1
A Gene al Pe spec i e o he Cha ac e iza ion and Quan i ica ion
o Nanopa icles. Imaging, Spec oscopic and Sepa a ion
Techniques
A. Lap es a-Fe nández,a* A. Salinas-Cas illo,a S. Ande son de la Llana,b J.M. Cos a-
Fe nández,c S. Domínguez-Meis e ,d R. Cecchini,e L.F. Capi án-Vall ey,a M.C. Mo eno-
Bondi, M.-Pila Ma co,g J.C. Sánchez-López,d and I. S. Ande son h
a ECsens, Depa men o Analy ical Chemis y, Campus Fuen enue a, Uni e si y o G anada,
18071 G anada, Spain
b Hôpi aux Uni e si ai es de Genè e, Se ice de Néona ologie e des Soins In ensi s
Pédia iques, Depa emen de l’En an e de l’Adolescen , ue Willy Donzé 6, Genè e ue
Micheli du C es 22, Gene e, GE, CH 1205 Swi ze land.
c Depa men o Physical and Analy ical Chemis y, Facul y o Chemis y, Uni e si y o
O iedo, 33006 O iedo, Spain.
d Ins i u o de Ciencia de Ma e iales de Se illa (CSIC – Uni . Se illa), A da. Amé ico
Vespucio 49, CIC Ca uja, 41092-Se illa, Spain
e Consiglio Nazionale delle Rice che - Is i u o pe lo, S udio dei Ma e iali Nanos u u a i,
(CNR-ISMN) - ia P. Gobe i 101, 40129 Bologna, I aly
Op ochemical Senso s and Applied Pho ochemis y G oup (GSOLFA), Depa men o
Analy ical Chemis y, Uni e sidad Complu ense, CEI-Moncloa, 28040 Mad id, Spain
g Nanobio echnology o Diagnos ics g oup (AMRg), IQAC-CSIC, CIBER de Bioingenie ía,
Bioma e iales y Nanomedicina (CIBER-BBN), Jo di Gi ona 18-26, 08034 Ba celona, Spain
h Neu on Sciences Di ec o a e. Oak Ridge Na ional Labo a o y. Building 8600, MS 6477,
Oak Ridge, TN 37831, USA
* Co esponding au ho : [email p o ec ed]
2
ABSTRACT
This e iew gi es an o e iew o he di e en echniques used o iden i y, cha ac e ize and
quan i y enginee ed nanopa icles (ENPs). The s a e-o - he-a o he ield is summa ized, he
di e en cha ac e iza ion echniques ha e been g ouped acco ding o he in o ma ion hey can
p o ide. In addi ion some selec ed applica ions a e highligh ed o each echnique. The
classi ica ion o he echniques has been ca ied ou acco ding o he main physical and
chemical p ope ies o he nanopa icles such as mo phology, size, polydispe si y
cha ac e is ics, s uc u al in o ma ion and elemen al composi ion. Mic oscopy echniques
including op ical, elec on and X- ay mic oscopy and sepa a ion echniques wi h and wi hou
hyphena ed de ec ion sys ems a e discussed. Fo each o hese g oups, a b ie desc ip ion o
he echniques, he speci ic ea u es and concep s, as well as se e al examples a e desc ibed.
Table o con en s
ACRONYMS AND ABBREVIATIONS .................................................................................. 3
1. In oduc ion ............................................................................................................................ 5
2. Mo phology, size and polydispe si y cha ac e is ics ............................................................. 6
2.1. Mic oscopy echniques .................................................................................................... 6
2.1.1 Hyd a ed, liquid o gas samples by elec on mic oscopy ........................................ 12
2.1.2 C yo-elec on mic oscopy ....................................................................................... 16
2.1.3 C yo-elec on omog aphy ...................................................................................... 16
2.1.4 Scanning p obe mic oscopy: AFM ......................................................................... 17
2.1.5 X- ay mic oscopy (XRM) ....................................................................................... 18
2.2 Spec oscopic echniques ............................................................................................... 20
2.2.1. X- ay and neu on spec oscopy ............................................................................. 24
2.3 Ch oma og aphy and ela ed sepa a ion echniques. Size exclusion ch oma og aphy
(SEC), capilla y elec opho esis (CE), hyd odynamic ch oma og aphy (HDC) and ield-
low ac iona ion (FFF). ...................................................................................................... 25
2.4 Cen i uga ion, il a ion and dialysis echniques. ......................................................... 30
3. S uc u al in o ma ion and chemical composi ion. Single o hyphena ed echniques ......... 31
3.1 X- ay spec oscopy ......................................................................................................... 32
3.1.1 Non hyphena ed X- ay echniques .......................................................................... 33
3.2 O he echniques coupled o EM .................................................................................... 35
3.3 X- ay mic oscopy (XRM) .............................................................................................. 36
3.5. AFM and B unaue -Emme -Telle echnique (BET) ................................................... 38
3.6. Spec oscopic echniques o chemical iden i ica ion ................................................... 39
4. Elemen al composi ion and concen a ion. .......................................................................... 41
4.1 Mass spec ome y (MS) ................................................................................................ 41
4.2 Ch oma og aphy and ela ed sepa a ion echniques. ...................................................... 43
Acknowledgemen s .................................................................................................................. 44
3
ACRONYMS AND ABBREVIATIONS
AFM A omic o ce mic oscopy
ADF Annula da k- ield
AE Auge elec on
AES Auge elec on spec oscopy
AgNPs Sil e nanopa icles
AUC Analy ical ul acen i uga ion
AuNPs Gold nanopa icles
BF B igh ield de ec o
BP B eakdown p obabili y
BSEs Backsca e ed elec ons
CE Capilla y elec opho esis
CEND Cohe en elec on nanodi ac ion
CFF C oss low il a ion
CFM Chemical o ce mic oscopy
CFUF C oss low ul a il a ion
CHA Concen ic hemisphe ical analyze
CLSM Con ocal lase scanning mic oscopy
CMA Cylind ical mi o analyze
CNT Ca bon nano ube
CRM Con ocal Raman mic oscopy
CuNPs Coppe nanopa icles
CXDI Cohe en X- ay di ac ion imaging
DLS Dynamic ligh sca e ing
DF Da k ield de ec o
DIMs Di ac ion imaging mic oscopes
DMA Di e en ial mobili y analysis
EDAX/ EDS/ EDX Ene gy dispe si e X- ay spec oscopy
EDXRF Ene gy dispe si e x- ay
EELS Elec on ene gy-loss spec oscopy
EFTEM Ene gy- il e ed TEM
EM Elec on mic oscopy
EPR Elec on pa amagne ic esonance
ESEM En i onmen al SEM
ESI Elec osp ay ioniza ion
ETEM En i onmen al TEM
EXAFS Ex ended X- ay Abso p ion Fine S uc u e
FCS Fluo escence co ela ion spec oscopy
FE Field emission
FFF Field- low ac iona ion
FIB-SEM Focused ion beam SEM
FTIR Fou ie ans o m in a ed spec oscopy
GISAXS G azing incidence SAXS
HAADF High-angle ADF
HDC Hyd odynamic ch oma og aphy
HPLC High pe o mance liquid ch oma og aphy
HRTEM High- esolu ion ansmission elec on mic oscopy
ICP-MS Induc i ely coupled plasma-mass spec ome y
LDI Lase deso p ion/ioniza ion
LIBD Lase induced b eakdown de ec ion
LIBS Lase induced b eakdown spec oscopy
LIF Lase -induced luo escence
LSPR Localized su ace plasmon esonance
MALDI Ma ix assis ed lase deso p ion/ioniza ion
MALS o MALLS Mul i angle (lase ) ligh sca e ing
MEMS Mic o-elec omechanical sys ems
MoNPs Molybdenum nanopa icles
MWCNT Mul i-walled ca bon nano ubes
4
NEXAFS Nea -edge X- ay abso p ion ine s uc u e
NMR Nuclea magne ic esonance
NPs Nanopa icles
NSOM Nea - ield scanning op ical mic oscopy
PCS Pho on co ela ion spec oscopy
PEELS Pa allel acquisi ion sys ems
PIMs P ojec ion imaging mic oscopes
POM Polyoxome ala es
P NPs Pla inum nanopa icles
QDs Quan um do s
RPLC Re e sed-phase liquid ch oma og aphy
SAED Selec ed A ea Elec on Di ac ion
SAM Scanning Auge mic oscopy
SANS Small-angle neu on sca e ing
SAXS Small angle X- ay sca e ing
SEC Size exclusion ch oma og aphy
SEM Scanning elec on mic oscopy
SERS Su ace-enhanced Raman sca e ing
SEs Seconda y elec ons
SLS S a ic ligh sca e ing
SPM Scanning p obe mic oscopy
SPR Su ace plasmon esonance
STEM Scanning TEM
STM Scanning unnelling mic oscope
STXMs Scanning ansmission X- ay mic oscopes
SWCN Single-walled ca bon nano ubes
SXM Scanning X- ay mic oscopes
TAD Thin annula de ec o
TADBF Thin annula de ec o used wi h b igh - ield
TADDF Thin annula de ec o used wi h da k- ield
TEM T ansmission elec on mic oscopy
TPL Two-pho on luminescence
TOF Time o ligh
TSEM SEM in ansmission mode
TXMs T ansmission X- ay mic oscopes
WAXS Wide angle X- ay sca e ing
WDS Wa eleng h dispe si e X- ay spec ome y
WDXRF Wa eleng h dispe si e Fluo escence X- ay spec ome y
XANES Nea -edge x- ay abso p ion spec a
XAS X- ay abso p ion spec oscopy
XFEL X- ays om upcoming ee-elec on lase s
XMCD X- ay magne ic ci cula dich oism
XPS X- ay pho oelec on spec oscopy
XRD X- ay di ac ion
XRF X- ay luo escence
XRM X- ay mic oscopy
XRR X- ay e lec ome y
Z A omic numbe
5
1. In oduc ion
Nowadays, enginee ed nanopa icles (ENPs) can be syn hesized ou o a la ge a ie y o
di e en ma e ials and possess a wide ange o p ope ies including size, shape, physical o
chemical cha ac e is ics and elemen al composi ion. Fu he mo e, in e ac ions wi h he
su ounding media can de e mine he colloidal s abili y o he pa icles, hence in luencing
hei inal s a e. The e o e, unexpec ed p ope ies and in e ac ions which a e no solely
dependen on elemen al composi ion and s a e o agg ega ion,1,2 bu a e mo e likely o be
go e ned by su ace a ea,3 su ace chemis y,4 cha ge pa icle numbe ,2 size and size
dis ibu ion,5 agg ega ion,6 s uc u e7 and shape5,8 can lead o complex nano-sys ems whose
p ope ies di e om simple model heo ies.
