, special ca e
should be gi en o he beam pola iza ion [82] Also, he wa eleng h o he STED beam is c ucial.
The s imula ed emission e iciency depends on he emission spec um o he luo escen
molecule and he lase wa eleng h should be chosen p ope ly. A wa eleng h ha aligns wi h he
maximum o he emission spec um should gi e he maximum o e iciency o his ansi ion.
Howe e , he abso p ion and emission spec um a e no enough spec ally sepa a ed (S okes
shi ) o a oid abso p ion in co espondence o he maximum o emission. Fo his eason, he
STED wa eleng h mus be mo e ed-shi ed and should align wi h he ail end o he emission
spec um o he luo opho es. A such wa eleng hs he s imula ed emission is less e icien and
occu s a highe powe s, equi ing he STED beam o be some o de o magni udes mo e
powe ul han he exci a ion lase .
Figu e 3.6: images o Abbe io STAR g een, luo escen beads (d = 40 nm) acqui ed wi h a Leica SP5. λex = 488 nm,
λSTED = 592 nm. We can no ice he imp o emen o la e al esolu ion wi h STED.
The esolu ion o STED mic oscopy can be uned wi h he powe o he STED beam and,
heo e ically has no limi s excep he molecule i sel (Figu e 3.7).
Ma hema ically, he esolu ion o a STED mic oscope can be w i en by modi ying he Equa ion
(26) as ollows:
𝑑= 𝜆
2𝑁𝐴√1+ 𝐼
𝐼𝑠𝑎𝑡
(5)
28 | P h D h e s i s - M a e o M a i a n g e l i
Whe e I ep esen s he maximum in ensi y o he STED lase and Isa is he sa u a ion in ensi y
ha co esponds o he in ensi y o he STED lase which is necessa y o quench he 50% o he
spon aneous emission o he luo opho es.
Figu e 3.7: simpli ied a chi ec u e o a STED mic oscope. The esolu ion imp o es wi h he powe o he STED beam.
Adap ed om [83]. Copy igh © 2013 Hie semenzel, B own and Duncan.
Equa ion (5) shows ha when he in ensi y I o he STED lase app oaches o su passes he
sa u a ion in ensi y Isa , he esolu ion d imp o es. The highe he powe o he STED lase , he
mo e i es ic s he a ea o luo escence, hus imp o ing he esolu ion o he mic oscope.
Howe e , he e is a p ac ical limi o how much he in ensi y I can be inc eased, as ex emely
high in ensi ies can lead o pho obleaching and pho odamage o he sample.
29 | P h D h e s i s - M a e o M a i a n g e l i
3.3 Fluo escence co ela ion spec oscopy
Fluo escence co ela ion spec oscopy (FCS) is echnique i s ly in oduced in he 70’s ([84]) ha
p o ides a way o s udy he dynamics and in e ac ions o molecules in solu ion a a single-
molecule le el. I ope a es by moni o ing luc ua ions in he in ensi y o ligh emi ed by
biomolecules as hey mo e in and ou o a ocused beam o ligh [85]. Typically, FCS is in eg a ed
in con ocal se ups, he e o e he olume in which he molecules di use is ep esen ed by he
con ocal olume, usually < 1 l. This olume is he minimum achie able olume gi en a ce ain
pinhole size, a wa eleng h λ and a nume ical ape u e, hence i is de e mined by he PSF o he
con ocal mic oscope. Howe e , among he many applica ions o FCS, he mos es ablished one is
he “single poin ” FCS, whe e he con ocal olume is no scanning o e he sample, bu i ’s in a
ixed posi ion.
Figu e 3.8: a) ep esen he ypical con ocal sys em on which FCS is implemen ed. The molecule mo e eely on he
sample, and hey a e de ec ed when hey di use on he con ocal olume. b) example o he in ensi y luc ua ions
δF( ) used o gene a e he au oco ela ion unc ion. c) ypical au oco ela ion cu e calcula ed om he luc ua ions.
1/N (ACF ampli ude) and τD (ACF wid h) a e depic ed. F om [85]. Copy igh © 2021 Yu, Lei, Ma, Liu, Zheng, Dan and
Gao.
These luc ua ions in luo escence in ensi y, while appea ing andom, a e ac ually spon aneous
a ia ions a ound a mean alue. They a e indica i e o he numbe o luo escen pa icles
unde going mo ion wi hin he con ocal olume. The analysis o hese luc ua ions is done h ough
an au oco ela ion unc ion, which co ela es he luo escence in ensi y a a gi en ime wi h he
one a a la e ime +τ .
This unc ion e eals se e al i al pa ame e s: he a e age numbe o luo escen pa icles N in
he ocal olume, he di usion ime τD and he di usion coe icien D, a pa ame e which
desc ibes how a a pa icle can a el ia di usion.
a
b
c

30 | P h D h e s i s - M a e o M a i a n g e l i
I we de ine a luo escence luc ua ion as:
𝛿𝐹(𝑡)=𝐹(𝑡)−〈𝐹(𝑡)〉
(6)
Whe e 〈𝐹(𝑡)〉 is he a e age in ensi y a ime , hen we can de ine he no malized
au oco ela ion unc ion (ACF) as:
𝐺(𝜏)=〈𝛿𝐹(𝑡)𝛿𝐹(𝑡+𝜏)〉
〈𝐹(𝑡)〉2
(7)
The ampli ude (Figu e 3.8) o he co ela ion cu e G(0), is in e sely p opo ional o he a e age
numbe o luo escen molecules N wi hin he obse ed olume:
𝐺(0)=1
〈𝑁〉
(8)
The ACF can also be w i en as:
𝑔(𝜏)=〈𝐹(𝑡)𝐹(𝑡+𝜏)〉
〈𝐹(𝑡)〉2=𝐺(𝜏)+1
(9)
Such heo e ical model has o be aligned wi h he expe imen al au oco ela ion unc ion ha
yields he key pa ame e s we wan o ex ac . I we model he di ac ion-limi ed obse a ion
olume as a 3D Gaussian p o ile ep esen ed by ou PSF, we can desc ibe he 𝑔(𝜏) by using a 3D
di usion model. This model o a single di using species yields:
𝑔(𝜏)=1
𝑁∙(1+ 𝜏
𝜏𝐷)−1∙(1+ 𝜏
𝛾2𝜏𝐷)−12
⁄
(10)
𝛾 akes accoun o he size o he con ocal olume. In o he wo ds:
𝛾= 𝑧
𝑤𝑥𝑦
(11)
Whe e 𝑧 equals o hal o he axial PSF and 𝑤𝑥𝑦 o he la e al PSF (Figu e 3.9).
31 | P h D h e s i s - M a e o M a i a n g e l i
F om Equa ion (10) we can e ie e τD ( he di usion ime) ha co esponds o he poin o
in lec ion o he co ela ion cu e (Figu e 3.8). In pa icula , τD is linked o he di usion ime D by
he ollowing equa ion:
𝐷=𝑤𝑥𝑦
2
4𝜏𝐷
(12)
Figu e 3.9: schema ic ep esen a ion o he exci a ion ocal olume, i.e. he 3D PSF o he sys em.
32 | P h D h e s i s - M a e o M a i a n g e l i
A omic Fo ce Mic oscopy
In he ea ly 80’s wo scien is s, G. Binnig and H. Roh e , wo ked on a e y sophis ica ed
mic oscope, called Scanning Tunneling Mic oscope (STM) [86] o which hey we e awa ded
wi h he Nobel P ize in Physics in 1986 [87].
In he e y same yea G. Binnig ( oge he wi h C.F. Qua e and C. Ge be ) published a pape
desc ibing a new mic oscope, which was closely ela ed o he STM, called he A omic Fo ce
Mic oscope [88]. Bo h mic oscopes belong o he class o Scanning P obe Mic oscopes (SPMs),
ins umen s used o ob ain opog aphical images and in o ma ion abou a su ace, a he
a omic le el. They ope a e by means o he physical scanning o a p obe o e he sample.
Figu e 4.1: schema ical ep esen a ion o he main componen s o an AFM mic oscope.
Speci ically, AFM de ec s he o ces be ween a e y sha p nanome ic ip, and he a oms o he
sample. The ip is a ached o a e lec i e iny can ile e ( ypically ~102μm long and ~101 μm
wide) ha wo ks as a senso o he in e ac ions and bends whene e a o ce is de ec ed. The
bending o he can ile e is quan i ied by means o a lase ha hi s he can ile e su ace and,
as a esul , is e lec ed on a ou -quad an pho odiode. The e ical o ho izon al de lec ion o
he lase is p opo ional o he bending o he can ile e which, in u n, is sensi i e o he o ces
be ween he ip and he sample. The e ical mo emen o he p obe is con olled by a closed
eedback-loop, which keeps he p obe-sample in e ac ion a a cons an o ce. In his way he
opog aphy o he sample can be eco ded.
In he ideal case, he ip-sample in e ac ion occu s be ween a single a om om he ip
ex emi y and a single a om on he sample.
33 | P h D h e s i s - M a e o M a i a n g e l i
The model ha desc ibes he in e ac ion be ween wo a oms a a ce ain dis ance is gi en by
he Lenna d-Jones po en ial:
𝑉(𝑟)=4𝜀[(𝜎
𝑟)12 −(𝜎
𝑟)6]
(13)
Figu e 4.2: ep esen a ion o he Lenna d-Jones po en ial. ε ep esen s he deepness o he po en ial well, while σ is
he in e sec ion o V( ) wi h he x axis.
The i s e m is posi i e and goes wi h -12 . I quan i ies he epulsion a sho dis ance, gi en
by he Pauli exclusion p inciple. The second e m ha goes wi h -6 is nega i e and desc ibes
he a ac i e o ces be ween he a oms ha domina e a long dis ances. The con ibu ion o
ha e m is gi en by he Van de Waals o ces. The minimum o he po en ial is an equilib ium
dis ance be ween he a oms and is ep esen ed by 0.
In p ac ical e ms, he in e ac ion be ween he ip and he sample is mo e complex han ha
desc ibed by his model, and i is also de e mined by he en i onemen in which he AFM is
ope a ing. Indeed, apa om i s g ea spa ial esolu ion, especially on he z-axis whe e i can
dis inguish sub-nanome ic ea u es [89], one o he mos impo an ea u es o he AFM is he
possibili y o be implemen ed in physiological condi ions.
In ac , AFM can ope a e ei he in ai o in liquid en i onmen s, whe e he la e condi ion is
necessa y o s udy biological samples unde na i e condi ions.
To ope a e in wo di e en en i onmen s, i ’s necessa y o ca e ully pick he igh can ile e s.
In ac , he o ce which he p obe exe s on he sample is dependen on he physical
cha ac e is ics o he can ile e , ha de e mine i s endency o bend.
40 | P h D h e s i s - M a e o M a i a n g e l i
subsequen ly placed on o a glass co e slip and obse ed using a con ocal mic oscope. Due o he
dis inc emission spec a o Hyp and Sy o13, i was possible o concu en ly isualize bo h wi hin
he same ield o iew. In bo h images, he emissions om Sy o13 (Figu e 1.1d− ) and Hyp (Figu e
1.1e) appea as b igh spo s o a size limi ed by di ac ion, and a signi ican numbe o hese
spo s o e lap in he wo de ec ion channels (Figu e 1.1 ). This o e lap p o ides e idence ha
Hyp p edominan ly a aches o in ac SARS-CoV-2 pa icles ha con ain RNA. The small
p opo ion o luo escen spo s ha do no o e lap may be due o he incomple e s aining o
i al RNA wi h Sy o13. Howe e , he possibili y o some pa icles being deb is o sligh ly damaged
canno be comple ely discoun ed.
Figu e 1.2: luo escence aniso opy on a solu ion o 50 nM Hyp and
∼
1 nM SARS-CoV-2 pa icles. The obse ed da a
indica e ha when Hype icin is mixed wi h he i al pa icles, i becomes solubilized and exhibi s i s dis inc i e
luo escence emission. The p esence o non-ze o aniso opy sugges s ha he o a ional mo emen o Hyp molecules
is es ic ed, which is likely a esul o hei in e ac ion wi h he la ge i al pa icles.

41 | P h D h e s i s - M a e o M a i a n g e l i
1.2 Hype icin A ini y o he i al en elope
To u he explo e how Hyp in e ac s wi h SARS-CoV-2, we used FCS o analyze SARS-CoV-2
pa icles a e exposu e o a ying concen a ions o Hyp, om 1 o 100 nM. Wi h FCS i ’s possible
o assess he le els o luo escen molecules in a solu ion and de e mines hei 3D di usion
p ope ies. As unbound Hyp lacks luo escence, he de ec ed di using species co espond o i al
pa icles loaded wi h Hyp. A ep esen a i e FCS co ela ion cu e is depic ed in Figu e 1.3a.
Fi ing he FCS cu es con i med he p esence o slow-di using pa icles (a e age di usion
coe icien D = (2.4 ± 1.2) μm²/s), wi h a hyd odynamic diame e o app oxima ely 150 nm,
aligning wi h he expec ed size o SARS-CoV-2. The concen a ion o Hyp-loaded i al pa icles,
de e mined om he ampli ude o co ela ion cu es, was oughly 1 nM, displaying some
a iabili y a ibu ed o he p esence o a e, challenging- o- emo e species wi h slowe
di usion, such as agg ega es o esidual deb is.
Figu e 1.3: a) his igu e shows a ep esen a i e FCS co ela ion cu e (black) ob ained om a solu ion con aining
SARS-CoV-2 pa icles ea ed wi h 5 nM Hyp. The i ing o his da a, depic ed in ed, was done using a model o a
single di using species. b) This pa illus a es he in eg a ed luo escence emission o Hyp a p og essi ely inc easing
concen a ions, while main aining a cons an amoun o SARS-CoV-2 pa icles (black ci cles). The i ing o hese da a
poin s, shown in ed, was conduc ed using a binding model. c) Displayed he e a e he no malized ime- esol ed
luo escence decays o Hyp when i is bound o DLPC liposomes (black) and o SARS-CoV-2 pa icles ( ed). The g een
lines indica e he i ing esul s, which we e done using a biexponen ial model o bo h se s o decay da a.
Subsequen ly, he same solu ions we e subjec ed o a luo ome e o measu e ensemble
luo escence emission. Figu e 1.3b illus a es he o al emission (black ci cles) ob ained by
in eg a ing he en i e emission spec um a inc easing concen a ions o Hyp (0−90 nM) and
cons an SARS-CoV-2 pa icle concen a ion (∼1 nM, as de e mined by FCS). The obse ed
luo escence sha ply ises be ween 0 and 20 nM o Hyp, appea ing o sa u a e a abou 30-40
nM. Beyond his concen a ion, he SARS-CoV-2 pa icles each hei capaci y o binding
addi ional Hyp, leading o he o ma ion o non- luo escen agg ega es. Using he concen a ion
o SARS-CoV-2 pa icles de e mined om FCS, we es ima ed he sa u a ion mola a io o be
a ound 30:1 (Hyp o SARS-CoV-2). Addi ionally, by i ing he expe imen al da a o a p e-
es ablished equa ion, we quan i ied he binding a ini y o Hyp o SARS-CoV-2 pa icles,
42 | P h D h e s i s - M a e o M a i a n g e l i
de e mining an appa en equilib ium dissocia ion cons an (KD) o 8.5 nM a his speci ic i us
concen a ion.