This e iew deals wi h he use o di e en echniques, used ei he indi idually o in
combina ion, o iden i y and cha ac e ize NPs depending on he desi ed p ope y o be
s udied. Conside ing size dis ibu ion, deg ee o agg ega ion and mo phology, imaging
echniques based on di e en ypes o mic oscopies (op ical, elec onic, X- ay) and sca e ing
(ligh , X- ay, neu on) echniques a e desc ibed along wi h ch oma og aphy and ela ed
sepa a ion echniques. Elemen al composi ion, s uc u al in o ma ion, and concen a ion a e
ypically p o ided by spec oscopic echniques (e.g., UV- is abso p ion, luminescence and X-
ay emission). Combining a ious cha ac e iza ion echniques (e.g., elec onic mic oscopy
coupled wi h X- ay de ec o s) can p o ide in o ma ion ha canno be ob ained om each o
he echniques indi idually. These so called hyphena ed echniques can lead o a mo e
comple e cha ac e iza ion o NPs.
In he ollowing sec ions a classi ica ion o di e en analy ical echniques ega ding he
p ope ies o he NPs, such as mo phology, size, polydispe si y cha ac e is ics, s uc u al
in o ma ion and elemen al composi ion is p esen ed along wi h some ela ed examples o
applica ion.

6
2. Mo phology, size and polydispe si y cha ac e is ics
As men ioned abo e, NPs show di e en p ope ies depending on hei mo phology and size.
Nowadays, he knowledge on ma e ials composed o nanome e -sized en i ies is la gely
ela ed o elec on mic oscopy (EM) and sca e ing phenomena de e mining size and shape
cha ac e is ics. Fu he mo e NPs sepa a ion has long been a key s ep in cha ac e izing
complex polydispe se nano/mic oma e ials. Al hough se e al analy ical echniques can be
used o hese cha ac e iza ion pu poses,9 all a ailable EM me hods a e limi ed by spa ial
esolu ion and/o he ela ed de ec ion limi s.
2.1. Mic oscopy echniques
Since he nanopa icle is a nano-objec wi h all h ee ex e nal dimensions in he nanoscale, he
de ec ion limi o op ical mic oscopy does no o en co e he NPs size since he isible
wa eleng hs (λ) a e compa able o he dimensions o he pa icles. To sol e his limi a ion, a
modi ied echnique called nea - ield scanning op ical mic oscopy (NSOM) can be used in i s
di e en con igu a ions. NSOM is a scanning p obe mic oscopy echnique (SPM)10 wi h a
ypical esolu ion be ween 50 and 100 nm, eaching 20 nm and 2-5 nm o la e al and e ical
spa ial esolu ion, espec i ely,11 and e en sub-20 nm o s ipped gold py amids.12 I can be
used o image agg ega es o nanopa icles wi h a heo e ical spa ial esolu ion limi ed o λ/2
(see below). To de ec luo escen NPs, con ocal lase scanning mic oscopy (CLSM) is
applied wi h esolu ions o he o de o 200 nm13 and i enables he isualiza ion o he
di e en accumula ion o polyme ic NPs in ime a (sub)-cellula esolu ion in umou s.14
Fluo escence imaging is es ic ed o in insically luo escen NPs o luo escen ly labelled
NPs. The la e echnique can in oduce po en ial p oblems such as: label ins abili y
(leaching), modi ica ion o physicochemical p ope ies o pho obleaching.15 Con ocal Raman
mic oscopy (CRM), which does no equi e labelling, allows, o example, he isualiza ion o
7
oxicological e ec s o polyme ic NPs in HepG2 cells and cellula NP up ake.16 Mo eo e , by
using he su ace plasmon esonance (SPR) o me allic NPs (e.g., gold nano ods) i is possible
o image indi idual pa icle loca ions wi h op ical mic oscopy by using Two-Pho on
Luminescence (TPL) and da k- ield op ical mic oscopes.17 The la e echnique p o ides a
means o cha ac e izing he size o gold NPs (AuNPs) by colou , since di e en colou s such
as iole , blue, g een, and ed a e co ela ed wi h a size o 30, 50, 70, and 90 nm,
espec i ely.18
Fluo escence co ela ion spec oscopy (FCS) uses con ocal op ics (a con ocal mic oscope)19
o e alua e di usion coe icien s o luo escen NPs20 o NPs wi h luo escen coa ings whose
luo escence luc ua ions depend on he NP shape.21 FCS allows o de e mina e size
dis ibu ions in a e y small lase illumina ed olume ( ocal olume o app ox. 10−15 µL) o
e y dilu ed solu ions (10−8 o 10−15 mol·L-1).
Elec on mic oscopy (EM) echniques including ansmission elec on mic oscopy (TEM),
and scanning elec on mic oscopy (SEM); and scanning p obe mic oscopies (a omic o ce
mic oscopy (AFM)22) a e pa icula ly sui able o he cha ac e iza ion o NPs due o hei
in insic subnanome e scale esolu ion. These echniques a e widely applied o NPs
isualiza ion.19,23-25 EM echniques equi e acuum condi ions (~10-4 Pa) o allow bo h he
ope a ion o he elec on sou ce and o minimize sca e ing o he han om he samples. The
wa eleng h o elec on beams depends on he accele a ion ol age and is much sho e han
he wa eleng h o ligh , hence allowing much highe spa ial esolu ion down o single a oms
in some cases. Thus, o an op ical mic oscope ope a ing a wa eleng hs, λ om 400 o 800
nm and nume ical ape u e (NA) o ~ 1, Abbe’s di ac ion limi d = λ/2NA, gi es a
esolu ion o app ox. 200 nm26 up o 10 nm wi h some imp o emen ,27 while o an EM
wo king wi h an accele a ion ol age om 100 o 1000 keV, he heo e ical alue o he
wa eleng h will be om 0.0037 o 0.00087 nm28 (See Table 1). Ne e heless, in he cu en
8
s a e-o - he-a , he bes TEM esolu ion, by using double abe a ion co ec ion, is 0.047 nm29
while SEM mic oscopes achie e esolu ions in he ange o 1 nm (high esolu ion) o 3 nm
(con en ional).28 In TEM, he elec on beam (80-200 keV) passes h ough he sample and
only NPs wi h a su icien combina ion o elec on densi y and size can be de ec ed, being
imp ecise o co es smalle han 2 nm. A his poin , high- esolu ion-TEM (HRTEM, 300
keV) eaches a 0.047 nm spa ial esolu ion (see abo e), can image he c ys allog aphic
s uc u e,30 e.g., edge leng h dis ibu ions o image 3D s uc u es by combining di e en
angula iews, ( he elec on di ac ion pa e ns can also p o ide s uc u e in o ma ion (see
sec ion 3)) and con ibu es o cla i y he di e en beha iou o NPs om he bulk and ee
molecula s a e.31 Fo biological ma e ials, accele a ion ol age is limi ed up o 100 keV, due
o he damage caused in he sample by highe ene gy beams.