Figu e 1.3c displays he no malized luo escence decay p o iles o 5 nM Hype icin (Hyp) bound
o app oxima ely 1 nM SARS-CoV-2 pa icles (shown in ed), and o 10 nM Hyp bound o
app oxima ely 2 nM DLPC liposomes (depic ed in black), which ac as a compa a i e model o
he memb ane con ex . These measu emen s a e made a concen a ions signi ican ly lowe
han sa u a ion le els, whe e indi idual Hyp molecules bound a e su icien ly spaced apa ,
making hei in e ac ions wi h o he Hyp molecules on he same pa icle negligible. Visually, he
decay cu es in Figu e 1.3c a e s ikingly simila , wi h no able di e ences p ima ily in he ini ial
phase o he decay. Bo h se s o da a we e mos accu a ely modeled using a biexponen ial
unc ion, esul ing in wo dis inc li e imes (τ1 and τ2) o Hyp bound o ei he SARS-CoV-2
pa icles o liposomes. While he longe li e imes (τ2) a e consis en ac oss bo h, he sho e
li e imes (τ1) show a ia ion. The longe li e ime is indica i e o a la ge popula ion o Hyp
molecules si ua ed in a mic oen i onmen less pola han wa e , common o bo h liposomes and
i uses, likely wi hin he phospholipid bilaye . In con as , he sho e li e ime componen s may
e lec molecules ha a e mo e exposed o he aqueous su oundings, possibly nea memb ane
p o eins.
In ac , Hyp migh bind o spike p o eins, pa icula ly o he exposed ecep o binding domain
(RBD) o he spike p o ein, on he su ace o SARS-CoV-2 pa icles. We u ilized a ecombinan
soluble e sion o RBD o in es iga e Hyp's po en ial speci ic binding. FCS measu emen s on Hyp
exposed o RBD e ealed no binding o monome ic RBD in he nanomola concen a ion ange.
Howe e , high-a ini y binding (KD ∼ 60 nM) was obse ed o la ge RBD agg ega es a low
concen a ions, which do no co espond o he biologically ele an monome ic s a e o hese
p o eins.
While he p ima y binding a ge o Hyp appea s o be he i al en elope, he possibili y o lowe -
a ini y in e ac ions wi h o he i al p o eins canno be comple ely uled ou , al hough hey a e
less likely a he employed concen a ions.
Hyp in:
τ1(ns)
τ2(ns)
SARS-CoV-2 pa icles
1.2±0.1
6.3±0.3
DLPC liposomes
2.3±0.5
6.8±0.4
43 | P h D h e s i s - M a e o M a i a n g e l i
1.3 Hype icin Dis ibu ion on SARS-CoV-2 Pa icles
Fluo escence emission in his s udy o igina es solely om Hyp bound o SARS-CoV-2 i uses,
meaning he emission in ensi y om a single i us pa icle is app oxima ely p opo ional o he
numbe o Hyp molecules i con ains. This allows o he disce nmen o a ia ions in Hyp con en
on indi idual SARS-CoV-2 pa icles using luo escence imaging. To acili a e his, i al pa icles
(a ound 1 nM) we e i s incuba ed wi h a ious Hyp concen a ions (5−90 nM) and hen placed
on a glass co e slip wi hou washing. A consis en amoun o PBS bu e was added be o e
imaging o ensu e op imal isualiza ion, comple e glass su ace co e age, and main enance o
he Hyp: i us a io. Figu e 1.4a shows a ypical image om his p ocedu e.
The analysis o hese images in ol ed quan i ying he emission in ensi y om single i al
pa icles. Ini ially, a h eshold was se o iden i y b igh spo s, co esponding o pa icles wi h an
in ensi y abo e he backg ound le el, which included de ec o noise and esidual Hyp s icking o
he glass. The in ensi ies o he iden i ied pa icles we e hen ca ego ized o cons uc he
p obabili y dis ibu ions shown in Figu e 1.4b.
These dis ibu ions p ima ily ea u e i al pa icles wi h in ensi ies be ween 430 and 500
a bi a y uni s (au), along wi h a ail o highe in ensi y alues. Pa icles wi h in ensi ies below
a ound 400 au we e indis inguishable om he backg ound (see Figu e S8), and hose jus abo e
his h eshold (400−430 au) showed mo e a iabili y ac oss di e en images. The dis ibu ions in
Figu e 1.4b lack a dis inc shape, indica ing a he e ogeneous dis ibu ion o Hyp ac oss indi idual
i al pa icles. This a ia ion likely esul s om he andom na u e o he binding p ocess and
po en ial di e ences in memb ane su ace accessibili y among indi idual pa icles.
While he me hod o sampling indi idual i al pa icles is di ec , i aces sensi i i y challenges a
e y low Hyp concen a ions (<10 nM), whe e emission om a single pa icle is due o only a ew
molecules, and some pa icles may emain unlabeled and in isible o luo escence imaging. Mo e
in ica e echniques, such as co ela i e ligh and elec on mic oscopy, would be equi ed o a
ho ough sampling ha includes unlabeled o ain ly emi ing pa icles. None heless, a end is
obse able by examining a e age single-pa icle in ensi y alues de i ed om he dis ibu ions
(Figu e 1.4c). These a e ages show a quali a i e inc ease in in ensi y wi h ising Hyp
concen a ion. Despi e conside able a iabili y, i ing hese da a wi h he same model as in
Figu e 1.3b yields an appa en KD o 7 nM (Figu e 1.4c, in ed), aligning wi h he bulk
measu emen esul s. Impo an ly, his kind o da a on d ug loading a he single- i al-pa icle
le el is una ainable wi h ensemble measu emen me hods.
44 | P h D h e s i s - M a e o M a i a n g e l i
Figu e 1.4: a) his is a ep esen a i e image showing SARS-CoV-2 pa icles a e exposu e o Hyp o single-pa icle
in ensi y analysis. The scale ba in he image is 5 μm. b) The image displays p obabili y dis ibu ions o single i al
pa icle luo escence in ensi ies o SARS-CoV-2 pa icles exposed o a ious concen a ions o Hyp: 5 nM (g ay), 10
nM ( ed), 20 nM (g een), 30 nM (blue), 40 nM (cyan), 50 nM (magen a), 70 nM (yellow), and 90 nM Hyp (o ange).
'N' indica es he o al numbe o i al pa icles analyzed in each dis ibu ion. The wid h o each dis ibu ion bin is se
a 10 a.u. c) The g aph illus a es he a e age luo escence in ensi y alues o single i al pa icles a inc easing
concen a ions o Hyp. The ed line in he g aph ep esen s he ou come o he i ing p ocess using a binding model.
The Hyp concen a ions men ioned co espond o hose used du ing incuba ion wi h he SARS-CoV-2 pa icles.
1.4 E ec i eness o Hype icin Agains SARS-CoV-2
To assess he e ec i eness o Hyp agains SARS-CoV-2, i al in ec i i y assays we e conduc ed. In
hese expe imen s, ac i e SARS-CoV-2 pa icles unde wen ini ial incuba ion wi h Hyp a
concen a ions o 3, 30, and 300 nM. Subsequen ly, samples we e subjec ed o ei he da k
condi ions o i adia ed wi h blue ligh o pho oexci e Hyp, enabling he di e en ia ion o bo h
da k and pho oinduced e ec s. Following ea men , i al samples we e se ially dilu ed and
incuba ed wi h a monolaye o Ve o E6 cells unde da k condi ions o de e mine esidual
in ec i i y. The 50% issue cul u e in ec i e dose pe mL (TCID50/mL), ep esen ing he i al i e
a he dilu ion whe e cell iabili y is educed by 50%, was hen measu ed.
Figu e 1.5 p o ides a summa y o he measu ed TCID50/mL alues o i uses incuba ed wi h Hyp
unde da k condi ions ( ed ba s) o pho oexci ed condi ions (blue ba s). Re e ence alues
(TCID50/mL ∼ 105, g ay ba s) we e ob ained o ully in ec i e i al samples ha we e no ea ed
wi h Hyp bu we e di ec ly incuba ed wi h Ve o E6 cells a e se ial dilu ion.
Unde he employed condi ions, a ema kable educ ion in in ec i i y (TCID50/mL om 105.5 o
<101) was obse ed o SARS-CoV-2 exposed o bo h 300 and 30 nM Hyp and pho o-i adia ed.
A clea an i i al e ec was e iden e en a 3 nM Hyp, upon ligh exposu e, causing a 2.2-log
educ ion in i al i e (TCID50/mL om 105.2 o 103) compa ed o un ea ed i uses. These esul s
45 | P h D h e s i s - M a e o M a i a n g e l i
a i m ha e en low concen a ions o pho oexci ed Hyp a e e ec i e agains SARS-CoV-2,
consis en wi h he obse ed high a ini y o Hyp o i al pa icles.
This e icacy o Hyp compa es a o ably wi h ecen ly epo ed pho oinac i a ion s udies on
SARS-CoV-2 using me hylene blue and adachlo in as pho osensi izing molecules.
An i i al ac i i y was also obse ed unde da k condi ions, albei o a lesse ex en han in
pho oexci ed samples. In he da k, in ec i i y educ ion was negligible o i uses exposed o 3
nM Hyp (TCID50/mL 105.5) and mode a e o i uses exposed o 30 nM Hyp (TCID50/mL om
105.5 o 104.6). Signi ican ly, a 300 nM Hyp, a 3-log educ ion in TCID50/mL alue ( om 105.5 o
102.5) was induced compa ed o un ea ed i uses. Impo an ly, no oxici y was obse ed in
con ol expe imen s conduc ed on Ve o E6 cells exposed o Hyp o DMSO, in he absence o
i uses and in he da k, con i ming ha cells a e no di ec ly damaged by Hyp o he small amoun
o DMSO added o deli e Hyp. To e alua e Hyp’s e ec i eness agains SARS-CoV-2, i al
in ec i i y assays we e pe o med. In hese es s, ac i e SARS-CoV-2 pa icles we e i s incuba ed
wi h Hyp a 3, 30, and 300 nM concen a ions. The samples we e hen ei he kep in da k
condi ions o exposed o blue ligh o ac i a e Hyp, allowing o he obse a ion o e ec s bo h
in he absence and p esence o ligh . Pos - ea men , hese i al samples we e dilu ed se ially
and added o a monolaye o Ve o E6 cells, main ained in da k condi ions, o assess emaining
in ec i i y. The i al concen a ion causing a 50% educ ion in cell iabili y (TCID50/mL) was
measu ed.
Figu e 1.5 p esen s he TCID50/mL alues o i uses ea ed wi h Hyp in he da k (shown wi h
ed ba s) and unde ligh ac i a ion (blue ba s). Fo compa ison, e e ence alues (TCID50/mL ~
105, g ay ba s) we e ob ained om ully in ec i e i al samples ha hadn' been ea ed wi h Hyp
bu we e di ec ly incuba ed wi h Ve o E6 cells a e dilu ion.
Unde hese expe imen al condi ions, a signi ican dec ease in in ec i i y was no ed o SARS-
CoV-2 exposed o 300 and 30 nM Hyp and hen pho o-i adia ed, wi h TCID50/mL alues
d opping om 105.5 o less han 101. E en a 3 nM Hyp wi h ligh exposu e, a no able an i i al
e ec was seen, e idenced by a 2.2-log educ ion in i al i e (TCID50/mL om 105.2 o 103)
compa ed o un ea ed i uses. These indings con i m he e icacy o pho oac i a ed Hyp agains
SARS-CoV-2, e en a low concen a ions, aligning wi h i s high a ini y o he i us.
The e ec i eness o Hyp in hese condi ions is compa able o o e en su passes o he
pho oinac i a ion s udies on SARS-CoV-2 using di e en pho osensi ize s like me hylene blue
and adachlo in.
An i i al ac i i y was also obse ed wi hou ligh exposu e, hough i was less p onounced han
in pho oexci ed samples. In da kness, he in ec i i y educ ion was minimal o i uses ea ed
wi h 3 nM Hyp (TCID50/mL 105.5) and mode a e o 30 nM Hyp (TCID50/mL om 105.5 o 104.6).
No ably, a subs an ial 3-log educ ion in TCID50/mL ( om 105.5 o 102.5) was seen wi h 300 nM
Hyp compa ed o un ea ed i uses. Impo an ly, con ol expe imen s wi h Ve o E6 cells exposed

46 | P h D h e s i s - M a e o M a i a n g e l i
o Hyp o DMSO in he absence o i uses and ligh con i med no di ec cellula oxici y, ensu ing
ha he obse ed e ec s we e speci ic o he i al in e ac ion wi h Hyp.
Figu e 1.5: he g aph illus a es he Vi al Ti e , measu ed in TCID50/mL, on Ve o E6 cells in ec ed wi h SARS-CoV-2
i uses ha we e p e iously exposed o ising concen a ions o Hyp. The samples we e ei he kep in he da k
(depic ed in ed) o i adia ed wi h 20 J/cm² o blue ligh (depic ed in blue). Re e ence TCID50/mL alues ob ained
om Ve o E6 cells in ec ed wi h SARS-CoV-2 bu no exposed o Hyp a e ep esen ed in g ay.
1.5 Conclusions
These esul s highligh he dual-ac ion na u e o Hyp as an an i i al agen , esol ing he
inconsis encies seen in p e ious epo s. When pho oac i a ed while bound o he i us, Hyp
likely induces subs an ial damage o he i al pa icles h ough he gene a ion o eac i e oxygen
species, a phenomenon no ed in simila s udies. Since a single Hyp molecule can abso b mul iple
pho ons du ing ea men , his p ocess equi es only a limi ed numbe o Hyp molecules on each
i al pa icle o be e ec i e. In con as , he absence o ligh sugges s an al e na i e mechanism
o he obse ed educ ion in i al in ec i i y. This da k mechanism seems o equi e a highe
concen a ion o Hyp on he i al pa icles o be signi ican . One hypo hesis is ha Hyp's
in eg a ion in o he i al en elope migh in e e e wi h i s luidi y, subsequen ly inc easing he
ene gy ba ie s needed o he i us o use wi h hos cell memb anes. Howe e , u he esea ch
is necessa y o ully unde s and hese mechanisms and hei po en ial combined e ec s.
47 | P h D h e s i s - M a e o M a i a n g e l i
1.6 Ma e ial and me hods
1.6.1 Fluo escence mic oscopy.
The colocaliza ion measu emen s shown in Figu e 1d– and he single-pa icle in ensi y analysis
illus a ed in Figu e 1.4 we e pe o med using a Nikon A1MP in e ed con ocal mic oscope. This
mic oscope is equipped wi h ou exci a ion lase s a 405, 488, 561, 640 nm wa eleng hs, and i
inco po a es h ee alkaline pho omul iplie ubes (PMTs) o de ec ion. Speci ically, o hese
expe imen s, Hyp was exci ed wi h he 561 nm lase and Sy o13 wi h he 488 nm lase . The 561
nm lase is a con inuous-wa e diode-pumped solid-s a e lase om Melles G io , while he 488
nm lase is a con inuous-wa e diode semiconduc o lase om Cohe en . Fluo escence emissions
we e de ec ed using wo di e en bandpass il e s: a 605/70 nm il e o Hyp and a 515/30 nm
il e o Sy o13.
STED imaging, as p esen ed in Figu es 1b and c, was conduc ed using he Leica Mic osys ems
STELLARIS 8 STED sys em. This mic oscope ea u es a supe con inuum Whi e Ligh Lase (WLL) in
exci a ion, and o hese pa icula measu emen s, an exci a ion wa eleng h o 561 nm was
selec ed using an Acous o-Op ical Tunable Fil e (AOTF). The STED wa eleng h was 775 nm.
Sample p epa a ion. The s ock suspension con aining ixed i al pa icles was ini ially cen i uged
o 3 minu es a 5000 pm o elimina e he la ges agg ega es. The i al pa icles we e hen
dilu ed 50- old in PBS bu e wi h a pH o 7.4.