SEM p oduces images o he su ace (o nea su ace) o a sample by scanning i wi h a
ocused beam o elec ons (1 - 30 keV) and de ec ing he a ious signals ha a e p oduced
when he elec on beam in e ac s wi h elec ons in he sample. The di e en signals ha a e
de ec ed (seconda y elec ons (SE), back-sca e ed elec ons (BSE), cha ac e is ic X- ays,
ligh (ca hodeoluminescence (CL)), and ansmi ed elec ons) p o ide complemen a y
in o ma ion conce ning he nea su ace s uc u e o he sample), hough a e a ely a ailable
all oge he . Acco dingly, in o ma ion o he su ace opog aphy is gi en by low ene gy
seconda y elec ons (SEs), which a e e y sensi i e o he sample cha ge and con amina ion.
Wi h high-ene gy backsca e ed elec ons (BSEs), a mapping con as based on di e ences in
a omic numbe o elemen s as low as 0.128 is p o ided; consequen ly good con as o hea y
and ligh elemen s (e.g., AgNPs in cells) is ob ained.32 Depending on he elec on sou ce used
i is possible o change he spa ial esolu ion, o ins ance, a low b igh ness ungs en elec on
sou ce ypically p o ides 50-100 nm esolu ion while b igh e sou ces such as LaB6 ilamen s
allow a esolu ion below 5-10 nm. Using ield emission guns (sha ply-poin ed Mülle - ype
9
emi e ), he elec on beam is mo e cohe en eaching mo e in ense beam o high ene gy han
in con en ional he mionic emi e s such as ungs en and LaB6 ilamen s. Table 1 shows he
usual ol age ange o SEM and TEM wi h he ela ed ela i is ic and non- ela i is ic
elec on wa eleng h calcula ions.
Table 1
Con en ional SEM can be ope a ed in ansmission mode (TSEM)33,34 a low ol age (10
keV) o dimensional cha ac e iza ion o NPs wi h a minimum size o he o de o 10 nm.
He e, he de ec ion o ansmi ed elec ons can imp o e he signal- o-noise a io, when
compa ing wi h de ec ion modes ha use only BSEs o SEs ( he la e elec on de ec ion is
less sensi i e o he cha ge o he NPs.35
In a ocused ion beam SEM (FIB-SEM), an ion beam subs i u es o he inciden elec on
beam and ei he seconda y elec ons o seconda y ions, o bo h, can be de ec ed and used o
imaging. The echnique is des uc i e in ha i spu e s he su ace. Resolu ion is no as good
as SEM, bu using he milling and analysis echnique i is possible o ob ain a dep h image in
p inciple, 3D.36 Focused cha ged pa icles (elec ons, ions, and mo e ecen ly clus e s) can be
used in scanning mode o each sub-10 nm esolu ion allowing he obse a ion o he milling
p ocess in si u. This echnique is applied in a wide ange o nanos uc u es o pho onic
pu poses (e.g., semiconduc o NPs, ca bon nano ubes (CNT)).37
Some common d awbacks a e known when using TEM o SEM:
(i) hey may be des uc i e echniques ei he due o sample p epa a ion o beam damage.
(ii) Only allow he sample o be analyzed once (pa icula ly in he case whe e he sample
may be deg aded by he beam, wi h he implica ion ha i may be necessa y o adjus
beam ene gy (and hence esolu ion) o mi iga e damage.
16
2.1.2 C yo-elec on mic oscopy
To a oid dehyd a ion a i ac , NPs a e kep ozen by c yo-TEM and c yo-SEM using a c yo-
s age in he mic oscope unde high acuum. Thus, he glu a aldehyde o sample
immobiliza ion o chemical ea men (bo h in ol ing sample al e a ion) a e no necessa y
because o he physical ixa ion by eezing.28 C yo-TEM can each a esolu ion o 0.2 nm
imaging he gold plane (111).68 In AgNPs, he la ice inges spacing (0.207 nm) is also
isualized in ionic liquid media (due o i s low apou p essu e).69 A esolu ion o 0.6-0.8 nm
70 can be eached when imaging AuNPs-p o ein a ays in 3D. Replacemen eac ions can also
be isualized, e.g., he elec os a ic ci a e shell is subs i u ed by sel -assembled
polyoxome ala es (POM) monolaye s on he 5 nm AuNPs su ace.71 Wo king wi h c yo-SEM
echnique72 (3-5 keV, -160 ºC, using SE and BSE imaging modes) i is possible o ob ain a
esolu ion om 1 o 3 nm28 imaging he cellula in e naliza ion o polyme ic NPs (250-300
nm).
2.1.3 C yo-elec on omog aphy
C yo-elec on omog aphy (c yo-ET) is an eme ging s uc u al echnique which uses a
combina ion o bo h c yo-TEM and omog aphic me hods. He e, he NPs a e imaged by
sec ions in h ee dimensions (3D) o an app oxima e 5 nm esolu ion.73 This echnique
p ese es samples a liquid-ni ogen empe a u es, so he cells eeze wi hou needing
chemical ixing o addi ional s aining.74 Ta eau e al., ha e used C yo-ET o obse e he
nuclea ion and g ow h o polys y ene (PS) nodules on 170 nm silica seeds 75 being use ul o
isualize he g ow h o AgNPs by ca hodic spu e ing in ionic liquid.69 C yo-ET p o ides high
esolu ion ega ding in o ma ion o pleiomo phic s uc u es76 o silica and AuNPs up ake by
liposomes.77 To obse e he NPs in biological samples ( equen ly 80 keV o less), highe -
han-no mal mic oscope ol ages (200–400 keV) a e ypically used o ob ain highe specimen

17
pene a ion wi hou losses in elec on de ec ion. The hickness o a 300 keV elec on in
o ganic ma e ials is app oxima ely 0.35 µm.73 Se e al examples o NPs measu ed wi h
di e en de ices a e shown in Figu e 1.
FIGURE 1
2.1.4 Scanning p obe mic oscopy: AFM
A omic o ce mic oscopy (AFM), is a echnique ha belongs o he b oade amily o
scanning p obe mic oscopies (SPM).78 In a can ile e (a e y sha p p obe ip) scans he
sample and allows imaging o NPs (su ace s uc u e) in hei na u al en i onmen wi h
a omic esolu ion (app ox. 0.5 nm o DNA heigh ).13 I is also possible o apply AFM o non-
conduc i e samples in a wide ange o media, including nanoma e ials in liquid condi ions
(en i onmen s), hence o e coming he limi a ion o scanning unneling mic oscopy (STM) in
which only conduc ing samples can be imaged.79 AFM esolu ion is limi ed by he size and
geome y o he ip, which can be la ge han he nanoma e ial being s udied and hence
leading o an o e es ima ion o he la e al dimension o he NP. The e a e se e al modes o
AFM, (i) con ac , (ii) non-con ac 80 and (iii) apping scanning o in e mi en mode.81 Con ac
mode ( equen ly used in s a ic mode: wi hou equency modula ion) is he mos
con en ional imaging mode whe e he can ile e is de lec ed as i mo es o e he su ace. The
ip is cons an ly adjus ed o main ain a cons an de lec ion o " ead" he sample opog aphy.
Due o he d agging mo ion o he ip, biomolecules o objec s ha a e weakly a ached o he
subs a e su ace can be damaged o emo ed82 and may e en ually s ick o he can ile e ,
hence leading o possibili y o imaging a i ac s. In an a emp o minimize hose undesi able
e ec s, (ii-iii) non-con ac and apping mode ( ypically wi h equency modula ion), whe e
he ip only ouches he su ace momen a ily, hence minimizing he physical con ac , we e
18
de eloped. In AFM di e en subs a es a e used such as mica, clean silicon (Si) and
chemically ea ed silicon o deposi NPs. AFM allows he analysis o pa icles in hei own
en i onmen 23 e.g., na u al aqua ic colloids83 in di e en ypes o wa e such as wa e shed
( apping mode),84,85 d inking wa e ,24 o o image a size ac iona ion o na u al aqua ic NPs.86
AFM is also success ully applied o he cha ac e iza ion o TiO2, Z O2 and Al2O3 NPs,87
semiconduc o NPs such as CdSe/ZnS wi h 5–8 nm diame e s and colloidal AuNPs wi h 15
nm diame e .88 Fu he mo e, AFM has been used o s udy he e ec s o unc ionalized NiNPs
on cance cells89 and he in luence in he mo phology o he c oss-linke agen in
he mo esponsi e Au@NIPAM NPs.90 AFM can also gi e chemical in o ma ion abou he
sample (see sec ion 3.5). Table 3 shows some o he p ope ies ela ed o he echniques used
o cha ac e ize NPs in nea na i e s a e by usual EM and AFM wi h some examples o
applicabili y.
Table 3
2.1.5 X- ay mic oscopy (XRM)
Mic oscopy echniques based on X- ay pho ons can no only p o ide in o ma ion on chemical
composi ion and binding (See sec ion 3) bu also on s uc u e and mo phology. As wi h EM
echniques, he spa ial esolu ion achie able wi h a mic oscope is limi ed by he wa eleng h
o he adia ion and he op ical elemen s used. We can classi y wo ypes o mic oscopes
depending on whe he ocusing op ical elemen s a e used o ocus he ligh by di ac ion o
no . The ypical ocusing elemen o X- ay wa eleng hs is a F esnel zone pla e. Mic oscopes
ha do no equi e ocusing op ics a e classi ied as lensless p ojec ion imaging mic oscopes
(PIMs) o di ac ion imaging mic oscopes (DIMs).91 As he e a e no lenses be ween he
sample and he de ec o , highe esolu ion can be ob ained, bu i is limi ed by bo h he
wa eleng h o he inciden beam (synch o on X- ay sou ce) and by he s ong decay o he
19
di ac ion in ensi y.92 In biological samples, hese echniques also allow good esolu ion (11-
13 nm) when labelling cells wi h AuNPs o 1.8 nm as used by Nelson e al..93 3D images can
also be aken by high-con as cohe en X- ay di ac ion pa e ns o Ag/Au nanoboxes (>100
nm) wi h spa ial esolu ion o 4.2 nm92 by Takahashi e al., while New on e al., ob ained 3D
images o ZnO nano ods wi h 40 nm o spa ial esolu ion.94 By using cohe en X- ay
di ac ion imaging (CXDI), whe e o e sampled di ac ion pa e ns can be in e ed o ob ain
eal space images, he ace ed mo phology o Pb nanoc ys als95 and single ZnO nano ods96
ha e been imaged. P e iously Isaacs e al., showed a di ac ion pa e n o a 160 nm sil e
cube.97 Recen ly, he same esea ch eam, ha e been able o measu e 400 nm diame e gold
nanoc ys al in highe esolu ion. The h ee-dimensional mo phology and e olu ion o he
s ain unde p essu es up o 6.4 GPa we e ob ained wi h be e han 30 nm spa ial esolu ion.98
Unde low p essu e, he edges o he nanoc ys al became s ained as expec ed, while unde
highe comp ession he s ains disappea ed, sugges ing ha he p essu ised ma e ial is
unde going "plas ic low".
Two ypes o mic oscopes using op ic elemen s a e a ailable: ull- ield ansmission X- ay
mic oscopes (TXMs) and scanning ansmission X- ay mic oscopes (STXMs). TXM is
mainly used o imaging while scanning X- ay mic oscopes (SXM) a e used as an elemen al
analysis echnique.99 By he end o he 90´s, he s uc u e o humic subs ances was isualized
in si u o liquid samples eaching a esolu ion o 43 nm.100 NPs p epa a ion such as ixa ion,
s aining o sec ioning a e no necessa y since he o ganic ma e o some NPs (e.g., i on
oxides NPs) p o ides a much highe X- ay abso p ion han wa e . Al hough he TXM beam
has highe ene gy han in STMX, causing mo e sample damage, i is mo e sui able han
STMX o omog aphic measu emen s since i akes as e images.91 (STXM acquisi ion akes
app oxima ely 2 min pe image). TXM is used o in-si u obse a ion o he sul ida ion
p ocess (hollowing e ec ) om Cu2O c ys als o Cu2S cages in o liquid phase o he i s
20
ime (pho on ene gy ange o 8–11 keV).101 On he o he hand STXM pe mi s he analysis o
s uc u al changes due o agg ega ion e ec s in magne i e NPs (50 nm)102 and he mo phology
o co e-shell semiconduc ing polyme NPs (shell hickness a ound 10 nm and a co e o 50–60
nm).103 Quan i a i e composi ional maps can be ob ained by nea -edge X- ay abso p ion
spec a (XANES) (see sec ion 3.1). Finally, 3D images wi h a esolu ion om 40 nm104 up o
20 nm105 a e ob ained by in e sion o he cohe en X- ay di ac ion.
Wi h lowe pho on ene gies ( om ~250 eV (~5 nm wa eleng h) o 1.8 keV (~0.7 nm)),
ypically called so -XRM, he X- ays each a condense zone pla e gi ing a pa ially cohe en
hollow-cone illumina ion. This zone pla e, in combina ion wi h a cen al s op and a pinhole,
p o ides a monoch oma ic beam on a CCD came a. A spa ial esolu ion below 15 nm is
o e ed.106 So -XRM in liquid media using a we sample chambe (pho on ene gy o 430 eV)
gi es a 40 nm esolu ion when imaging CdTe nanowi es and hype b anched PbS
nanoc ys als.107 Ex emely low ene gies (ca. 100 eV), used in So X- ay STXM, a e use ul
o in es iga ions o ac inide dioxides o u anium (UO2), nep unium (NpO2) and plu onium
(PuO2). This echnique also allows nea -edge X- ay abso p ion ine s uc u e (NEXAFS) o
elemen al mapping o ac inide elemen s (see sec ion 3.1) and imaging wi h 30 nm spa ial
esolu ion.108 Finally, esolu ion a in e a omic scale is possible by applying in ense bu b ie
pulses o X- ays om upcoming ee-elec on lase s (XFEL)105 ex ending he X- ay
mic oscopy o he em osecond ime domain. Ne e heless, FEL pulses a e e y b igh and
could in ol e sample des uc ion.
2.2 Spec oscopic echniques
The in e ac ion be ween NPs and he elec omagne ic adia ion (elec omagne ic wa es,
pho ons o elec on beams) p o ides unequi ocal cha ac e is ics. These cha ac e is ics in ol e
composi ion in o ma ion (See sec ion 3), as well as NPs size and size dis ibu ion. La e wo
21
a e p esen ed in his sec ion and can be s udied by spec oscopy (mos ly h ough sca e ing).
Di e en sou ces o ligh such as lase , X- ays o neu ons can be used. By using a lase , ligh
sca e ing helps de e mine pa icle size in a sho ime (minu es). The con as in sca e ing
ligh a ises om he di e ence o he e ac i e index be ween he pa icle and wa e . To
de e mine he size dis ibu ion p o ile and NPs agg ega ion in suspension, dynamic ligh
sca e ing (DLS),109 also called pho on co ela ion spec oscopy (PCS) o quasi-elas ic ligh
sca e ing, is used. Fluc ua ions in he sca e ed ligh measu ed in DLS, can be o igina ed
om bo h (i) he B ownian mo ion and (ii) om he cons uc i e o des uc i e in e e ences
o he sca e ed ligh om he neighbou ing pa icles. Al hough i is a apid and simple
me hod, his echnique should be used in combina ion wi h ano he echnique such as SEM o
TEM110 because o agg ega es o dus , ha can lead o unde es ima ion o o e es ima ion o
esul s, in ol ing a limi a ion in he in e p e a ion, (especially o polydispe se sys ems).111
The e o e, i is no possible o sepa a e ou he con ibu ions coming om NPs o di e en
sizes in he o al popula ion o he o al co ela ion unc ion. This is because he signal om
la ge pa icles domina es o e ha o smalle ones. Hence ma hema ical con e sions o
olume o numbe dis ibu ions om he in ensi y da a, de i ed o DLS, can be e oneous and
should only be p ope ly p o ided wi h good knowledge o he pa icle shapes, polydispe si y
and unde lying assump ions.112 DLS is widely applied o he de e mina ion o a e age size,
size dis ibu ion and polydispe si y o e.g., AgNPs,113 AuNPs below 2 nm,114 coa ed i on
oxide NPs115,116 o luo escen polyac ylamide NPs.117 Mo eo e , DLS measu emen s can be
used o moni o he a ia ion o he hyd odynamic diame e o he e mo esponsi e polyme ic
NPs (see igu e 2).