Fo single-pa icle in ensi y measu emen s ( e e enced in Figu e 1.4), p e-dilu ed i al pa icles
we e combined wi h a ious concen a ions o Hyp ( anging om 5 o 90 nM). 20 μL o each
mix u e was hen placed on a s e ile cell cul u e dish wi h a glass bo om. Following a 10-minu e
incuba ion pe iod, 100 μL o PBS was added di ec ly, wi hou any in e media e washing s eps, o
achie e he desi ed olume o imaging. This olume ensu es uni o m co e age o he glass
su ace wi h he solu ion. The sample was hen sealed o p e en e apo a ion and imaged
immedia ely. This p ocedu e was consis en ly applied o all samples o ensu e compa abili y.
Fo he colocaliza ion measu emen s (as in Figu e 1.1d– ), p e-dilu ed i al pa icles we e i s
mixed wi h Hyp ( o a inal concen a ion o 50 nM), hen added o a s e ile cell cul u e dish and
combined wi h Sy o13 (also o a inal concen a ion o 50 nM). These samples we e imaged
immedia ely wi hou any in e media e washing s eps, using a sequen ial scanning mode o a oid
bleed- h ough du ing he acquisi ion p ocess.
In all samples, he inal concen a ion o DMSO was kep negligible (<2%).
48 | P h D h e s i s - M a e o M a i a n g e l i
1.6.2 Spec oscopy
Abso p ion spec a we e ob ained using a Jasco V-650 spec opho ome e om Jasco Eu ope.
Fo s eady-s a e luo escence exci a ion, emission, and aniso opy measu emen s, we u ilized
he SF5 spec o luo ome e (Edinbu gh Ins umen s L d., Li ings on, U.K.) This ins umen is
i ed wi h bo h exci a ion and emission pola ize s, which acili a e he de ec ion o luo escence
aniso opy.
Sample p epa a ion. We i s cen i uged he s ock suspension o ixed i al pa icles o 3
minu es a 5000 pm o emo e la ge agg ega es. The pa icles we e hen dilu ed 50- old in PBS
bu e wi h a pH o 7.4. A small olume o Hyp concen a ed in DMSO was added o he
suspension, achie ing a concen a ion 50 imes highe han he desi ed inal concen a ion,
esul ing in a inal DMSO concen a ion o 2%. A e a 5-minu e incuba ion pe iod a oom
empe a u e in da k condi ions, 50 μL o his mix u e was ans e ed in o a qua z cu e e wi h
a pa h leng h o 0.3 cm o luo ome ic analysis. Emission spec a we e acqui ed by exci ing he
sample a 553 nm, while o exci a ion and aniso opy spec a, emission was collec ed a 650 nm.
1.6.3 Fluo escence Co ela ion Spec oscopy.
Fluo escence Co ela ion Spec oscopy (FCS) expe imen s we e conduc ed using a Mic o ime 200
sys em om PicoQuan . This sys em in eg a es an in e ed con ocal mic oscope (Olympus IX71)
and is equipped wi h wo Single Pho on A alanche Diodes (SPADs) ope a ing in c oss-co ela ion
mode. Fo he exci a ion o Hype icin (Hyp), we employed a 475 nm picosecond diode lase ,
which was ope a ed a a equency o 20 MHz. The luo escence emission om Hyp was cap u ed
h ough a 675/25 nm bandpass il e and hen equally di ided be ween wo de ec ion channels
using a 50/50 beam spli e . This con igu a ion enabled he simul aneous eco ding o bo h
co ela ion cu es and ime- esol ed luo escence decays. These decays we e measu ed using
he echnique o ime-co ela ed single pho on coun ing (TCSPC), p o iding de ailed insigh s in o
he luo escence cha ac e is ics o Hyp when in e ac ing wi h he a ge .
Sample p epa a ion.
A ound 40 μL o he same solu ion p epa ed o spec oscopic analysis was employed in he FCS
sys em. Each measu emen was conduc ed o e a pe iod o 5 minu es, wi h mul iple epea s (3–
5 imes) o each sample o ensu e eliabili y. The p epa a ion o all samples ollowed he same
me hod, and measu emen s we e ca ied ou unde uni o m condi ions. The use o low sample
concen a ions was c i ical o ensu e ha he measu emen s we e no a ec ed by po en ial
a i ac s a ising om he op ical densi y o he samples a he exci a ion wa eleng h.
The analysis o he collec ed da a was pe o med using SymphoTime so wa e, de eloped by
PicoQuan . The ime- esol ed luo escence decays we e e ec i ely modeled using a
biexponen ial i , p o iding de ailed insigh s in o he luo escence cha ac e is ics o he sample.
49 | P h D h e s i s - M a e o M a i a n g e l i
Simila ly, he co ela ion cu es we e accu a ely i ed wi h a model ha assumes a single ype
o di using species. This app oach was in line wi h he heo e ical amewo k and equa ions used
in he s udy, ensu ing a comp ehensi e and accu a e in e p e a ion o he FCS da a.
1.6.4 Single-Pa icle In ensi y Analysis
The single-pa icle in ensi y da a p esen ed we e ob ained using consis en pa ame e s,
including a 1024 × 1024 image size, a pixel dwell ime o 6.2 μs, a pixel size o 80 nm, and 4 line
a e ages. The acquisi ions we e pe o med wi h a compa able axial posi ion o he ocal plane.
Fo each sample, 8–10 acqui ed images unde wen analysis using ImageJ.
The analysis in ol ed a sequen ial p ocess. Ini ially, a Gaussian il e wi h a s anda d de ia ion
(σ) o 180 nm was applied o diminish unco ela ed backg ound noise and enhance he
disc imina ion o luo escen pa icles. Ins ances whe e ce ain po ions o he images we e
no ably ou o ocus, likely due o i egula i ies on he glass su ace, we e excluded om he
analysis.
Subsequen ly, a pa icle analyze ool was employed, ecognizing pa icles wi h a ci cula i y in
he ange o 0.9-1, a minimum in ensi y abo e a h eshold alue ( anging om 400 o 420
a bi a y uni s), and a minimum a ea (8–10 pixels). The esul s o his analysis we e subjec ed o
isual inspec ion, and pa icles (<1%) displaying un ealis ic ea u es, such as hose indica i e o
agg ega es, we e excluded.
Finally, he mean in ensi y alues o each selec ed pa icle we e s o ed and u ilized o he
econs uc ion o dis ibu ions.
1.6.5 Vi al In ec i i y Assays and Vi us Fixa ion
The isola ion o he SARS-CoV-2 i us and subsequen expe imen s o assess he an i i al e icacy
o Hyp agains SARS-CoV-2-in ec ed cells we e conduc ed wi hin biosa e y le el 3 (BSL3)
labo a o ies a he i ology depa men in Is i u o Zoop o ila ico Spe imen ale o Emilia
Romagna and Lomba dia. The i us isola ion p ocess in ol ed he inocula ion o an in ec ed
biological human sample in o Ve o E6 cell lines. The SARS-CoV-2 HCoV-19/I aly/310902/46/2020
s ain (GISAID code: EPI_ISL_9011947), belonging o clade 20A (Nex s ain naming), was
p opaga ed in he same cell line and incuba ed a 37 °C wi h 5% CO2. The i al i e (TCID50/mL)
was e i ied using he Reed-Muench assay. To in es iga e he an i i al e icacy o Hyp, di e en
Hyp concen a ions we e p epa ed by dilu ing Hyp in DMSO.
Th ee expe imen s we e pe o med a concen a ions o 300, 30, and 3 nM o Hyp. Fo each Hyp
concen a ion es ed, i e samples we e p epa ed: SARS-CoV-2 + Hyp ligh , SARS-CoV-2 + Hyp
da k, SARS-CoV-2, Hyp, and DMSO.
In he expe imen al se up, 900 μL o i al suspension was mixed wi h 100 μL o Hyp o achie e
he desi ed Hyp concen a ion. One aliquo o his mix u e unde wen lamp illumina ion o 15
minu es wi h an in ensi y o 22 mW/cm2, co esponding o a luence o 20 J/cm2 (SARS-CoV-2 +
56 | P h D h e s i s - M a e o M a i a n g e l i
2.2.1 AFM imaging wi h Hyp+ligh
We s a ed by analyzing he pho odynamic e ec s o Hyp on he bilaye a di e en
concen a ions, by using a me cu y a c lamp o exci e he molecule and p oduce ROS. We we en’
absolu ely su e abou wha o expec wi h espec o he mo phology o he bilaye , none heless,
due o he dis up i e e ec s o single oxygen, i is likely ha Hype icin can cause i egula i ies
on he bilaye , possibly nanoscopic holes [96] Be o e p oceeding on he ac ual expe imen we
pe o med one con ol expe imen o assess ha ligh i sel is no a ec ing somehow he
mo phology o he memb ane.
Figu e 2.3: bilaye in da k condi ion (le ) and wi h ligh i adia ion, using a me cu y a c lamp ( igh ). No Hyp was
added in ei he measu emen .
The con ol measu emen is shown in Figu e 2.3. We can clea ly see ha no app eciable e ec is
caused by he p esence o he sole ligh . In ac , he wo images a e almos iden ical excep o
some disca dable di e ences ha a e mos likely due iny mo emen s o he bilaye .
In gene al, e e y ime we we e making AFM acquisi ion on he e y same a ea o ou bilaye s
and in he same condi ions, some iny di e ences showed up o e ime. They could in ol e he
appea ance (o disappea ance) o iny holes, as well as hei change in shape, coalescence o lipid
a s and a ia ion on hei bo de s. All o hese (sligh ) di e ences a e p opo ional o he ime
o acquisi ion equi ed o a s anda d AFM image (0.7-1 Hz, no mally 256x256 px).
The lipids a e ee o mo e, ei he on he lipid a s o in he mo e luid phase; also, he bilaye is
lying on a e y iny laye o wa e which can a o an o e all d i ing o he s uc u e.
The image in ligh was made a e 5 min o con inuous i adia ion, he e o e he e was some ime
o he lipids o mo e and o he bilaye o slowly ea ange.

57 | P h D h e s i s - M a e o M a i a n g e l i
Figu e 2.4: images o he bilaye a he same ROI. The images we e acqui ed while i adia ing a 0 nM (a), 25 nM (b),
50 nM (c) and 100 nM (d).
The image in Figu e 2.4 ep esen he bilaye a di e en concen a ions o Hyp. We can see ha
he bilaye wi hou Hyp al eady exhibi s he h ee phases, as well as some de ec s. By adding Hyp
he e is an ex ensi e e ec on he imaged a ea, pa icula ly on he amoun o lipids in he Lo
phase. Also, he numbe and he size o he de ec s a e inc easing, in line wi h he pho odynamic
e ec induced by he molecule.
Simila ly, he lipid a s in he gel phase a e also ge ing bigge , possibly indica ing ha a
edis ibu ion o he local concen a ion o choles e ol could be igge ed by Hyp.
58 | P h D h e s i s - M a e o M a i a n g e l i
Mo eo e , in many Lβ lipid a s we ound a beha io like he one shown in Figu e 2.5, ha is
many iny holes appea ing as soon as he concen a ion o Hyp inc eased.
Figu e 2.5: he e ec on he gel phase a 100 nM concen a ion is isualized he e. The inse images b) and c) clea ly
show he nanoscopic holes on he su ace o he Lβ a s. We can app ecia e ha hey possess a peculia shape,
almos ound. Those i egula i y a eas in his phase we e ne e de ec ed in measu emen wi hou Hyp+ligh .
2.2.2 AFM imaging wi h Hyp in he da k
Because we we e aiming o s udy i Hyp a ec s he memb ane e en in absence o ligh , we
pe o med some se o expe imen s wi h he e y same condi ions as hose employed in he
measu emen s shown in Figu e 2.4 and Figu e 2.5; he only di e ence was ha we didn’ employ
any ligh i adia ion.
We a e no able o app ecia e any ele an di e ence in he mo phology and he gene al
a angemen o he bilaye as shown in Figu e 2.6: we can’ see ei he any holes appea ing in he
image, o edis ibu ion o he bilaye phases. In ac , we can only app ecia e wo phases
in ol ed, Ld and Lo in all he 4 images wi h subs an ially he same occupancy a io on he a ea o
he image.
The e a e some di e ences in he shape o he lipid a s in he 4 images, bu hey a e mos
likely due o he egula di usion o lipids and o he d i o he bilaye .
a
b
c
59 | P h D h e s i s - M a e o M a i a n g e l i
Figu e 2.6: images o he bilaye a he same ROI. The images we e acqui ed wi hou ligh i adia ion a 0 nM (a), 25
nM (b), 50 nM (c) and 100 nM (d).
A i s glance, his esul could sugges ha he mo phological e ec s induced by he PS in he
da k a e negligible. Howe e , i is a ional o expec ha imaging echniques may no be sensi i e
enough o de ec sub le di e ences be ween ligh and da k condi ions.
In ac , i an e ec in he da k is p oduced, i is mos p obably linked o he mechanical p ope ies
o he memb ane, as i should be o i al pa icles.
One o AFM ad an ages among o he s, is ha i o e s he possibili y o ob aining spec oscopic
in o ma ion; by measu ing he o ces as he ip app oaches and e ac s om he su ace we can
ha e access o he nano-mechanical p ope ies o a sample, such as s i ness, adhesion, and
elas ici y.
60 | P h D h e s i s - M a e o M a i a n g e l i
2.3 AFM mechanical p ope ies
2.3.1 Measu emen s o he Young Modulus
Since we we e mainly in e es ed in he a ia ion o s i ness ha could be induced by Hyp, we
ob ained alues o he Young Modulus (E), also called he elas ic modulus, o he model
memb ane. E can be ega ded as a measu e o he s i ness o a ma e ial: a high Young modulus
means high s i ness, i.e. low de o ma ion unde load. Con e sely, a ma e ial wi h a low Young
modulus is mo e lexible o elas ic. Ope a i ely, i is de ined as he a io o s ess ( o ce pe uni
o a ea) o s ain (change o leng h pe uni o leng h) in a ma e ial unde ension o comp ession:
𝐸=𝜎
𝜀
(15)
Whe e σ is he s ess, and ε is s ain [97], he Young Modulus is dimensionally exp essed in uni s
o p essu e, speci ically Pascal (Pa) o (N m2
⁄).
To ex ac he alues o E om AFM we pe o med nanoinden a ion measu emen s om which
we ob ained Fo ce-Dis ance (F-D) cu es. Nanoinden a ion means ha a e y small and poin ed
inden e (ou ip) is p essed in o he su ace o a ma e ial o measu e i s mechanical p ope ies.
We chose o i ou cu es using he He z-Sneddon model [98] [99][100].
This is a e y common model o his kind o applica ions [101], and is based on a ce ain se o
assump ion and app oxima ions. These include [102]:
1. The de o ma ion occu s wi hin he elas ic egime, meaning ha he ma e ial will e u n
o i s o iginal shape a e he emo al o he o ce.
2. The magni ude o he de o ma ion is small compa ed o he adius o he con ac a ea
whe e he o ce is applied.
3. The applied o ce is pe pendicula o he su ace, indica ing he absence o any shea
o ces in he in e ac ion.
4. The sample is conside ed o be semi-in ini e, implying ha i s dep h is signi ican ly la ge
han he ex en o he de o ma ion o con ac a ea.
The p essu e applied by he ip is dependen on i s shape, he e o e i s cha ac e is ics is included
in he model. In ou case he ip can be conside ed a ci cula pa aboloid, which is unambiguously
dependen on only one geome ical pa ame e , which is i s adius, R. The o mula o he o ce
can be exp essed as ollow:
𝐹=4
3𝐸√𝛿3𝑅
(1−𝜈2)
(16)
61 | P h D h e s i s - M a e o M a i a n g e l i
Whe e E is he Young Modulus, R is he adius o he ip, δ is he inden a ion and 𝜈 is he Poisson
a io. This alue is de ined as he a io be ween ans e se s ain and longi udinal s ain. Typically
o sligh ly comp essible ma e ials like liquids and ubbe s, whe e s ess p edominan ly leads o
changes in shape, he Poisson's a io app oaches 0.5 [103][97].