FIGURE 2

22
S a ic ligh sca e ing (SLS), also called mul i angle (lase ) ligh sca e ing (MALS o
MALLS), equi es cleane o su icien ly dilu ed samples compa ed o DLS. He e, he
expe imen al a iable is he ime-a e age in ensi y o sca e ed ligh while in DLS i is he
luc ua ions in ligh in ensi y. SLS p o ides in o ma ion on pa icle mo phology o ins ance
in suspension o iaxial cellulose nanoc ys als isola ed om co on.118
Lase induced b eakdown de ec ion (LIBD) allows o de ec bo h mean pa icle size and hei
concen a ion (See Sec ion 3.6) in aqueous samples. He e, a lase beam wi h a ixed pulse is
ocused on he colloids eaching high empe a u e in ol ing he dissocia ion o he ma e ial
(b eaks down) in o exci ed ionic and a omic species. The analy ical signal ( he b eakdown
p obabili y (BP)) can be ela ed wi h he size o he NPs.119 LIBD esolu ion is down o he
10 nm ange.120 In o de o no sa u a e he b eakdown p obabili y, mos samples ha e o be
dilu ed, making i impossible o disc imina e be ween di e en ypes o NPs. The mos
equen ly used de ec ion sys ems a e CCD came as (in ensi ied (ICCD), non-in ensi ied
(CCD121)) o piezoelec ic c ys als. As example, colloidal pa icles o hexa alen u anium
(205-215 nm) can be de e mined.
Su ace-enhanced Raman sca e ing (SERS) is a su ace sensi i e echnique which measu es
he enhancemen o Raman signals o Raman-ac i e species due o he p esence o NPs.
Di e ences in he in ensi y o SERS signals e eal di e ences in mo phologies and sizes
(e.g., be ween Ag nanosphe es, nano ods and nanos a s122 o be ween Au nanop isms
showing a highe Raman enhancemen han nano ods).123 SERS was also applied o de ec
biocompa ible and non oxic PEGyla ed AuNPs o in i o umou a ge ing.124 No iceable is
he con ibu ion o he Liz-Ma zán g oup in SERS ield.125-127
UV- is spec oscopy allows o de e mine no only composi ion in o ma ion bu also he NPs
size and shape om he abso bance spec a.128 The NPs size can be de e mined by s udying
he shape and posi ion o he localized su ace plasmon esonance (LSPR) peak.129 Some
23
examples a e now discussed. In he case o gold, he SPR o sphe ical AuNPs smalle han 2
nm is placed a 505 nm130 while o 15-20 nm in size i is placed a 520 nm and o 100 nm i
is a ound 575 nm. The longi udinal SPR mode o di e en Au nano ods aspec a io (leng h:
wid h) is placed om 650 o1000 nm. On nano ods he SPR band obse ed spli s in wo
modes due o he di e en o ien a ions o he od in espec o he elec ic ield o he inciden
ligh . Thus elec on oscilla ion is p oduced ac oss ( ans e se mode) and along (longi udinal
mode) he long axis o he nano od. The ans e se SPR mode (a a ound 500 nm) only shows
a mino shi when he Au nano od aspec a io a ies. In con as , he longi udinal SPR mode
is signi ican ly a ec ed by he modi ica ion o he aspec a io.131 Simila ly, sphe ical AgNPs
p esen an abso bance band placed a 420 nm while Ag nano ods o e wo bands placed a
420 and 615 nm.122 Mo eo e , UV- is has been used o de ec biomolecula in e ac ions
aking place a he su ace o he noble me al nanopa icles.132 Thus, he SPR peak is also
in luenced by he e ac i e index a he adjacen su ace in e ace o he nanopa icle, and his
is a ec ed by bio uncionaliza ion o by binding in e ac ions. An o e iew o unc ionalized
gold nano ods used in a a ie y o analy ical and biomedical applica ions is e iewed in
de ails by Mannelli e al..133
UV- is spec oscopy can be easily combined wi h di e en echniques such as
ch oma og aphic sys ems o enable size sepa a ion134,135 o e en wi h MALLS o he same
pu pose.136 In combina ion wi h SEM, SAXS and XANES (see sec ion 3), UV- is
spec oscopy has been applied o obse e he size beha iou o AuNPs du ing di e en
syn hesis p ocedu es.137
Finally, nuclea magne ic esonance (NMR) can be used o measu ing he size o me al NPs
encapsula ed wi hin dend ime s (di e ing in size by jus a ew ens o a oms)138 as well as
he size o colloidal ma e .13
24
2.2.1. X- ay and neu on spec oscopy
O he sou ces o ligh such as neu ons and X- ays a e used o de e mine sizes nea (0.01–1
nm) and (1–1000 nm), espec i ely. Small o wide angle X- ay sca e ing (SAXS o WAXS)
use lexible X- ay sca e ing o p o ide in o ma ion o monodispe se samples abou shape,
size and s uc u e. The size dis ibu ion is possible only in polydispe sed samples ia he o m
ac o , P(q). SAXS does no show e y good con as be ween elemen s ha a e close in he
pe iodic able since he con as in X- ay sca e ing a ises om he a ia ion in elec on
densi y wi hin he sample. In ha case, i is possible o cause damage o he sample as a esul
o he la ge amoun o ene gy applied (pa icula ly a synch o on sou ces). The NPs diame e
and he size dis ibu ion a e ob ained om a sca e ing pa e n. Wi h nea monodispe sed
pa icles, he sca e ing pa e n o he X- ay shows concen ic ings wi h a diame e di ec ly
ela ed o he mean diame e o he NPs. SAXS has been employed o obse e how he silica
NPs in e ac ions a e dec eased by he p og essi e dissolu ion o he e ap opylammonium
hyd oxide unc ionalized su ace (dec easing he su ace cha ge when he NPs size
dec eases).139 This echnique is also use ul o e eal co e-shell s uc u es, e.g., in polyme
coa ed i on oxide nanoc ys als due o he high di e ence in elec on densi y be ween he co e
and he polyme shell.140 Suspensions o polyme hyl me hac yla e (PMMA) NPs wi h low
polydispe si y and diame e s o 108 nm and 192 nm also ha e been imaged by SAXS.141 X-
ay e lec ome y (XRR) combines small angle sca e ing wi h he e lec ion geome y o
ob ain dimensional p ope ies o nanos uc u ed su aces. He e, by g azing incidence SAXS
(GISAXS) s uc u al pa ame e s o 10 nm AuNPs142 a e de e mined. X- ay sca e ing
coupled wi h UV- is128 allow o measu e he mean size o AuNPs om he sca e ing da a
wi hin 10% e o .143 SAXS also enables online analysis o he nuclea ion and g ow h o
AuNPs a oiding unce ain ies in he in e p e a ion o UV- is spec a.137 This echnique is also
used o ollow he g ow h o hiol-co e ed AuNPs a di e en eac ion imes.144 Long
25
exposu e imes a e used o ob ain high-quali y da a se s (≈1 h) while sho e imes
p edomina e in case o high sca e ing in ensi y o he sample (e.g., sizing dis ibu ion o
AuNPs ( e e ence ma e ials)).145
By small-angle neu on sca e ing (SANS), he con as a ises om he di e en changes in
he ene gy o he neu ons om di e en NPs (densi y and composi ion) and he magne ic
momen s o a oms in ol ing a possible de e mina ion o bo h he chemical and he a e age
spa ial dis ibu ions o magne ic, e.g., Fe3O4 NPs.146 Mo eo e , i has been applied o he in-
si u de e mina ion o he mean co e diame e , he ligand leng h, and ligand sol a ion om
dodecano hiol-AgNPs in gas expanded liquids.147 Combining SANS and SAXS, i is possible
o measu e he size o ca boxylic acid modi ied zi conia (Z O2) NPs in di e en sol en s.148
Table 4 displays some o he ea u es ela ed o he echniques used o cha ac e ize NPs
depending on o size, s uc u e and mo phology among wi h some use ul applica ions.