The alue o he Poisson a io ha we will hen conside om now on is 0.5, he e o e we can
ew i e Equa ion (17) as:
𝐹=16
9𝐸√𝛿3𝑅
(17)
The assump ions in oduced in he He z-Sneddon model migh esul in no able inaccu acies in
he calcula ion o Young modulus. Anyway, ou in e es doesn’ lie in inding he absolu e alue
o he Young moduli, bu a he on he dis inc ion be ween he case wi h and wi hou Hyp.
No ably, employing solid suppo s has many ad an ages, o example hey p ima ily con ibu e
o he inc eased obus ness and longe i y o he phospholipid bilaye memb ane. Howe e , a
signi ican limi a ion is ha he suppo ed memb ane does no exis in comple e sepa a ion om
he subs a e benea h i [6].
The e o e, among all o he assump ions, he numbe 4 is he one ha could a ec ad e sely
e en compa a i e measu emen s. In ac , he lipid phases in ol ed possess di e en heigh s,
making i necessa y o in oduce a co ec ion ha accoun s o he hickness o he sample, hus
o i s icini y o he solid subs a e.
The solu ion o exclude he also called “bo om e ec a i ac ” [104] has been ex ensi ely
discussed [105] [106] and i was ecen ly p oposed a model ha comple ely co ec s i [107]. Fo
he same ip geome y, i yields he ollowing o ce–inden a ion exp ession:
𝐹𝐺=𝐹0 (1
ℎ0+1.133√𝛿𝑅
ℎ1+1.497𝛿𝑅
ℎ2+1.469𝛿𝑅√𝛿𝑅
ℎ3+0.755(𝛿𝑅)2
ℎ4)
(18)
Whe e h is he heigh o he sample and 𝐹0 is he o ce exp essed in he Equa ion (17).
By using his o mula o each o ce cu e, we we e able o econs uc he co ec ed alue o
he Young moduli.

62 | P h D h e s i s - M a e o M a i a n g e l i
In o de o ob ain he mos accu a e possible alue o h, we pe o med some measu emen s
wi h pa ially in ac bilaye . I he de ec s in he bilaye a e no wide enough, he e is no
ce ain y ha he ip is e ec i ely ouching he solid subs a e unde nea h. Wha we did o
ensu e he p esence o su icien ly wide holes, was o gene a e hem by ou sel es, by insing
he bilaye s e y s ongly a e he measu emen s. We acqui ed 5 images wi h plen y o holes
o each ield o iew, o which we had a heigh dis ibu ion. The dis ance be ween he peaks
ga e us he alues o he heigh o he phases.
Figu e 2.7: a) ep esen a i e image o opog aphy o he sample we exploi ed o quan i y he heigh o he
bilaye . The holes a e clea ly isible, and hey we e ob ained by s ongly insing he sample wi h ul apu e
wa e . The image is 512 px x 512 px. b) Heigh dis ibu ion ob ained om one o he images we analyzed.
We can dis inguish h ee peaks whose alues we e ob ained by means o a “peak i ”. They a e be e
isualized on he inse on he op igh , whe e he y-axes is log-scaled. The dis ance be ween he peaks
de ines he heigh o Ld and Lo . In pa icula , he able is showing he mean alues ob ained by means o
he 5 images men ioned abo e.
Heigh (nm)
Ld
4.84 ± 0.06
Lo
6.02 ± 0.06
a
b
63 | P h D h e s i s - M a e o M a i a n g e l i
We pe o med Quan i a i e Imaging (QI) [108] measu emen s in o de o ex ac mechanical
p ope ies om he sample di ec ly om he images. QI allows o eco d F-D cu es a each pixel
o he image, gi ing in o ma ion abou he local in e ac ion be ween he ip and he sample.
The e o e, o all he measu emen s we ob ained 16,384 o ce cu es (128x128 pixels), enough
o ha e a ele an s a is ic abou he mechanical p ope ies o he sample.
Ope a i ely, we i ed he F-D cu e using he model o Equa ion (18) on he e y i s nm a e
he con ac o he ip o he sample; we will deepen he desc ip ion o he analysis on he ma e ial
and me hod sec ion.
In o de o be as accu a e as possible, we acqui ed QI images o a phase pe ime, using small
ields o iew o 500 nm – 1 μm. Fo each sample, we analyzed sepa a ely he phases in ol ed,
especially he Lo and Ld phases. In ac , ypically a eas wi h lipids in he Lβ phase whe e no always
p esen and, i so, hei size we e insu icien o selec a ROI and disc imina e om he adjacen
phases.
Fo each sample we di e en ia e he cases o : No Hyp, Hyp in da k, Hyp wi h ligh .
We used a much highe concen a ion o Hyp wi h espec o hose desc ibed in 2.2 o his kind
o measu emen s. The easons o ha we e dependen on he sys em ha we used o he QI:
he mic oscope was equipped wi h a weake lamp, and he e o e he concen a ion o Hyp was
aised o a easonably high alue, in o de o balance he loss in pho odynamic e ec s induced
by he low ligh powe . Mo eo e , we belie ed ha whe he Hyp was p oducing an e ec on he
igidi y in da k, his should ha e been p ac ically unde ec able in a bilaye a he concen a ions
used in he 2.2 pa ag aph. Ac ually, we showed ha o a single i al pa icle, because o i s
dimension and geome y, only 30-40 molecules o Hyp could be accommoda ed.
I is easonable o expec ha he e ec on he s i ness happens when sa u a ion condi ion is
eached. This means ha a highe concen a ion is needed o a much mo e ex ended sys em
such as a lipid bilaye .
Figu e 2.8: his og ams o he dis ibu ion o he Young moduli co ec ed o he heigh o he bilaye in he h ee
di e en condi ions, i.e. wi hou Hyp ( ed), wi h 1 μM o Hyp in da k (g een), wi h 1 μM Hyp du ing i adia ion (blue).
We sepa a ed he wo phases in wo di e en g aphs, he liquid o de ed phase on he le , he o de ed phase on he
igh .
0 5
0
5000
Coun s
Young modulus (MPa)
Ld phase
Coun s
0 5
0
5000
Coun s
Young modulus (MPa)
Lo phase ( a s)
64 | P h D h e s i s - M a e o M a i a n g e l i
Hyp]
E o Ld phase (MPa)
E o Lo phase (MPa) - a s
0 μM
0.68 ± 0.11
1.13 ± 0.03
1 μM in da k
1.31 ± 0.14
2.49 ± 0.08
1 μM in ligh
1.89 ± 0.16
2.66 ± 0.05
Table 2: he able ou lines he alues o he Young moduli (E) ex ac ed by i ing he dis ibu ions wi h a gaussian
unc ion.
The esul s o he measu emen s a e summa ized in Figu e 2.8 and in Table 2: he able ou lines
he alues o he Young moduli (E) ex ac ed by i ing he dis ibu ions wi h a gaussian unc ion.
Appa en ly, he e is a end indica ing he s i ening ac ion o Hyp on he bilaye , bo h in he da k
and du ing ligh i adia ion. This is in acco dance wi h he p e iously discussed hypo hesis.
Also, he di e ences in he Ld and Lo phases a e signi ican , indica ing ha he Lo phase has a
mo e p onounced s i ness.
The QI measu emen s om which we ob ain hese esul s a e no i ial o be pe o med. E en
hough he numbe o o ce cu es acqui ed should be good enough o ha e a ele an s a is ic,
he measu emen s could be a ec ed by some sys ema ic e o s.
The chosen pa ame e s can be c i ical o a p ope ou come, also, he di iness ha he ip can
collec du ing imaging can a ec he eal s i ness. This issue is also bo h icky o quan i y and o
p e en . In ac , he e is no way o comple ely disca d his issue, because o how he
measu emen s a e ca ied ou : we analyzed wi h he same pa ame e s and he same ip be o e
and a e addi ion o Hyp.
The inc ease o he Young modulus induced by Hyp is conside able, bu we can’ comple ely
disca d he possibili y ha i has also been a ec ed by di iness on he can ile e .
In o de o ha e mo e e idence abou he e ec s o he PS, we decided o employ ano he AFM
me hodology o assess he mechanical p ope ies o he bilaye .
2.3.2 AFM mechanical p ope ies: b eak h ough measu emen s
Besides accessing he in o ma ion abou he elas ic modulus, an essen ial app oach o
comp ehending he s i ness o a bilaye is o s udy i s b eak h ough p ope ies. When acqui ing
o ce cu es he e can be a no iceable jump o he AFM ip h ough he ilm, which occu s when
i su passes a speci ic h eshold o ce, ha we will call b eak h ough o ce (Fb) [109][110].
This alue ep esen s he maximum o ce ha a memb ane can endu e be o e being pene a ed
by he sha p ip; i is a key me ic o assessing he mechanical s abili y o he SLB in ques ion,
and i ’s a pa ame e closely associa ed wi h he a angemen and o ganiza ion o lipids wi hin
he memb ane [93].
Fb can be di ec ly de e mined by he o ce cu es. A up u e e en esul s in a no iceable
discon inui y in he o ce-dis ance g aph, indica ing a local b eakdown o he bilaye and he
65 | P h D h e s i s - M a e o M a i a n g e l i
pene a ion o he ip in o he SLB. Following his e en , he ip p esses agains a solid
subs a e, main aining a s eady dis ance om he sample [111].
Ano he in o ma i e me ic ha can be ex ac ed by he b eak h ough discon inui y in he
o ce cu es is he dis ance he ip a els du ing bilaye pene a ion, which we name he
b eak h ough leng h (Lb). Assuming ha he ip con ac s he unde lying subs a e pos -bilaye
up u e, Lb indica es he hickness o he SLB a i s poin o maximum de o ma ion.
Mo eo e , like o any o he kind o o ce cu e, he inden a ion (I) can be ex ac ed. Based on
wha we discussed abou Lb , we can say ha : I+Lb= o al bilaye hickness.
Figu e 2.9: ep esen a i e image o a F-D cu e wi h a up u e e en , and ela i e schema ic pic u e. a) Pa o he
cu e whe e he ip is app oaching he sample; b) inden a ion o he ip on o he bilaye ; c) he ip is now ouching
he subs a e; hence he slope is p ac ically e ical. Zoomed inse o he F-D cu e, highligh ing he discon inui y
egion. The pa ame e s I, Fb , Lb a e di ec ly ex ac ed om he spec a.
In his case he F-D cu es o ex ac he b eak ough pa ame e s we e ob ained by using a
di e en expe imen al appa a us and AFM modali y; as a ma e o ac we didn’ apply QI
app oach, bu a a he simila mode called Jumping mode (JM), also called pulse o ce mode
[112] [113] JM is used o imaging biomolecules unde physiological condi ions, and i is
pa icula ly ad an ageous when imaging samples ha a e weakly bonded o he subs a e. I has
been e ec i ely applied o a a ie y o biological sys ems such as DNA [114], Alzheime pai ed
helical ilamen s and di e en kinds o i uses [115][116][117], memb ane, demons a ing i s
e sa ili y and e ec i eness in a ious con ex s [118] In ac , in his modali y la e al and no mal
o ces a e minimized, making i p e e able o e o he modes o ce ain samples. The a ionale
behind choosing his pa icula me hod will be u he elucida ed, bu p ima ily i ela es o ou
72 | P h D h e s i s - M a e o M a i a n g e l i
Mo eo e , we ook he oundness o he de ec s o he bilaye in Figu e 2.7a as a e e ence
measu emen . The p ocess o o ma ion o holes in his case was independen on he
pho odynamic ac ion o Hyp, since he holes we e “a i icially” made by a s ong insing.
They o med by mechanical s ess induced by he wa e , which is a comple ely di e en p ocess
wi h espec o he Hypo he ic single-lipid ex ac ion induced by Hyp and ROS; as a esul , we
should expec a di e en alue o oundness.
Figu e 2.15: box plo s ep esen ing he oundness o po es c ea ed by he pho odynamic ac ion o Hyp ( ed) and he
con ol (blue) ob ained by he images a e he s ong insing, in Figu e 2.7.
In he cha (Figu e 20), he da ase ep esen ed by he ed box exhibi s conside able a iabili y,
which is e idenced by he wide sp ead o da a poin s and he e ical ex en o he ed box. This
could be a ibu ed o a ying po e sizes a ec ing hei oundness, wi h smalle po es ending
owa ds g ea e ci cula i y. The cen al endency o his da ase is indica ed by a median alue o
app oxima ely 0.6, sugges ing ha he shapes a e ela i ely less ound, which aligns wi h ou
quali a i e obse a ions.
Con e sely, he con ol da ase , as an icipa ed, p esen s less a ia ion wi h da a poin s mo e
closely g ouped a ound a median alue ha is nea 0.9. This indica es ha he po es in his g oup
a e nea ly ound, despi e being a popula ion dimensionally e y di e se.
The second obse a ion abou hese po es is abou hei peculia deepness.
Wi h e e ence o he measu emen s we pe o med o unco e he heigh o he bilaye (2.3.1),
we ob ained he alue o ~5-6 nm, o espec i ely he Ld and Lo phase. Addi ionally, om he
plo p o iles in he bilaye s wi h de ec s depic ed in he 2.2 pa ag aph, he same heigh can be
app ecia ed.
oundness holes oundness con ol
0.0
0.2
0.4
0.6
0.8
1.0
Range

73 | P h D h e s i s - M a e o M a i a n g e l i
Meanwhile, in his expe imen he dep h disclosed is much less han he expec ed one, as
depic ed in he heigh dis ibu ion o Figu e 2.16.
A gaussian dis ibu ion cen e ed a ound 2 nm dep h (wi h e e ence o he Ld phase) is ound
om he images.
Figu e 2.16: dis ibu ion o heigh s o Figu e 2.12 wi h Gaussian i s in blue and g een ep esen ing he Ld and Lo
phases o lipids, espec i ely. An inse wi hin he g aph shows he dis ibu ion o po e dep h, wi h i s i illus a ed in
ed.
Appa en ly, his alue is consis en wi h he hickness o a monolaye emo ed, which anyway
yields o a di icul in e p e a ion based on he s uc u e o he bilaye . The small laye o wa e
p esen o e he mica subs a e (0.5-1 nm hick) and he olume o wa e o e he memb ane,
makes he bilaye e y s able, exposing he hyd ophilic heads on he opposi e sides o he
s uc u e. The emo al o one laye is ins ead exposing he hyd ophobic ails o he wa e , which
could cause ins abili y o he s uc u e.
Theo e ical and expe imen al s udies ha e p edic ed ha up u e in phospholipid bilaye s begins
wi h he o ma ion o a po e illed wi h wa e molecules [120], and his could be ou case.
Ou hypo hesis is ha he s abiliza ion o he po e s uc u e could be s angely gi en by Hype icin
i sel , which is possibly packing in co espondence o he wa e laye abo e he hyd ophobic ails.
This e ec could be a o ed by he highe concen a ion o Hype icin ha we used in hese
expe imen s wi h high-speed AFM.
74 | P h D h e s i s - M a e o M a i a n g e l i
Fo ma ion o he dense Lβ phase
Due o he insu icien sampling in he ini ial s age o he i s expe imen , a second expe imen
was pe o med o desc ibe be e he dynamics o he o ma ion o he hi d, mo e ele a ed
phase.