Table 4
2.3 Ch oma og aphy and ela ed sepa a ion echniques. Size exclusion ch oma og aphy
(SEC), capilla y elec opho esis (CE), hyd odynamic ch oma og aphy (HDC) and ield-
low ac iona ion (FFF).
NPs size cha ac e iza ion can be made by di e en sepa a ion echniques such as size
exclusion ch oma og aphy (SEC), hyd odynamic ch oma og aphy (HDC), capilla y
elec opho esis (CE) and ield- low ac iona ion (FFF). All hese echniques can be applied
wi h o he echniques o de ec ion de ices o u he sample analysis.
SEC is a widely used echnique o NPs isola ion. The column is packed wi h po ous packing
ma e ials, which o m he low channels. Pa icles which ha e a diame e smalle o equal
han he po e size o he packing ma e ials can pe mea e deep inside he column, while la ge
pa icles can only ans e h ough bigge po es o be excluded o ex a-pa icula egion. This
32
imaging echniques o gi e elemen al and s uc u al in o ma ion. In he ollowing sec ions,
se e al indi idual o hyphena ed echniques a e discussed along wi h some applica ions o
cha ac e ise NPs by hese echniques.
3.1 X- ay spec oscopy
The gene a ed X- ays om a sample a e cha ac e is ic o each elemen o a e ela ed o he
a omic s uc u e. The e o e, he use o X- ay spec oscopic echniques enables us o de ec
sub le di e ences in a omic s uc u e o chemical bond. These X- ays a e ob ained when an
ou e elec on placed in a highe -ene gy shell is ans e ed o a lowe ene gy le el o ill he
inne elec on hole ha has been exci ed by an inciden elec on beam. Ene gy dispe si e X-
ay spec oscopy (EDS, EDX, XEDS o EDAX,) iden i ies he a omic composi ion o he NPs
by he gene a ion o X- ays. EDX can be hyphena ed o TEM o SEM o analyze he chemical
composi ion o e.g., dus NPs,193 P @Fe2O3 co e-shell NPs194 o e en o de e mine he
possible an ibac e ial e ec o sil e ions.195
The combina ion o STEM and HAADF is sui able o iden i y s uc u al a ia ions o he
AgPd@P NPs composi ion196 o he concen a ion o gold in AuNPs using, in e es ingly, hai
ib e as nano eac o .197
In ully liquid condi ions, i is also possible o de elop elemen al analysis by using liquid
sample holde s o mic ochips. EDS coupled wi h liquid-TEM helps con i ming he g ow h o
P nanoc ys als by coalescence as an al e na i e o simple g ow h54 and he in si u o ma ion
o ellu ium NPs by mic oo ganisms (see sec ion 3.1.1.). In special condi ions such as high
empe a u es and gaseous a mosphe es, he isualiza ion o P NPs on Al2O3 is also possible.66
Coupled wi h we -SEM, he elemen al iden i ica ion o Au, TiO2, ZnO and Fe2O3 NPs is
eached.6 Mo eo e , EDS coupled wi h ESEM has been used o moni o he biomedical

33
e ec s o se e al NPs such as me als (Ni, Co), ce amics (TiO2 and SiO2), and PVC64 in a s
muscles.198 Figu e 4 displays he EDX analysis o o Au/Ag alloy NPs imaged by TEM.
FIGURE 4
Wa eleng h dispe si e X- ay spec ome y (WDS, also called WDXRF when using X- ay
luo escence) measu es he X- ays coming om a speci ic and di ac ed wa eleng h o he
NPs. WDS is mo e p ecise han EDX and shows se e al imp o emen s such as (i) a be e
ene gy esolu ion (ii) a peak- o-backg ound capabili y o de ec smalle amoun s o
elemen s199 and (iii) a be e de ec ion o ligh a omic numbe (Z) elemen s. Combined wi h
STEM,200 he composi ion o Molybdenum NPs (MoNPs) can be gi en. As single echnique,
i is use ul o he de e mina ion o Bismu h composi ion as esidual syn hesis componen o
magne ic me al oxide nanoc ys als.201 Fu he mo e, i is used o iden i y he esidual B – and
Cl– capping agen s on palladium nanowi es in he ca alysis o ace ylene.198
3.1.1 Non hyphena ed X- ay echniques
X- ay pho oelec on spec oscopy (XPS) measu es he kine ic ene gy o pho oemi ed
(ejec ed) elec ons when i adia ing a sample wi h ocused so X- ays ( ypically below 1,5
keV).202 As he X- ay pho on ene gy is known, i is possible o de e mine he elec on binding
ene gy and iden i y he a omic co e-le el whe e he elec on was ex ac ed. XPS is use ul o
cha ac e ize NPs su aces and coa ings (e.g., he e olu ion in he composi ion o Au/Cu NPs
along he ime in ca alysis).202 I is also used o he iden i ica ion o encapsula ed AuNPs in o
Cu2S nanocages101 and e en o checking di e en chemical s a es o elemen s p esen in NPs
(e.g., he concen a ion o Au(I) in Au@Au(I) NPs and Ag in silica nanocomposi es).203,204
In X- ay di ac ion (XRD),140 X- ay beams a e p ojec ed o e he NPs in a de e mined angle,
called he a “θ”, and he di ac ed X- ays a e collec ed in an angle o 2θ (See Table 5). XRD
34
allows s uc u al phase iden i ica ion o he NPs c is allini y (e.g., be ween magne i e and
maghemi e NPs)115,116 gi ing also he a e age pa icle size by using he Sche e equa ion.
Fu he mo e, di e en chemical s a es and elemen al composi ion (e.g., Ag in o silica
nanocomposi es204 o in nanocubes205) can be p o ided.
X- ay luo escence (XRF)206 esul s om he a om-localized emission when inciden X- ays
ha e highe ene gy han he ioniza ion po en ial o he a oms in he NPs. Di e en ypes such
as Wa eleng h X- ay sepa a ion (WDXRF) and ene gy dispe si e X- ay (EDXRF)13 can be
used. Elemen al composi ion o nanoma e ials such as Ru-P bime allic NPs207 o ZnO
nanopla es208 is ob ained.
(Table 5)
X- ay abso p ion spec oscopy (XAS) s udies local a omic a angemen s when in ense and
collima ed X- ay beams (synch o on adia ion) each he sample (See Figu e 2). XAS can be
di ided in ou ene gy egions: (i) p e-edge egion < (ii) " ising edge" e e ed o XANES <
(iii) NEXAFS < (i ) Ex ended X- ay Abso p ion Fine S uc u e (EXAFS) (co esponding o
he sca e ing o he ejec ed pho oelec on o neighbou ing a oms). The combina ion o
XANES and EXAFS is called XAFS. NEXAFS measu emen s allow u he cha ac e iza ion
o he o ganic ma e ial, iden i ying phenolic, ca boxylic and ca bonyl ca bons. This speci ic
cha ac e iza ion is possible due o he ac ha each ype o ca bon is assigned o di e en eV
peaks.24 This imp o es he TEM-EDX analysis whe e all hese analy es a e g ouped as only
one ca bon peak. XAS is used o de ec small di e ences in he s uc u e o colloidal
nanoc ys als209 which can be di icul o disce n using EM and XRD.206 Ne e heless,
s uc u al e alua ion o bime allic NPs (Ru-P NPs)207 is cla i ied by using EXAFS, XANES
and XRD.7 Mo eo e , i is possible o moni o di e en eac ion s eps137 by checking bo h he
35
NPs su ace passi a ion and he di e en oxida ion s a es o CdSe quan um do s.210 Mo eo e ,
la ex pa icles a e also imaged.211
In e es ingly, using a pho oemission elec on mic oscopy, elec onic and magne ic p ope ies
o single FeNPs (6-25 nm) can be measu ed wi h XAS and X- ay magne ic ci cula dich oism
(XMCD) spec a.212
3.2 O he echniques coupled o EM
Elec on ene gy-loss spec oscopy (EELS) is based on he ene gy analysis o he sample’s
inelas ically sca e ed elec ons ( ansmi ed elec on beam). EELS p o ides in o ma ion on
he elec onic s uc u e, su ace p ope ies, oxida ion s a es and chemical composi ion a an
a omic o subnanome e scale. EELS spec a a e in luenced by bo h he coo dina ion
chemis y (en i onmen su ounding he a om) and he alence s a e o he a omic species
(a omic co e elec on exci a ions). Pa allel acquisi ion sys ems (PEELS) allow he
simul aneous collec ion o da a o e a ange o ene gy losses. Elemen al analysis is possible
o mos elemen s, al hough in p ac ice, he quan i ica ion is mos ly applicable o he ligh e
elemen s wi h Z g ea e han 3. PEELS has supe io de ec ion e iciency o low Z elemen s
compa ed wi h EDX (gene ally be e sui ed o de ec ing elemen s o high Z). Bo h
echniques show clea ad an ages o iden i ying and analysing highe Z componen s. EELS
and EDX a e complemen a y echniques and can be used o map composi ion in 2D o 3D.213
Coupled wi h TEM o STEM hey a e success ully applied o elemen al analysis (e.g., o he
oxide shell on he su ace o FeNPs)214 o o di e en ia e ca bon nano ubes by me allic o
semiconduc ing na u e.215 Wi h he aim o inc easing he ene gy- esolu ion in elemen al
analysis by SEM-EDX (usually limi ed be ween 100 and 150 eV and nea ly wo o de s o
magni ude la ge han he ene gy esolu ion o EELS in TEMs/STEMs) a success ul
36
combina ion be ween SEM and EELS is de eloped o acqui e s uc u al in o ma ion wi h 4
eV o ene gy- esolu ion.216
EELS hyphena ed o STEM38 is use ul o ob ain maps o he SPR modes o single molecule
SERS-ac i e nanos uc u es.217
Ene gy- il e ed TEM (EFTEM) o ms images using only elec ons coming om a pa icula
ene gy-loss. EFTEM is use ul o ligh e elemen s218 as well as o dis inguish di e en
ma e ials in composi es (e.g., Nylon (22 eV)) om he nano ube (27 eV).213
Rega ding nanodi ac ion echniques, wi h STEM i is possible o ob ain nanodi ac ion
pa e ns which p o ide nanos uc u e and mo phology in o ma ion om indi idual
nanocomponen s by cohe en elec on nanodi ac ion (CEND). CEND is he only echnique
ha gi es ull di ac ion in o ma ion abou indi idual NPs, and is applied o ins ance o
AgNPs smalle han 3 nm.38
Selec ed A ea Elec on Di ac ion (SAED) pai ed wi h TEM o STEM gi es in o ma ion
abou c ys alline p ope ies o NPs,23 e.g., pa e n o a omic Fe3O4 magne ic laye deposi ion
on ca bon nanocoils,219 and allows o ob ain a omic o de in o ma ion. Mo eo e , compa ing
wi h X- ay di ac ion, bigge sample ields can be moni o ed.150 All echniques desc ibed
om sec ions 3.1 o 3.2 a e shown in Table 6, among wi h se e al cha ac e is ics and
applica ions.
3.3 X- ay mic oscopy (XRM)
XRM uses so X- ay beams o image he samples, allowing bo h abso p ion and luo escence
signals o map he elemen al and chemical composi ion o he NPs. XRM eaches a esolu ion
be ween op ical mic oscopy and EM in he so-called wa e window. Two ypes o
mic oscopes a e used in X- ay mic oscopy (i) ull- ield ansmission X- ay mic oscopes o
s andalone and synch o on sou ces (TXM and labo a o y TXM (LTXM) whe e expe imen s
37
can be pe o med in he use 's home lab) mainly used o imaging and pa icula ly well sui ed
o omog aphy, and he (ii) scanning X- ay mic oscope (STXM), mainly used o
mic oanalysis and magne iza ion wi h high spa ial esolu ion. In bo h echniques, F esnel
zone pla es lenses o di ac i e and spa ially ocused cohe en X- ays a e used. By TXM, i
is possible o each 3D images o Ag/Au nanoboxes (up 100 nm) no ob ained by X- ay
omog aphy wi h lenses no a om p obe mic oscopy o elec on omog aphy.92
STXM can be used o moni o he p esence o zinc in human cells exposed o ZnO NPs,220 as
well as o p o ide spa ially esol ed chemical s a e in o ma ion o FeNPs221 o o cha ac e ize
ac inide pa icles (100 o 1000 nm).108 When combined wi h NEXAFS, he elec onic and
s uc u al p ope ies ob ained by chemical mapping allow o dis inguish co e-shell s uc u es
in polyme NPs.103 Di e en CNTs ( om onion like o ca bon NPs) and nano ubes
syn hesized by di e en g ow h me hods222 can also be dis inguished. No ice ha he
analy ical signal is 100-1000 imes smalle o STXM han o echniques based on
spec omic oscopy such as TEM-EELS, dec easing he adia ion damage (See Table 6).222
Figu e 5 displays a schema ic ep esen a ion o elec onic and X- ay mic oscopes.
FIGURE 5
3.4. Auge elec on and Mössbaue spec oscopies.
A highly su ace-sensi i e echnique such as Auge elec on spec oscopy (AES) o e s
in o ma ion abou he su ace opog aphy and he su ace composi ion o he NPs by
collec ing o analyzing Auge elec ons (AE) emi ed om he NPs su ace (simila
in o ma ion is ob ained wi h seconda y elec ons, SE). When an inciden elec on beam
exci es an inne elec on, an ou e elec on can mig a e o he c ea ed oid in he inne shell.
In consequence, ene gy is emi ed, which is di e en o X- ay emission, and i is ans e ed

38
o a hi d elec on on a u he ou e shell, leading o i s ejec ion. AES was used o e alua e
su ace oxida i e chemis y o AuNPs3 and combined wi h SEM o STEM allows also
chemical-composi ion analysis.150 In STEM, sub-nanome e su ace de ails can be obse ed a
high- esolu ion SE images. AES can gi e quali a i e and, in some cases, quan i a i e
in o ma ion abou he su ace composi ion o NPs consis ing o mul iple componen s.38 Fo
ins ance, i is applied o di e en ypes o chemical deposi ions such as diamond-like ca bon
on me al subs a es (used as ca alys empla es o he g ow h o CNTs)223 and P ussian-blue
analogues (PBA) NPs ( om 6 o 25 nm) on silicon su aces.224 In Scanning Auge
mic oscopy (SAM)38 an image esolu ion <1 nm can be ob ained which allows o de ec as
ew as 15 sil e a oms in AgNPs o a diame e smalle han 1 nm.225
Mössbaue spec oscopy is based on he esonan abso p ion and emission o gamma ays
gi ing in o ma ion abou physical, chemical and magne ic p ope ies o NPs. Gold-coa ed
magne ic nanopa icles show he abili y o yield high magne ic momen s wi h he simplici y
o bioconjuga ion on he gold su ace. This ype o NPs a e sui able a ge s o be measu ed by
Mössbaue spec oscopy,226 which p o ides de ailed in o ma ion abou magne i e NPs
o ma ion.227
3.5. AFM and B unaue -Emme -Telle echnique (BET)
AFM allows o measu e su ace o ces o he NPs o physicochemical cha ac e iza ion. AFM