Figu e 2.17: images o he Hyp-loaded bilaye du ing ligh i adia ion a empo al in e als o 50 seconds. We can
no ice he inc ease o a ea in he Lβ. The acquisi ion pa ame e s he e we e 1024 x 1024 px, 20 lines/s, 5 x 5 μm.
The images we e collec ed wi h a smalle ield o iew, o allow as e imaging and o limi
possible a i ac s de i ed om he scanning a high a es.
The expe imen was no i ial o pe o m, because we i s had o ind a egion whe e an e en
o hi d phase o ma ion whe e beginning, as shown in Figu e 2.17, and be as o swi ch o a
smalle egion ha was allowing a as e acquisi ion wi hou losing spa ial esolu ion. In
pa icula , he acquisi ion pa ame e s o he subsequen images we e 512 x 512 px, 50 lines/s, 3
x 3 μm, allowing a ime o 10 seconds o each acquisi ion. A ep esen a i e image acqui ed in
hese condi ions is shown in Figu e 2.18.
Figu e 2.18: le ) one o he 15 images o he bilaye used o measu e he a ia ion o he a ea o he dense phase.
igh ) cu e ep esen ing he ime-dependen dimension o he Lβ pa ches.
75 | P h D h e s i s - M a e o M a i a n g e l i
The a ea alues we e ob ained by image analysis on ImageJ and a e plo ed in he g aph o Figu e
2.18. The end highligh s a as dynamic ha eaches a sa u a ion alue, when, in his case, he
lipid a ha was p e iously in Lo phase (Figu e 2.19 a) phase becomes (almos ) all Lβ (Figu e 2.19
b). The dis ibu ions o he co esponding heigh s in Figu e 2.19c is in ac con i ming ha he
main phases a he beginning o he measu emen s we e sepa a ed by ~1 nm in heigh , in
acco dance wi h he coexis ence o Ld and Lo phases; while by he end o he expe imen almos
all he lipids in Lo phase became Lβ , as he di e ence aised o ~2 nm.
Figu e 2.19: a) image a ime ze o om ligh i adia ion, as p e iously depic ed in Figu e 2.17. b) image a e 90
minu es a e he ligh i adia ion began. c and d ep esen he espec i e heigh dis ibu ions.
The ed Gaussian i in bo h g aphs ep esen s he dis ibu ion o he Ld phase o he ini ial and la e s a es. The
g een i s illus a es he ansi ion om he Lo o Lβ phase, p o iding a isual ep esen a ion o he phase change.
Quali a i ely, he lipid a s in gel phase depic ed in Figu e 2.19b look e y simila o hose we
showed in Figu e 2.5, con i ming he ep oducibili y o his esul in di e en mic oscopes, a
di e en concen a ion o Hyp and a ied ligh dose; mo eo e , in his expe imen we e en
showed he dynamics o he o ma ion o he gel phase.
In any case, he in e p e a ion o he esul conce ning he ansi ion Lo → Lβ is no
s aigh o wa d, bu i could be ela ed o some e ec on he local concen a ion o choles e ol.
d
c
a
b
76 | P h D h e s i s - M a e o M a i a n g e l i
Indeed, i has been in es iga ed ha Hyp and choles e ol in memb anes s ands in close
ela ionship. Mo eo e , as highligh ed by imaging, he ansi ion begins om he Lo phase, which
is he iche in choles e ol.
Hyp p edominan ly in eg a es in o lipid s uc u es wi h highe concen a ion o choles e ol,
highligh ing i as a c i ical ac o o Hype icin's selec i e associa ion, sugges ing ha choles e ol
signi ican ly in luences Hype icin's selec i i y in memb ane en i onmen s. I has also been
implied ha Hype icin may o m compac s uc u es wi h choles e ol [46]. Lβ is ypically
cha ac e ized as a phase de oid o choles e ol. Howe e , i seems imp obable ha choles e ol
simply anishes due o he pho odynamic ac ion o Hyp. The e o e, he exac mechanism o Hyp
in e ac ion emains unclea . I is unce ain whe he Hype icin o ms he p e iously men ioned
compac s uc u es ha migh hide choles e ol molecules om su ounding lipids, he eby
mi iga ing choles e ol's e ec s, o i a complex in ol ing Hype icin and choles e ol di uses ac oss
he bilaye , leading o a dec ease in choles e ol wi hin lipid a s. Ano he hypo hesis is ha
single oxygen, gene a ed du ing he p ocess, could al e choles e ol molecules in a way ha
diminishes hei e ec i eness.
A po en ial solu ion o his issue would be o ack he loca ion o choles e ol du ing he ansi ion
phase. Un o una ely, we we e unable o conduc such measu emen s in ou s udy.
The ime-dependen cu es ha showed he wo dynamics in he bilaye e ealed a end ha
can be in e p e ed as a kine ics e lec ing he ac ion o a pa icula ligand and a esponse
dependen on i s concen a ion. As ime p og esses, i can be assumed ha he p oduc ion o
1O2 and/o i s ac ion on lipids is g owing, eaching a poin when Hyp has exhaus ed he possibili y
o igge new ROS.
𝑂2
3+3𝐻𝑦𝑝→ 𝑂2→𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒
1
(20)
This esponse could be ei he he o ma ion o he gel phase, o he occu ence o po es on he
bilaye .
The wo dynamics can in ac be well i ed by a Hill equa ion [121] ha highligh s he ela ionship
be ween concen a ion o a molecule
A
and e ec
E
p oduced by i [122]:
𝐸=𝐸𝑚𝑎𝑥 [𝐴]𝑛
[𝐴]𝑛+[𝐴]50
𝑛
(21)
Whe e
n
deno es he Hill coe icien , which desc ibes he s eepness o he subs ance's dose-
esponse cu e, and [𝐴]50 ep esen s he concen a ion o subs ance
A
ha p o ides an e ec
equal o 50% o he maximum e ec (Emax).
77 | P h D h e s i s - M a e o M a i a n g e l i
Howe e , we a e no able o access he di ec in o ma ion abou he eal concen a ion o 1O2;
we can only in e ha he e is a ime-dependency o i s p oduc ion, he e o e:
[ 𝑂2
1]∝𝑡
(22)
And Equa ion (21) can be w i en as:
𝐸=𝐸𝑚𝑎𝑥 𝑡𝑛
𝑡𝑛+𝑡50
𝑛
(23)
This equa ion is no linked o a p ecise eac ion scheme; hence we can conside his as an empi ical model ha can
desc ibe quali a i ely he phenomenology o he expe imen s. In any case, he i wi h such equa ion is isualized in
Figu e 2.20 and he pa ame e s i yields a e epo ed in
Table 4.
Figu e 2.20: ends o ime-dependen dimension o he Lβ pa ches (le ) and o he newly o med holes ( igh ). The
black line ep esen s he i wi h a Hill equa ion
Fi ing pa ame e s
Dense phase
Po es
50
144 ± 13 s
83.1 ± 1.5 minu es
n (Hill coe icien )
1.74 ± 0.17
3.15 ± 0.11
Table 4: esul s o he i ing wi h he empi ical model o Equa ion (23) de i ed om he Hill equa ion.
The quali y o he wo i s in Figu e 2.20 may indica e ha he concen a ion o ROS indeed
inc eases o e ime.
In gene al, he Hill coe icien is a pa ame e used in he Hill equa ion, which models how ligands
bind o a mac omolecule, such as an enzyme o a ecep o . In his case i can indica e how
e ec i ely 1O2 exe s i s ac ions; bo h i s yield a alue o he Hill coe icien , n, g ea e han 1 (
Table 4).

78 | P h D h e s i s - M a e o M a i a n g e l i
A Hill coe icien g ea e han 1 is an indica ion o posi i e coope a i i y in he ligand-binding
p ocess and e lec s a scena io whe e he binding o one ligand inc eases he likelihood o
subsequen ligand binding. The e o e, i can be assumed ha he lipids a e mo e inclined o be
a ec ed by he pho odynamic ac ion o 1O2, which in u n inc eases i s a ini y o he
phospholipids.
79 | P h D h e s i s - M a e o M a i a n g e l i
2.4.2 AFM imaging o Hyp + da k
Gi en he highe empo al and spa ial esolu ion p o ided by he Fas Scan AFM, some
measu emen o imaging in da k we e a emp ed. Ou goal was o highligh i he e was some
e e sible modi ica ion ha wasn’ de ec able wi h con en ional AFM.
Howe e , e en wi h he as e acquisi ion a e, no changes we e de ec ed in he da k o he
un ea ed bilaye (da a no shown). Howe e , we did no ice some peculia di e ence be ween
he un ea ed bilaye s, and hose wi h Hype icin in da k.
Quali a i ely, whene e we added Hyp, we saw he bo de s o he lipid a s being inc easingly
mo e ugged.
Figu e 2.21: same egion o he bilaye in he da k wi hou he p esence o Hyp (le ) and a e he addi ion o Hyp
( igh ).
We conduc ed a quan i a i e analysis o he edges o lipid a s, which appea ed o ha e ac al-
like cha ac e is ics. Fo his pu pose, we u ilized a plugin in ImageJ designed o assess he ac al
dimension o an image.
F ac al dimension is a way o desc ibe he complexi y o a ac al [123], which is a geome ic
shape ha can be spli in o pa s, each o which is a educed-scale copy o he whole. This
cha ac e is ic is known as sel -simila i y.
F ac al dimensions a e applied in many ields, o example hey a e used o desc ibe phenomena
like he i egula i ies in coas lines [124].
Unlike he dimensions we a e amilia wi h in e e yday li e (such as one-dimensional lines, wo-
dimensional squa es, and h ee-dimensional cubes), ac al dimensions a e no necessa ily
in ege alues. They can be ac ional, which is whe e he e m " ac al dimension" comes om.
80 | P h D h e s i s - M a e o M a i a n g e l i
This ac al dimension is linked o how jagged he s uc u e is, and gi es us a way o quan i y how
comple ely a ac al pa e n ills he space as i s size inc eases.
Ma hema ically, he ac al dimension is de ined as i ollows.
A s aigh -line segmen possesses an inhe en cha ac e is ic ha , while ob ious, me i s explici
men ion o he sake o b oade gene aliza ion. Gi en an in ege N, he leng h o a segmen L is
he sum o N segmen s o leng h =L/N, he ac al dimension D o he segmen L is [125]:
𝐷= log(𝑁)
log(1 𝑟)
⁄
(24)
Which can be gene alized o all shapes, yielding in ege alues o Euclid shapes and non-in ege
o mo e complex objec s.
Ope a i ely, wha we did o calcula e D o ou lipid a s, was o apply he box-coun ing me hod
[126] which is a p ac ical and common me hod, e y o en used in physics and en i onmen al
science [127].
I in ol es coun ing he numbe N(ε) o boxes o a ce ain la e al size ε needed o co e a ac al,
and hen seeing how his numbe changes as he size o he boxes is educed (Figu e 2.22):
𝐷=lim
𝜀→0log𝑁(𝜀)
log(1 𝜀
⁄)
(25)
Figu e 2.22: example o applica ion o he box coun ing me hod on he well-known Koch cu e. Adap ed om [128].
Copy igh © 2018 Ian Pilg im and Richa d P. Taylo . Licensee In echOpen.
ε
N(ε)= 10
N(ε)= 36
ε
ε
N(ε)= 84
81 | P h D h e s i s - M a e o M a i a n g e l i
The esul s o his analysis, in ol ing 7 di e en ROIs, a e shown in Figu e 2.23a.
A ep esen a i e ROI, be o e and a e he injec ion o Hyp, is depic ed in Figu e 2.23b.
The i s hing o no ice om his analysis is ha o ei he he con ol and he measu emen wi h
Hyp, we ha e a ac al dimension, which e lec s he complexi y o he objec s. Secondly, we do
no ice a di e ence in he alues o D, in pa icula he e is a end o a sligh dec ease o i s alue
by adding Hyp. Al hough he a ia ion is small (a ound 1-2%), i may indica e a di e ence in
shapes, as also no ed in p io s udies [95]. Occasionally, he e o ba s o e lap, which migh be
a ibu ed o he algo i hm used o calcula e he D alues, as well as po en ially inadequa e image
edge quali y o an e ec i e plugin applica ion.
Figu e 2.23: a) esul s o he analysis o ac al dimension using he Box coun ing me hod. The Roi numbe ep esen
he a ea o a speci ic lipid a . b) ep esen a ion o he images p ocessed om he so wa e. The ROI is he same o
Figu e 2.21: same egion o he bilaye in he da k wi hou he p esence o Hyp (le ) and a e he addi ion o Hyp
( igh ). The espec i e alues o he ac al dimensions a e depic ed.
a
b
88 | P h D h e s i s - M a e o M a i a n g e l i
This o ma ion could dec ease Hype icin's a ini y, possibly due o he lack o choles e ol in hese
egions. Un o una ely, we don’ ha e he concu en AFM da a ha would con i m his
possibili y.
Du ing he same se o measu emen s, we ealized ha a e a ew minu es om he addic ion
o he pho osensi ize , i s luo escence pa e n was changing p e y much wi h espec o wha
was disclosed in Figu e 2.26, as e ealed by Figu e 2.28. I is unequi ocal ha mos o he
luo escence is coming om he diso de ed phase Ld and his e en may be caused by ei he one
o he ollowing explana ions, o a combina ion o hem.
Figu e 2.28: bilaye seen by using DPPEA o647N (le ) and 1 μM Hype icin ( igh ). 512 x 512 px images. These images
we e acqui ed app ox. 20 minu es la e hose shown in Figu e 2.26.
The molecule may highly pack inside he lipid a s, and o ha eason he e could be some
sel -quenching e ec o he PS. This e en occu s when luo opho es a e in close p oximi y o
each o he , leading o non- adia i e ene gy ans e be ween hem, and as a esul limi ing he
luo escence emission. Ano he explana ion is possibly in acco dance wi h he p e ious AFM
da a. In ac , we saw ha he e ec s o Hyp had wo dis inc dynamics, a as e one in ol ing
he lipid a s and a slowe one in ol ing he p oduc ion o holes in he diso de ed phase. In
ligh o he new con ocal da a, such empo al di ision can be explained as a p esence o Hyp in
a speci ic lipid phase. In he i s minu es a e he injec ion o Hyp and i adia ion, ep esen ed
in Figu e 2.26, Lo is he p e e en ial phase whe e we ind he pho osensi ize , e lec ing he
speci ici y Hyp possesses o choles e ol. La e on (Figu e 2.28) he speci ici y mo es owa ds
he diso de ed phase, e lec ing an exchange be ween he wo phases. The possible o ma ion
o compac and bulkie s uc u es wi h choles e ol [46], as we s a ed abo e, could be he
d i ing o ce o his mechanism.

89 | P h D h e s i s - M a e o M a i a n g e l i
2.5.3 Fluo escence co ela ion spec oscopy - p elimina y measu emen s
Mo e insigh s ega ding he dynamics o Hype icin could be p o ided by Fluo escence Co ela ion
Spec oscopy. The o ma ion o compac s uc u es o Hyp wi h choles e ol would be possible o
ace due o an inc ease o he hyd odynamic adius ha e lec s in a educed di usion
coe icien o Hyp when bound o choles e ol.
Mo eo e , FCS could be e y in o ma i e on he e ec s o he PS on he en i onmen o he
bilaye . As s a ed abo e, many s udies ha e highligh ed he possibili y o analyze lipid bilaye
models sys ems wi h FCS [138], and sensi i e enough o dis inguish dis inc lipid phases based on
he di usion coe icien [134].
The S okes-Eins ein equa ion ela es he di usion coe icien D wi h he iscosi y η and he
hyd odynamic adius H
𝐷= 𝑘𝐵𝑇
6𝜋𝜂𝑟𝐻
(27)
Whe e kB is he Bol zmann cons an , and T is he empe a u e in K.
E en a small al e a ion in he iscosi y o he en i onmen is de ec able h ough FCS. The e o e
i he pho osensi ize s IMPACT on he iscosi y o he bilaye , i become obse able using a
p ope luo escen p obe, such as DPPEA o647N. This molecule is pa icula ly app op ia e
because i can be exci ed wi h wa eleng hs in he ed whe e Hyp does no abso b any ligh . In
his way i is possible o di e en ia e be ween he case o Hyp wi h and wi hou ligh i adia ion.
Howe e , when FCS is applied o measu e luo opho es in memb anes, we aces mo e challenges
han when used in solu ion. In memb ane en i onmen s, di usion ends o be slowe ,
necessi a ing addi ional p ecau ions. To p e en pho obleaching, he use o e y low exci a ion
powe s is essen ial. Addi ionally, o ensu e a s a is ically signi ican numbe o independen
e en s, long measu emen a e equi ed. Ul ima ely, he leng h o he measu emen s is limi ed
by he s abili y o bo h he expe imen al se up and he sys em unde in es iga ion.
Scanning FCS [139] se es as a signi ican example in his con ex . I s undamen al concep
e ol es a ound mo ing he exci a ion olume by scanning o e he sample, e ec i ely
sho ening he esidence imes on he luo opho es and enhances he s a is ical accu acy o he
measu emen s.
Hence, i is essen ial o pe o m his kind o measu emen s on a lase scanning mic oscope such
as ou Leica SP5 con ocal/STED se up.
In ou applica ion, we pe o med line-scan FCS measu emen s in which, ins ead o analyzing a
ixed small olume, a line is apidly scanned by he mic oscope. Apa om he educed
pho obleaching, his me hod p o ides enhanced spa ial in o ma ion compa ed o adi ional FCS.
I cap u es molecula dynamics ac oss a line, gi ing a mo e comp ehensi e iew o molecula
mo emen s and in e ac ions o e ime.
90 | P h D h e s i s - M a e o M a i a n g e l i
We chose line-scan FCS since he expec ed di usion a e is a ew μm2/s [134], accu a ely
esol able using he esonan scanne a 8 kHz. This app oach also o e s he ad an age o ime-
esol ed spa ial in o ma ion, enabling us o de e mine whe he he a is mo ing slowly du ing
he acquisi ion p ocess o i he e has been a change in ocus. Such insigh s a e c ucial o
dis inguishing ac ual da a om a i ac s. Addi ionally, his me hod allows o he simul aneous
measu emen o he di usion coe icien s o bo h componen s (Lo and Ld phases) in a single
expe imen . This is highly bene icial, ensu ing ha he ocal olume emains consis en o bo h
componen s, he eby gua an eeing he same poin sp ead unc ion (PSF) o each.
Un o una ely, ou expe imen s wi h hese measu emen s encoun e ed nume ous issues,
leading o inconclusi e esul s, which is why hey will no be p esen ed. Mo e speci ically, we
aced challenges in di e en ia ing he wo phases based on di usion coe icien s, e en in he
absence o Hype icin. In mos cases, a dominan as componen o e shadowed he slowe one,
he la e associable wi h lipid di using on he memb ane.
P e ious s udies ha e indica ed ha excessi e subs a e hickness can be he cause o poo
esul s [132], causing sphe ical abe a ions and dis o ion o he PSF, u he exace ba ed by he
use o mica. Indeed, in he ocal plane, he PSF de ia es subs an ially om a Gaussian shape,
complica ing he applica ion o s anda d FCS o mulas ha co ela e he cha ac e is ic ime wi h
he di usion coe icien . Addi ionally, he sca e ing e ec o mica leads o a u he educ ion in
he quan i y o de ec able luo escence.
Clea ly, e ining he sample p epa a ion and subs a e enginee ing is a i al s ep o add ess hese
encoun e ed p oblems. Op ing o glass as a subs a e seems o be he mos sui able choice o
such measu emen s. While ou goal was o es ablish a obus and eliable pla o m o s udying
he e ec s o Hype icin on suppo ed lipid bilaye s, hese e ec s should also be obse able in
o he model lipid bilaye sys ems. Fo example, gian unilamella esicles (GUV), which ha e been
ho oughly in es iga ed using FCS [140] [141], a e simple o place on glass. Taking all hese
ac o s in o accoun , ou u u e FCS s udies will mos likely ocus on his di ec ion.
91 | P h D h e s i s - M a e o M a i a n g e l i
2.6 Conclusions
In his chap e we ex ensi ely analyzed he beha io o SLBs mimicking i al en elopes when he
pho osensi ize Hype icin is loaded. Th ough con en ional AFM imaging, we obse ed a
concen a ion-dependen de imen al impac o Hyp on SLBs wi h p e-exis ing de ec s upon ligh
i adia ion, a ibu ed o he gene a ion o 1O2. Addi ionally, high-speed measu emen s
conduc ed in Mad id enabled us o delinea e he dynamics o he deg ada ion induced by Hyp in
de ec - ee bilaye s, p o iding p ecise insigh s in o he lipid ex ac ion mechanism om he SLB.
A dense lipid phase eme ged when Hyp and ligh we e in ol ed, a phenomenon whose
mechanism emains unclea ; ou hypo hesis sugges s ha choles e ol edis ibu ion in he
bilaye could be a con ibu ing ac o . Co ela i e AFM-Fluo escence imaging p o ided aluable
insigh s in o he ex en o he o e lap be ween Hype icin luo escence signals and lipid a s,
once again highligh ing he mul i ace ed and dynamic na u e o he pho osensi ize .
The impac o Hyp in he absence o ligh appea ed less conspicuous in he images, wi h he
p ima y di e ence being he al e ed shape o he edges o lipid a s, appea ing mo e ugged.
A omic o ce spec oscopy was employed o un eil he e ec o Hyp on he nanomechanical
p ope ies o he SLB, h ough he measu emen o he Young modulus and he b eak h ough
o ce. We obse ed an inc ease in he igidi y o he bilaye induced by Hyp, consis en wi h he
a o emen ioned hypo hesis. This e ec was obse able bo h wi h and wi hou ligh o he case
o he Young modulus bu no o he b eak h ough expe imen s. This ins ance p o ided e idence
ha Hyp has a dis inc ac ion owa ds elas ic (inden a ions) and plas ic (pene a ion) p ope ies
o he bilaye .
92 | P h D h e s i s - M a e o M a i a n g e l i
2.7 Ma e ials and Me hods
2.7.1 SLBs sample p epa a ion
The bilaye s p epa a ion adhe ed o he same p o ocol consis en ly in all he measu emen s
p esen ed in his chap e . I ollows he s eps ha I am going o discuss.
Figu e 2.29: 2D-S uc u e images o a) DOPC, b) DOPS, c) Sphingomyelin and d) Choles e ol. Images ob ained om
PubChem; a) h ps://pubchem.ncbi.nlm.nih.go /compound/10350317#sec ion=2D-S uc u e - CID 10350317,
b) h ps://pubchem.ncbi.nlm.nih.go /compound/6438639#sec ion=2D-S uc u e - CID 6438639,
c) h ps://pubchem.ncbi.nlm.nih.go /compound/44260124#sec ion=2D-S uc u e - CID 44260124,
d) h ps://pubchem.ncbi.nlm.nih.go /compound/5997#sec ion=2D-S uc u e - CID 5997.
The cons i uen s we used we e:
• DOPC (1,2-Dioleoyl-sn-Glyce o-3-PhosphoCholine), Mw = 786.1 g/mol - 66% (unsa u a ed)
• DOPS (1,2-Dioleoylphospha idylse ine), Mw = 788 g/mol - 13% (unsa u a ed)
• SM (Sphingomyelin), Mw = 20% (sa u a ed)
• Choles e ol, Mw = 388.5 - 1%
The a ionale behind he choice o he componen s o he bilaye was a comp omise be ween
choosing he eal componen s o he i al en elope and ha ing a su icien ly complex bu s able
sys em. Pa icula ly, we ca e ully chose 4 ou o 6 componen s o he i al en elope [52], i s by
main aining a concen a ion a io o 2:1 be ween DOPC and DOPS, which assu es a ce ain
s abili y o he Ld phase. Secondly, we chose he exac concen a ion o DOPS conside ing i s
nega i e ne cha ge a neu al pH. In he i al en elope, he pe cen age o PS plus ano he
nega i ely cha ged phospholipids PI (Phospha idylinosi ol) is exac ly 13%; hence by keeping DOPS
a ha concen a ion we ensu ed he co ec pola i y o he bilaye .
The emaining concen a ions o Choles e ol and SM we e chosen o secu e he o ma ion o
lipid a s wi h su icien ly la ge a eas, o allow he in es iga ion wi h imaging me hods.
93 | P h D h e s i s - M a e o M a i a n g e l i
P epa a ion o he lipid suspension. We s a ed wi h he s ock suspensions o he single
componen s al eady dissol ed in chlo o o m and we p epa ed he solu ion wi h he chosen
concen a ions inside a glass ial, ha ing a o al o 1 mg o mixed lipids dissol ed. We le he
sol en e apo a e wi h he help o a con inuous low o ni ogen, o abou 10 minu es. A e his
p ocess, a ilm o lipids was o med a he bo om o he ial which was eady o be used. We
added 1 mL o ul apu e MilliQ wa e , in o de o ha e a inal concen a ion o 1 mg/mL. The
addic ion o wa e allowed he o ma ion o LUV (la ge unilamella esicles) wi h e e ogenous
diame e s.
Vesicle ex usion. In o de o ha e he o ma ion o a good homogeneous bilaye wi hou de ec s
i is necessa y o ha e esicles wi h he same size. A e y well-es ablished p o ocol o ul ill his
is o pe o m a esicle ex usion.
Figu e 2.30: A an i Mini-ex ude , A an i Pola Lipids, Inc. The images on he igh ep esen DLS spec a o he size
o he esicles depending on he numbe o passes h ough he memb ane. Copy igh © 2024 C oda In e na ional
Plc. All igh s ese ed.
This p o ocol was based on he use o a Mini ex ude (Figu e 2.30). Wi h he help o wo
Hamil on sy inges o 1 mL olume, we le he suspension pass h ough a memb ane wi h 100 nm
size diame e po es. To educe any a iabili y in he p o ocol, we pe o med 19 passes h ough
he memb ane in each sample we p epa ed. E en hough he company ensu es ha he mean
size o he esicles a e 11 passes has al eady he desi ed alue (Figu e 2.30, igh ). The ex usion
was pe o med o e a ho pla e a a empe a u e o abou 60°, o ha e a gel-sol phase ansi ion
o he sa u a ed lipids and o acili a e he o ma ion o esicles wi h mul iple componen s.
A e he ex usion, he lipid esicles a e eady o be used, a a conse ed concen a ion o 1
mg/mL.

94 | P h D h e s i s - M a e o M a i a n g e l i
Deposi ion o he esicles and o ma ion o he bilaye . We ollowed a me hodology in which
he bilaye o ms a e he esicles up u e and use on o he subs a e [92], Figu e 2.31.
Figu e 2.31: schema ic ep esen a ion o he SLBs o ma ion by esicle usion. Adap ed wi h pe mission om [142].
Copy igh © 2008, Sp inge Na u e Limi ed.
Fo each bilaye we used a ce ain olume o he esicle suspension in combina ion wi h esh
CaCl2 solu ion, which acili a es he deposi ion. The exac olumes o he wo solu ions was
chosen conside ing he a ea o he suppo , bu in any case we kep cons an hei olume a io:
• Lipid suspension (50 μL, 0.5 mg/mL) and CaCl2 (8 μL, 10 mM) - Fo measu emen s o
sec ions 2.1 - 2.4 wi h he excep ion o 2.3.2.
• Lipid suspension (25 μL, 0.1 mg/mL) and CaCl2 (4 μL, 10 mM) - Fo measu emen 2.3.2.
• Lipid suspension (25 μL, 0.5 mg/mL) and CaCl2 (4 μL, 10 mM) - Fo measu emen s o
sec ions 2.5.
E e y sample we e deposi ed on eshly clea ed mica oil, p e iously glue o a s anda d
mic oscope slide (75 mm x 25 mm) o , o he case o 1 measu emen s, on a glass co e slip (ø =
25 mm, 100 μm hickness).
Musco i e Mica is a na u ally occu ing mine al, a silica e c ys al wi h a peculia , laye ed s uc u e
o shee s weakly bound oge he ha exhibi s pe ec clea age, i.e. hey can be easily spli in o
hin oils.
Using mica as a subs a e is ad an ageous o se e al easons. The i s is ha , by exe ing a
clea age o each expe imen , a esh laye can be exposed, ensu ing a consis en ly clean su ace,
eady o be used. Secondly, i p o ides a highly smoo h and uni o m su ace, wi h a omic le el
la ness, (0.37 ± 0.02) nm [143] which ensu es ha he lipid bilaye s s ably o m and sp ead
e enly ac oss i s su ace.
Following he deposi ion, he samples we e placed in a humid chambe o inhibi e apo a ion
du ing he p epa a ion p ocess.
They we e kep a oom empe a u e o 10 min and hen incuba ed o 15 min a 60°C in a
labo a o y s o e. The samples we e hen main ained a oom empe a u e o 2 hou s and hen
gen ly insed wi h MilliQ ul apu e wa e , o emo e non deposi ed esicles.
In he expe imen shown in Figu e 2.7a, we delibe a ely ampli ied his p ocess o in en ionally
de e mine he bilaye 's heigh .
95 | P h D h e s i s - M a e o M a i a n g e l i
2.7.2 S anda d AFM imaging
AFM s anda d imaging ha we epo ed in he sec ion 2.2 was pe o med wi h a JPK NanoWiza d
II (B uke , MA, USA) moun ed on a Leica SP5 (Leica, Ge many) in e ed lase scanning
mic oscope, placed on an ac i e ib a ion isola ion able. The s age o he ligh mic oscope has
been subs i u ed wi h a s age compa ible wi h AFM, wi h minimal mechanical noise.
The chosen modali y o AFM imaging was apping (o in e mi en ) mode in liquid. In apping
mode, he can ile e oscilla es a i s esonance equency. As he ip app oaches he sample
su ace, i ligh ly " aps" he su ace a he bo om o i s oscilla ion cycle. This echnique o e s
ad an ages o e adi ional con ac mode AFM, whe e he ip con inuously d ags ac oss he
su ace, po en ially damaging delica e samples such as he bilaye s we used.
We used V-shaped DNP silicon ni ide can ile e s (B uke , MA, USA) wi h nominal sp ing cons an
k=0.24 N/m and ip adius =20 nm, while he esonance equencies in ai a e in ange (40 - 75)
kHz, wi h a nominal peak a 56 kHz. In liquid condi ions esonance equencies shi o lowe
alues, ypically one qua e o hose in ai ; indeed, he alues in which we ope a ed we e in he
ange (13 - 15) kHz. We ypically acqui ed 256 x 256 px o 512 x 512 px images a line a es 0.5-1
Hz; all hose shown in he sec ion 2.2 a e 256 x 256 px images. All he images we e analyzed wi h
he JPKSPM so wa e.
The ligh i adia ion was ob ained by means o a me cu y a c lamp (Leica EL6000) and a bandpass
il e a (515-560) nm ocused on he sample wi h a Leica PLAN APO 10x objec i e, NA=0.4. The
ligh powe ha was ~ 29 mW (wi h e e ence o i s peak a 546 nm) and based on he
illumina ion a ea we es ima ed a densi y o ~ 0.2 W/cm2. Typically, he measu emen s s a ed
a max a e 5 minu es o i adia ion and hen hey we e pe o med wi h con inuous illumina ion.
Figu e 2.32: schema ical ep esen a ion o he con igu a ion used (le ). Ac ual pic u e o he se up ( igh ).
NanoWiza d
head
Leica SP5 s age
96 | P h D h e s i s - M a e o M a i a n g e l i
2.7.3 Mechanical p ope ies by AFM: Young Modulus
AFM expe imen s o measu e he Young Modulus we e pe o med on a JPK NanoWiza d III
(B uke , MA, USA) moun ed on a Axio Obse e D1 (Ca l Zeiss, Ge many) in e ed op ical
mic oscope Figu e 2.33, wi h a EXFO X-Ci e 120Q 120W Me al Halide Lamp wi h a 550/20
il e o exci a ion. The measu ed powe densi y was ~ 2 𝑚𝑊/𝑐𝑚2.
The measu emen s wi h ligh s a ed a e 20 minu es o i adia ion and hen hey we e
pe o med wi h con inuous i adia ion. Di e en ly om he NanoWiza d II, his mic oscope
allowed o imaging in QI (Quan i a i e Imaging), whose heo e ical amewo k we discussed
abo e. We used he same V-shaped can ile e s DNP men ioned ea lie .
Fo he acquisi ion o eliable spec oscopic da a, also compa able ac oss di e en
measu emen s, i was essen ial o p ecisely calib a e he can ile e . This ensu ed accu a e
knowledge o i s sensi i i y (a pa ame e ha de e mines how e ec i ely he can ile e can
de ec o ces, in uni [nm]/[V]) and sp ing cons an alues:
• Sensi i i y: a e app oaching he ip o he sample, we pe o med a o ce-dis ance cu e
o 1 μm o al excu sion in 1 second. The slope o he linea pa o he cu e yields he
alue o he sensi i i y. Knowledge o he sensi i i y is undamen al because i allows o
ela e he elec ical signal ou pu , measu ed by he pho ode ec o s, o he eal de lec ion
o he can ile e . We ound ypical alues o 12-15 nm/V.
• Sp ing cons an : in o de o ob ain he eal alue o his pa ame e we pe o med he
con ac - ee he mal noise calib a ion me hod [144]. We ypically ound alue in he
in e al 0.3-0.4 N/m.
We acqui ed 128 x 128 px images o e y small egions (0.5-1 μm), ei he inside o ou side lipid
a s. The se poin co esponding o he maximum o ce load was 1 nN, and he z-leng h o each
F-D cu e was 200 nm, and he esidency ime o each poin was (3.8-4.2) ms, yielding a scanning
speed o 2 Hz.
Figu e 2.33: pic u e o he se up used o he nanomechanical measu emen s. I is placed on an ac i e ib a ion
isola ion op ical able as well as in an acous ic enclosu e.
97 | P h D h e s i s - M a e o M a i a n g e l i
Da a analysis. To p ocess he F-D cu es (128 x 128 = 16384) we used he JPKSPM Da a P ocessing
so wa e. Fo each cu e we i s applied some da a smoo hing and backg ound sub ac ion.
Subsequen ly we pe o med a i on he linea pa o he cu e o se he ze o, which would be
he inal poin o he elas ici y i . Ul ima ely, we calcula ed he e ical ip posi ion which
au oma ically co ec s he heigh signal coming om he displacemen o piezo in o he eal
posi ion o he ip wi h espec o he sample. A e his p elimina y s age, we pe o med a ba ch
p ocessing o all he cu es, se ing he pa ame e s o he He z-Sneddon i (Equa ion (17)) o
ex ac he Young Modulus. We se he adius o he ip as he heo e ical alue o R=20 nm,
sphe ical shape, and we pe o med he i on he ini ial pa o he cu e (Figu e 2.34), ha is
when he de lec ion o he can ile e s a s o ise, e lec ing he con ac wi h he bilaye . Fi ing
he whole cu e wouldn’ be meaning ul because, a e a ce ain a e o inden a ion, he
con ibu ion o he subs a e would be p ominen . Ope a i ely, a e se ing he ze o wi h he
linea i p ocess, we an he i in he in e al [0,5] nm, which we can see om he (Figu e 2.34).
The i ing p ocess also de e mines he con ac poin alue, which is iden i ied as he poin whe e
he cu e's slope begins o inc ease, ma king he s a o inden a ion. This alue coincides wi h
he inden a ion dep h, δ.
Figu e 2.34: image om he JPKSPM so wa e. The pu ple line ep esen s an acqui ed F-D cu e, while he g een is
ep esen ed he He z-Sneddon i . The ex emi ies o he i a e shown as e ical black lines, as well as he con ac
poin .
Accu a ely knowing he inden a ion dep h is c ucial o wo easons: i s ly, o e i y he i 's
quali y; ypically, a dep h o 1-1.5 nm sugges s an accu a e i , whe eas la ge alues migh
indica e ha he i e oneously includes excessi e subs a e in luence. Secondly, he inden a ion
dep h is necessa y o applying he Ga cia co ec ion o he He z Model (Equa ion (18)).
We al eady discussed ha he unco ec ed He z-Sneddon model p o ides a Young modulus
ha we can call “appa en ”, Eapp. Such alue should be co ec ed o he hickness o he
sample, yielding he inal alue E ue. Un o una ely, in he JPKSPM so wa e we can’ i by using
Con ac poin
End o
he i
Beginning o
he i
104 | P h D h e s i s - M a e o M a i a n g e l i
AFM measu emen s o SARS-CoV-2
Th oughou his hesis, we ex ensi ely discussed he in e ac ion o Hype icin wi h SARS-CoV-2
and i s pho odynamic ac ion on he i al pa icles, in an a emp o unde s and he unde lying
molecula mechanisms, and elucida e he an i i al ac i i y in ligh - and da k condi ions, by using
model memb anes. Howe e , an en eloped i us ep esen s a mo e complex sys em han a
phospholipid bilaye , and i is c ucial o s udy i in i s en i e y o con i m he p e iously ob ained
esul s. Fo he applica ion on i uses, AFM was p ima ily used o examining he mo phology in
a liquid en i onmen [148], e en hough i s capabili ies ex end a beyond jus su ace imaging.
The AFM can ile e enables manipula ion o ma e a an a omic le el and he pulling o
indi idual biomolecules. Recen yea s ha e seen signi ican esea ch in o he mechanical
p ope ies o i us shells. S udies ha e linked i us mechanics o ac o s like densely packed
genomes, he ma u a ion p ocess, inco po a ion o a i icial ca go, and s uc u al changes [149]
[150] [151]. Among he mo e in o ma i e expe imen s in ol ing AFM on i uses we can men ion:
• Mechanical Fa igue expe imen s: in gene al, a igue measu emen s e e o he
me hods and echniques used o quan i y he le el o a igue expe ienced by a ma e ial.
Mechanical a igue in i us shells e e s o hei abili y o wi hs and a maximum o ce
be o e unde going s uc u al collapse [152]. No only his b eaking o ce is c i ical, bu
also he way he i us disassemble can be e y in o ma i e; o ins ance, he uncoa ing
pa hway o i uses can be eplica ed. I is o his eason ha his me hodology is applied
in he con ex o AFM imaging. Indeed, i is pe o med by applying con inuous AFM
imaging o obse ing any s uc u al changes in he p o ein shell unde epea ed low-
o ce load cycles (usually 100 - 150 pN pe pixel), demons a ing he s abili y o he shell
agains mul iple de o ma ions a such o ces.
• Nanoinden a ion: his spec oscopic me hod, in ol es obse ing he de lec ion o he
AFM ip when i in e ac s wi h indi idual p o ein shells. Once he ip makes con ac
wi h he pa icle, a Fo ce-Dis ance (F-D) cu e is egis e ed, he e o e indica ing he
mechanical p ope ies o he i us. Usually, a linea beha io co esponds o he elas ic
esponse o he shell, ha unde goes a de o ma ion as i ’s being comp essed.
None heless, when a c i ical poin is exceeded, he inden a ion leads o pa icle
b eakage. The maximum bea able o ce can be called, also in his case, he
b eak h ough o ce. Once his o ce is applied, he i us ypically unde goes la ge and
uncon ollable changes in i s s uc u e. The s uc u e changes a e b eaking can be
isible om he di e ence in he F-D cu es, e.g. he de o ma ion shi s om linea o
He zian i he i us shell is illed (Figu e 3.1). In any case he cu es can be i ed o
ob ain alues o he Young modulus. In e p e ing b eak h ough and Young modulus is
in e es ing o e alua e he s abili y p ope ies o he i us. Finally, nanoinden a ion also
e eals he po en ial sel - eco e y abili ies o p o ein cages a e b eakage.

105 | P h D h e s i s - M a e o M a i a n g e l i
Figu e 3.1: a) di e en momen o an inden a ion expe imen , 1-be o e con ac , 2-de o ma ion, 3-a e b eaking. b)
Fo ce-Dis ance cu e ep esen ing he s ages desc ibed in a). c) images o a i us in di e en momen s du ing a eal
nanoinden a ion measu emen (le ), and ela i e F-D cu es ( igh ). I ’s no iceable he beha io al change in he
mechanics a e b eakage (happening in cu e n°3) om he cu es, and he co esponding disassembly in he
images. Adap ed wi h pe mission om [116]. Copy igh © 2017 Else ie L d. All igh s ese ed.
All hese hings conside ed, we ini ia ed a s udy ocusing on nanomechanical p ope ies and
imaging o single SARS-CoV-2 i al pa icles by using AFM o unco e key cha ac e is ics. Ou aim
was, once again, o e eal he changes induced by he p esence o Hyp, wi h and wi hou ligh
i adia ion, wi h pa icula in e es on he mechanical p ope ies o he ex e nal laye , he i al
en elope. Ou hope was o be able o ob ain cohe en esul s wi h espec o hose p e iously
ob ained wi h lipid bilaye s.
All he expe imen s discussed in he ollowing sec ion we e pe o med wi h he Nano ec
Ce an es ha we desc ibed abo e. This se up p o es o be highly ad an ageous because o he
possibili y o applying jumping mode, especially use ul o handling delica e and easily de achable
samples i al pa icles [153], since i educes d as ically he la e al/d agging o ces.
Ano he key aspec o his mic oscope is i s scan head, which is ailo ed o op imal pe o mance
wi h ex emely small ields o iew (less han 500 nm). This makes i especially sui able o
cap u ing images o indi idual i al pa icles.
a
c
b
106 | P h D h e s i s - M a e o M a i a n g e l i
Due o sa e y equi emen s, wo king wi h he ac i e SARS-CoV-2 i us was no possible as i
equi es Biosa e y Le el 3 condi ions. As a esul , we decided o use an inac i a ed i us, which
was di e en om he one discussed in chap e 1. The change was necessa y because he
chemical inac i a ion wi h pa a o maldehyde in oduces c osslinks ha i e e sibly al e he
i us's mechanical s uc u e. An al e na i e me hod we conside ed was hea inac i a ion. This
app oach pa ially deg ades he i al RNA and memb ane p o eins, bu i migh also comp omise
he i us's s uc u al in eg i y, including he en elope, as indica ed in p e ious s udies [154].
Howe e , he e is exis ing esea ch on hea -inac i a ed i uses [155] ha sugges s i is possible
o main ain in ac i al pa icles, al hough his equi es ca e ul p epa a ion o he sample.
3.1 AFM imaging
The ini ial expe imen s ocused on assessing he basic cha ac e is ics o he i uses, including
hei shape, heigh , and o e all condi ions. In gene al, we expec ed in ac and ounded objec
[155], cohe en wi h he s uc u e o SARS-CoV-2.
Imaging he spike glycop o eins on he i us's su ace and de e mining hei heigh ela i e o
he en elope is imp obable. This limi a ion s ems om he imaging echnique used, whe e a
scanning ip likely sweeps o e and displaces he p o eins ins ead o accu a ely eco ding hei
heigh s. Ne e heless, i is easible o iden i y po en ial deb is o hese p o eins a ound he i us's
ounded s uc u e [156] (Figu e 3.2).
Addi ionally, i 's c ucial o no e ha he obse ed heigh o he pa icles no only e lec s hei
inhe en s uc u e bu also hei in e ac ion wi h he unde lying subs a e. This aspec is
pa icula ly ele an o en eloped i uses, whe e he en elope impa s addi ional lexibili y o
he s uc u e.
Figu e 3.2: image o an in ac i al pa icle deposi ed on mica. The o ange a ows indica e possible spike le o e s.
92.00 nm
0.00 nm
107 | P h D h e s i s - M a e o M a i a n g e l i
Figu e 3.2 p esen s a ypical A omic Fo ce Mic oscopy (AFM) image o an in ac i al pa icle,
which con o ms o ou expec a ions in e ms o shape. Nea he pa icle, he e appea s o be
emnan s o spike p o eins. Fu he away, we obse e o he i egula o ma ions ha could be
deb is om damaged pa icles, such as p o eins, RNA, o agmen s o he i al en elope, possibly
esul ing om he inac i a ion p ocess. I is unlikely ha hese i egula objec s a e caused by
he poly-L-lysine subs a e, as we ha e con i med i s su ace o be qui e la and smoo h, wi h
e y ew impu i ies, ypically measu ing less han 5 nm in heigh .
The expe imen o Figu e 3.3 I am going o desc ibe was aimed o in es iga e he esis ance o
samples o epea ed imaging, i.e. a a igue expe imen . The ou comes o nume ous a igue
expe imen s we e consis en wi h hose p esen ed in Figu e 3.3, which e ec i ely ep esen s he
indings om se e al simila es s. Speci ically, Figu e 3.3a shows he ini ial image, and Figu e
3.3b displays he inal image om he a igue expe imen . These images we e cap u ed a a o ce
o 50 pN, and he expe imen spanned app oxima ely 70 minu es, du ing which 24 images we e
ob ained. Analysis o he images and he line p o ile in Figu e 3.3c e eals ha he i us did no
comple ely disassemble. Ins ead, i appea s o ha e los some ma e ial, as indica ed by a heigh
di e ence o abou 10 nm. This obse a ion is pa icula ly no ewo hy as i uses wi h p o ein
shells ypically disassemble mo e clea ly in such expe imen s. On he con a y, he p esence o a
phospholipidic en elope is belie ed o impa a deg ee o pleomo phism o he i ions, allowing
hem o al e hei s uc u e unde mechanical s ess, no collapsing comple ely.
Figu e 3.3: an example o a igue expe imen is isualized. The heigh di e ence be ween a) he i s image and b)
he las is plo ed on he line p o ile c).
The e is an inc easing e idence ha such s uc u al adap abili y is ele an om an e olu iona y
poin o iew. Indeed, i ions can be exposed o physical o ces ha ha e he po en ial o de o m
o ac u e hem, whe he hey a e ou side o inside cells. These o ces encompass shea s esses
encoun e ed as hey mo e h ough iscous luids, o du ing ex usion h ough nuclea po es o
simila cons ic ions, osmo ic in luences, p essu e esul ing om he packaging o double-
92.00 nm
0.00 nm
73.55 nm
0.00 nm
050 100 150 200 250
0
20
40
60
80
Heigh (nm)
x (nm)
a
b
c
108 | P h D h e s i s - M a e o M a i a n g e l i
s anded DNA, hyd os a ic p essu es, and capilla y o ces, e c expe ienced du ing desicca ion,
among o he s.
I is plausible o sugges ha i uses may ha e de eloped dis inc mechanical cha ac e is ics as
an e olu iona y esponse o he a ious selec ion p essu es exe ed by di e en o ces. This
adap a ion would se e o ei he endu e hese o ces o po en ially le e age hem o hei own
bene i [157].
Addi ionally, he s i ness o some en eloped i uses like MuLV o HIV-1 ha e been s udied in
di e en condi ions and di e en s ages o he ma u a ion cycle, cen al o unde s anding he
i us's in ec i i y and in e ac ion wi h hos cells. In e es ingly, i has been disclosed ha he
imma u e i ions a e much s i e han he ma u e ones , which a e in u n mo e in ec i e [158]
[159]. This e eals ha he mechanical p ope ies o i uses en elopes/shells a e me as able,
and hei unc ion is go e ned by he biolophysical p ocess he i ion mus endu e.
The e o e, as we al eady epo ed, he beha io o he SARS-CoV-2 i ions exposed o a igue
measu emen s ha we showed in Figu e 3.3 may e eal he s uc u al adap abili y and so ness
o such i us. As a consequence, we migh ha e conside ed o pe o m some mechanical
measu emen s ex ac ing he alue o he Young modulus, by adding Hype icin as well. Anyway,
ou goal was o unde line he di e ences ha Hyp is in oducing in ela ion only o he i al
en elope, by i sel .
The ques ion ha we asked ou sel es a his poin was: can we say ha he adap abili y o hese
i uses is gi en only by he p esence o he i al en elope, and mo e speci ically o he
phospholipidic bilaye ? The answe is, mos likely, ha he con o ma ional changes ha he
i ions unde go a e mo e complex. Hence, i would be an o e simpli ica ion o say ha he
mechanical p ope ies a e go e ned only by he p esence o he en elope. The RNA packing, as
well as memb ane p o eins [159] ha e a ole.
Addi ionally, as we epo ed in he pa ag aph 2.3.1, co ec e alua ion o he Young modulus
equi es he knowledge o he hickness o he sample. Gi en he a iabili y o heigh s o he i al
pa icles (40 - 100 nm), would ha e equi ed a lo mo e s a is ics han he a ailable one.
These a e he easons why we didn’ pe o m any measu emen wi h he pu pose o knowing
he Young moduli o ou i uses.
3.2 AFM nanomechanical p ope ies
A mo e di ec measu emen o assess he mechanical p ope ies (only) o he i al en elope is
he e alua ion o he b eak h ough o ce. Such o ce is, in ac , ela ed o he p ope ies o he
mo e ex e nal laye o he i us, i.e. he en elope, and i ’s no dependen on he heigh o he
dimension o he i us. The measu emen s we e pe o med in he h ee condi ions o no Hyp,
Hyp in da k and Hyp unde ligh i adia ion, wi h he aim o de ec ing he possible changes
induced by he molecule, simila ly o wha we measu ed in 2.7.4. Being able o compa e he Fb
alues o he i al en elope wi h wha we ob ained wi h he bilaye model sys em would be ideal.
109 | P h D h e s i s - M a e o M a i a n g e l i
Howe e , ou s udy used can ile e s ha di e ed om hose used in he abo e ci ed
measu emen s, ea u ing sha pe ips and a wi h a dis inc shape.
Consequen ly, di ec ly compa ing he absolu e o ce alues was no easible. Addi ionally, o
ensu e e ec i e Hyp inco po a ion in o i al pa icles, we mixed he PS and he i us in an
Eppendo and hen applied he solu ion on o he subs a e, as de ailed in he Ma e ials and
me hods sec ion. This app oach p ecluded he possibili y o di ec ly compa ing he same i al
pa icle be o e and a e Hyp adminis a ion. A he same ime, e en i we had chosen o
adminis e Hyp di ec ly on o he sample, i is highly imp obable ha we would ha e been able o
iden i y and selec he exac same i ion o obse a ion.
The measu emen s we e no easy o ca y ou , because we didn’ always ob ain “clea ”
b eak h ough, e ealing ip pene a ion on an in ac s uc u e. Tha ins ance was due o
inden a ion on al eady b oken i ions, no immedia ely dis inguishable om imaging, mos likely
due o he inac i a ion p ocess ha in oduced many inhomogenei ies on he sample. Mo eo e ,
e en wi h in ac pa icles, we ob ained b eaking o he s uc u e a e se e al inden a ion cycles
( ypically 5-10 inden a ions) in acco dance wi h p e ious esul s [155], and once again e ealing
he esis ance o he i al en elope o SARS-CoV-2.
Figu e 3.4: a) ypical F-D cu e in which a pene a ion in he en elope is isible; b) dis ibu ions o he de ec ed
b eak h ough o ces in he h ee condi ions, [Hyp]=1μM; c) image o he i al pa icle (indica ed wi h he o ange
a ow) be o e inden a ion and d) a e 15 inden a ion cycles.
88.00 nm
0.00 nm
88.00 nm
0.00 nm
-60 -40 -20 0 20 40 60 80
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Fo ce (nN)
Inden a ion (nm)
B eak h ough
no hyp hyp da k hyp ligh
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
B eak h ough o ce (nN)
a
b
c
d

110 | P h D h e s i s - M a e o M a i a n g e l i
Typical F-D cu es we used o he analysis wi h a clea b eak h ough a e ep esen ed in Figu e
3.4a; analogous cu es se ed o he analysis depic ed in he cha o Figu e 3.4b. Appa en ly
he Fb o he pu e i us yielded a alue o a ound 500 pN (Table 5) e lec ing a e y so laye , in
acco dance wi h he phenomenology o en eloped i uses which was p e iously discussed.
[Hyp]
N° o cu es
B eak h ough o ce
0 μM
34
(0.54 ± 0.03) nN
1 μM in da k
71
(1.05 ± 0.06) nN
1 μM in ligh
36
(0.48 ± 0.04) nN
Table 5: he esul s o he analysis showed in Figu e 3.4b a e summa ized he e.
In he p esence o Hype icin, we obse e a change in he sample's p ope ies, which a ies unde
ligh and da k condi ions. In iguingly, his end seems o align wi h ou p e ious indings o
bilaye s (sec ion 2.3.2), whe e Hype icin only caused he i us o s i en in he absence o ligh .
Howe e , he e is conside able a iabili y in he sample, as e idenced by he da a dispe sion o
he "Hyp in da k" condi ion shown in Figu e 3.4b, and on he numbe o cu es analyzed, as
de ailed in Table 5. As men ioned ea lie , b eaking he i uses, o inding in ac i al pa icles was
no always possible, leading o a disc epancy in he numbe o cu es ac oss he h ee condi ions.
Ideally, we would ha e a simila numbe o cu es o each condi ion o equally accoun o da a
luc ua ions.
Howe e , he dispe sion o he da a showed in he Hyp in da k condi ion, could also be ela ed
o he une en dis ibu ions o he numbe o Hyp molecules in he i ions, simila ly o wha we
epo ed in Figu e 1.4b. In ha scena io, he dis ibu ion o luo escence in ensi y was
inhomogenous, indica ing ha ce ain i ions con ained a highe numbe o Hyp molecules, while
o he s had ewe , o a same concen a ion. The obse ed dec ease in dispe sion upon
illumina ion migh be connec ed o he phenomena desc ibed in Sec ion 2.3.2 ega ding he
up u e o bilaye s. I is possible ha Hyp has a mo e signi ican s uc u al impac in da kness
han unde ligh exposu e. In he la e case, p ocesses such as lipid pe oxida ion and he
b eaking o double bonds migh come in o play, po en ially al e ing he plas ic ma e ial's
p ope ies o a s a e simila o when Hyp is absen .
Las ly, by conduc ing imaging be o e and a e he inden a ion cycles, we ound ha he pa icles
did no disassemble pos -b eak h ough, which is qui e ema kable Figu e 3.4c, d). This migh
sugges ha he mechanical damages in lic ed on he ex e nal laye o he SARS-CoV-2 i uses
could be e e sible [160].
111 | P h D h e s i s - M a e o M a i a n g e l i
3.3 Conclusions
F om he da a collec ed in ou expe imen s, we ha e ob ained some ini ial insigh s in o he
beha io o hea -inac i a ed SARS-CoV-2 i al pa icles when subjec ed o mechanical s ess. The
a igue expe imen s sugges ha he i us has an ex emely adap able s uc u e, which aligns
wi h cha ac e is ics ypical o highly in ec ious i uses. The ini ial esul s upon applying Hype icin
indica e he po en ial o moni o induced changes in he i us's s i ness, hough hese indings
a e s ill a a p elimina y s age. The ex en o which hea inac i a ion may nega i ely a ec ou
measu emen s emains unclea ; he sample's a iabili y cu en ly p e en s us om making
conclusi e s a emen s abou he e ec s o Hyp on i s nanomechanical p ope ies. Thus,
addi ional da a a e needed o de elop a mo e comp ehensi e unde s anding and o se he
sample's he e ogenei y.
3.4 Ma e ials and me hods
Sample p epa a ion. The SARS-CoV-2 s ock o i al pa icles we e p epa ed in he BSL3
labo a o ies a Is i u o Zoop o ila ico Spe imen ale della Lomba dia e dell’Emilia Romagna in
B escia, simila ly o wha we discussed in 1.6.5. The inac i a ion was pe o med by hea -
inac i a ion a 65°C o 30 minu es in MEM cul u e medium. The samples we e nei he ul a-
cen i uga ed no esuspended in PBS. The i al suspensions we e s o ed a -80°C un il he
measu emen s. The p o ocol used o he p epa a ion o he samples was he ollowing:
1. Applica ion o 40 μL o poly-L-lysine on o a eshly clea ed mica piece (0.5 cm x 0.5 cm),
which was p e iously a ached o a ci cula me allic suppo wi h a diame e o 18 mm.
2. Wai ing o 15 minu es, hen insing ho oughly wi h ul apu e MilliQ wa e h ee imes.
A e each inse, a ni ogen gas low was used o d y he su ace.
3. To dispe se any agg ega e, he Eppendo con aining he i al pa icles was o exed i e
imes, wi h each o exing las ing o 5 seconds.
4. Placemen o 50 μL o he o exed pa icles on o he mica su ace.
5. A e ano he 15 minu es, addic ion o 20 μL o TRIS bu e (5 mM) mixed wi h NaCl (150
nM) a pH 7.4, o main ain app op ia e osmo ic p essu e and pH le els.
6. Following 15 minu es, h ee inses wi h he same bu e o emo e all una ached i ions
and deb is, and addic ion o he igh amoun o liquid o he measu emen (usually 40-
50 μL).
Fo he expe imen s wi h Hyp, we p epa ed he Eppendo wi h he i us by adding he PS so ha
he o al concen a ion would ha e been 1 μM. We le i incuba e in Eppendo o 15 minu es.
a e his passage, we p oceeded wi h he same p o ocol.
112 | P h D h e s i s - M a e o M a i a n g e l i
3.4.1 Imaging
The jumping mode in wa e was used o he expe imen s. We chose he ec angula qp-BioAC
can ile e s om NanoAndMo e, cha ac e ized by a nominal sp ing cons an o 0.1 N/m and ip
adii o less han 10 nm. The calib a ion p ocess ollowed was he same as desc ibed in sec ion
2.7.4, esul ing in ypical sensi i i y alues a ound 6-7 nm/V. As wi h p e ious expe imen s, bo h
da a acquisi ion and analysis we e pe o med using he WSxM ee so wa e. The images we e
cap u ed a a esolu ion o 128 pixels wi hin e y small ields o iew (200-500 nm) and using e y
low o ces (less han 100 pN) o p ese e he in eg i y o he i ions. We conduc ed he a igue
measu emen s unde he same condi ions wi h o ces anging om 50 o 150 nN.
3.4.2 Nanomechanics
Fo he nanomechanical measu emen s, we began by selec ing an app op ia e i ion and
cen e ing he ROI on he midpoin o he pa icle. We hen pe o med F-D cu es wi h he aim o
ob aining a leas one b eak h ough e en . These cu es s a ed 40 - 60 nm away om he
sample. The o al z-piezo displacemen leng h a ied depending on he i al pa icle’s size,
ypically anging be ween 100 – 160 nm. We eco ded hese a esolu ions o ei he 128 o 256
pixels, wi h speeds be ween 100 – 150 nm/s. A e he se o F-D cu es, we pe o med an image
o he i al pa icle o assess i s in eg i y, checking i damage, collapse, e c. had occu ed.
The analysis in ol ed selec ing only he clea b eak h ough o ces, which cons i u ed less han
50% o he o al cu es acqui ed. We i s calib a ed he cu es in WSxM o ob ain he ac ual ip-
sample dis ance. Then, he b eak h ough o ce alues (FB) we e plo ed in O iginP o o analyze
he dis ibu ions.
Fo hese measu emen s, we used he same sou ce o i adia ion as in he expe imen s wi h he
bilaye s ( e e o 2.7.4).
113 | P h D h e s i s - M a e o M a i a n g e l i
Conclusions and u u e pe spec i es
O e all, his hesis p esen ed a comp ehensi e s udy on he in e ac ion and he e ec s o he
pho osensi ize Hype icin wi h a model o en eloped i us, i.e. SARS-CoV-2, and lipid bilaye s
esembling he i al en elope. We employed mul iple biophysical echniques, ei he op ical o
scanning p obe me hods, also used in a co ela i e way.
When Hyp is bound o SARS-CoV-2 and exposed o ligh , i demons a es signi ican an i i al
e ec s by gene a ing eac i e oxygen species. In iguingly, e en in he absence o ligh , Hype icin
induces an an i i al esponse, sugges ing an addi ional mechanism. This seconda y mechanism,
while equi es a highe concen a ion, appea s o in ol e Hype icin embedding in o he i al
en elope, po en ially al e ing i s luidi y.
Ul ima ely, Hyp eme ges as an e ec i e an i i al agen ope a ing bo h ligh -dependen and
independen mechanisms, each equi ing u he cha ac e iza ion o a comp ehensi e
unde s anding.
Fo his pu pose, we employed Suppo ed Lipid Bilaye s (SLBs) ha simula e i al en elopes.
We obse ed concen a ion-dependen de imen al e ec s o Hype icin on SLB mo phology,
pa icula ly in p e-exis ing de ec egions unde ligh exposu e. We also no ed simila e ec s in
de ec - ee bilaye s, bu a highe concen a ions. We p o ided de ailed insigh s in o he
deg ada ion p ocess, including lipid ex ac ion and he appea ance o a dense lipid phase,
h ough high-speed AFM imaging. Such e ec consis ed in he appea ance o a dense lipid
phase, likely due o he edis ibu ion o choles e ol wi hin he bilaye , al hough he exac
mechanism is no ully unde s ood.
We e ealed he spa ial o e lap o Hype icin luo escence signals and lipid a s wi hin he bilaye
was e ealed h ough co ela i e AFM and con ocal imaging. In his way we e ealed he spa ial
o e lap be ween Hype icin luo escence signals and lipid a s. The di usion o Hype icin in he
bilaye was emphasized as he luo escence signal an ico ela ed wi h lipid a s o e ime. In he
absence o ligh , mo phological e ec s we e less p onounced, p ima ily al e ing he shape o lipid
a s o appea mo e ugged and ac al-like.
Nanomechanical p ope ies o he bilaye we e analyzed by measu ing he Young modulus and
he b eak h ough o ce. Such expe imen s e ealed ha Hyp inc eases he igidi y o he bilaye ,
which aligns wi h he hypo hesis men ioned ea lie . This inc ease in igidi y was obse ed wi h
and wi hou ligh in he case o he Young modulus, bu no in he b eak h ough o ce
expe imen s. This disc epancy sugges s ha Hyp a ec s he elas ic (inden a ion) and plas ic
(pene a ion) p ope ies o he bilaye di e en ly.
To u he cha ac e ize he e ec s o Hyp on he mechanical p ope ies, we aim o es i s impac
on iscosi y using FCS o FLIM wi h lipid esicles as a model sys em. In ac , FLIM can be ca ied
120 | P h D h e s i s - M a e o M a i a n g e l i
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