helps o iden i y chemical elemen s placed on he NPs su ace by compa ing wi h a omic
o ce pa e ns228,229 o by chemical o ce mic oscopy (CFM)22 whe e unc ionalized p obe ips
a e used.230
BET me hod is used o cha ac e izing he po osi y and su ace a ea o solids, allowing he
de e mina ion o he speci ic su ace a ea by means o gas adso p ion. Fo ins ance, i is
39
applied o measu e he su ace a ea o FeNPs221 and he po e size dis ibu ion in SWCNTs231
as well as o conduc oxici y s udies.232,233
3.6. Spec oscopic echniques o chemical iden i ica ion
LIBS belongs o he a omic emission spec oscopy echniques and consis s in a sho lase
pulse i adia ed on a sample c ea ing highly ene ge ic plasma ha emi s ligh a a speci ic
wa eleng h peak acco ding o he ma e ial. By iden i ying di e en peaks o he analyzed
samples, hei chemical composi ion can be apidly de e mined. The numbe o plasmas
(dielec ic b eakdowns) pe numbe o o al lase pulses and hei spa ial dis ibu ion in he
lase ocus can e eal bo h he colloidal concen a ion and size.24 The LIBS plasmas depend
s ongly on he ambien condi ions, he ma ix e ec s and he signal o noise a io.234 LIBS
eaches a low limi o de ec ion (LOD) in he ange o pp (ng·L-1) allowing o analyze he
NP size be ween 10 nm and 1 μm235,236 as well as he pa icle numbe densi y (concen a ion)
(see sec ion 4) o colloids237 and NPs (20 nm).24
By using double-pulse LIBS, some signals can be enhanced, such as o Al and Ca lines, up o
i e imes compa ed o he single-pulse signal.238
Lase -induced luo escence (LIF) and Raman spec oscopy allow he molecula s uc u e
cha ac e iza ion.13 Elec on pa amagne ic esonance (EPR) de ec s unpai ed elec ons exis ing
in a sample239 and o e s he possibili y o analyze he pa icle su ace eac i i y. Due o he
high sensi i i y o EPR owa ds su ace a ia ions, i is possible o de e mine ligands in mixed
monolaye s which a e co e ing AuNPs and also impu i ies on CdSe QD.240 EPR is la gely
applied o NPs oxici y s udies.241
UV- is spec oscopy allows o ob ain he NPs size and concen a ion (see sec ion 4)
depending on he posi ion o he su ace plasmon peak as Haiss e al., demons a ed o
AuNPs.242 Se e al chemical e ec s, ha modi y he op ical beha iou o small me al
40
pa icles, can be s udied by op ical measu emen s ocusing on he SPR displacemen .243 This
displacemen depends on bo h he su ounding en i onmen o he NPs and he di e en ypes
o su ace unc ionaliza ion.244,245 Mo eo e , he pa icle concen a ion can be measu ed by
using he mola ex inc ion coe icien a he wa eleng h o he maximum abso p ion band
(e.g., in gold colloids).246 He e, due o he di e ence in abso bance i is possible o de e mine
Langmui iso he ms o he eplacemen o ci a e by POM anions a he gold su ace.71 Fo
AgNPs, he SPR shi s owa ds a longe wa eleng h as he sol en e ac i e index
inc eases.247 Depending on he composi ion o Ag/Au nanocages, di e en posi ions o he
SPR o he me al nanocages248 and hollow me al nanos uc u es249 can be obse ed a he
nea -in a ed egion. Simila ly, sil e and gold can be used o o m alloyed NPs which o e
di e en SPR peaks a he isible ange o wa eleng hs (See igu e 6).
Finally, he e ec o bo h humic acids a ached o ci a e-s abilized AuNPs and he pH can be
obse ed by he educ ion/displacemen o he gold plasmon peak, leading o NPs
agglome a ions.245
FIGURE 6
Fou ie ans o m in a ed spec oscopy (FTIR) is use ul o iden i y chemicals o o de e mine
s uc u al p ope ies e.g., in sil e nanocomposi es.204 FTIR is used o iden i y he ypical
peaks o o ganic and ino ganic NPs,250,251 bu o en p esen ing an o e lap be ween peaks ha
makes di icul o iden i y p ope ly he di e en species o unc ional g oups.115 Changes in
he abso bance bands o he NPs gi e de ails abou composi ion. Fo ins ance, he p esence o
bo ine se um albumin (BSA) shows a ed shi o he AgNPs plasmon peak and he oxida ion
o AgNPs p oduces a b oadening o he band wid h and a dec ease in abso bance.252
41
SERS allows de ec ing single molecules by he enhancemen o he Raman sca e ing signal
( om a molecule) when i is loca ed nex o a nanos uc u ed me al. Gold and sil e
nanoagg ega es wi h a ached epo e molecules can be iden i ied by he Raman
signa u e32,253,254 as well as he ligand con o ma ion on AuNPs.255 In his ield he con ibu ion
o Liz-Ma zán e al. is o ele an in e es .256,257 All hese echniques a e displayed in Table 6.
Table 6
4. Elemen al composi ion and concen a ion.
F om a oxicological poin o iew, in addi ion o all he explained abo e cha ac e is ics,
chemical composi ion is an essen ial issue o NPs iden i ica ion. Speci ic echniques a e
ela ed o cha ac e ize NPs, p o iding chemical composi ion o a ge ed NPs including i any,
he co e ma e ial and/o he su ace laye composi ion. Mo eo e , se e al cases based on he
NPs cha ac e iza ion by hese echniques a e also discussed.
4.1 Mass spec ome y (MS)
I is an analy ical echnique o elemen de e mina ion in which he sample is apo ized,
ionized and measu ed by mass- o-cha ge a io o quan i a i e pu poses. Di e en ioniza ion
modes such as elec osp ay ioniza ion (ESI) and lase deso p ion/ioniza ion (LDI) can be
used. ESI can be used e.g., o he cha ac e iza ion o Au nanoclus e s258 while lase
deso p ion/ioniza ion o indi idual pa icles by so ioniza ion called ma ix assis ed lase
deso p ion/ioniza ion (MALDI) can be applied o de ec p oduc s o ligand exchange
eac ions o he nanopa icles.259 Induc i ely coupled plasma (ICP) uses high empe a u es o
d y, apo ize, a omize, and ionize he sample and gene ally is used o me al analysis. LDI
can be applied o ae osol cha ac e iza ion by ae osol ime-o - ligh mass spec ome e
(ATOF-MS),13 p o iding aluable insigh s o unc ionalized NPs also by using ime o ligh
48
Table 4. X- ay and spec oscopic echniques o cha ac e ize NPs in e ms o size, size
dis ibu ion, s uc u e and mo phology. Some selec ed examples a e discussed.
Technique Main cha ac e is ic
measu ed phenomenon Spa ial
esolu ion commen s Applica ions in
ma e ials science
X-Ray Mic oscopy
XRM X- ays pass h ough he sample owa ds a CCD
de ec o . Fo hyd a ed samples, bo h phase and
ampli ude con as a e maximized when wo king
in he “wa e window,” he spec al egion be ween
he ca bon and oxygen K-shell ene gies.
[O (K: 543 eV) ~ C (K: 284 eV)]
La ge pene a ion dep h o so X- ays in o he
liquid media in compa ison o elec ons.
Be e han 15 nm; spec al ange om a pho on ene gy o 250 eV
(~5 nm λ) o 1.8 keV (~0.7 nm), so p ima y K and L a omic
esonances o elemen s such as C, N, O, Al, Ti, Fe, Co and Ni can
be p obed106.
Resolu ion: 40 nm in s udies o he g ow h o CdTe nanowi es and
hype b anched d ied PbS nanoc ys als 107.
So - XRM
TXM
Nanos uc u e e olu ion om Cu2O c ys als o Cu2S cages in liquid
phase, using a pho on ene gy ange o 8–11 keV 101. Resolu ion:
Cu2S wall hickness o 10–20 nm.
STXM
I ocuses a highly monoch oma ic synch o on X-
ay beam by using F esnel zone pla es. Sample
imaging by scanning he ocal spo o e he sample
and measu ing he ansmission poin by poin
sub-10 nm
S uc u al changes in magne i e NPs (50 nm) due o
agg ega ion a 1 keV 102
Co e (50-60 nm)-shell (10 nm) polyme NPs 103
So X- ay STXM
I ope a es a ambien p essu e emo ing in luence
o acuum, uses ully sealed sample holde s,
equi es a small amoun o sample
Resolu ion a ound 500 nm o ac inide dioxides o U, Np, and Pu
108. Ne e heless, di e ences be ween he appa en size and he
images a e due o sligh ly di e en STXM ocal se ings
CXDI
Sca e ing om he en i e olume o he c ys al a
he B agg e lec ion condi ion will in e e e in he
a - ield, p oducing a h ee-dimensional (3D)
di ac ion pa e n
Measu ed (111) o Pb and (010) o ien a ion o
ZnO nano ods
40 nm G owing Pb nanoc ys als inside he acuum
chambe 95
4.2 nm Ag/Au nanoboxes 92
40 nm ZnO nano ods (7.2 keV co esponding o a
wa eleng h o 1.72 Å) 94,96
30 nm 400 nm diame e gold nanoc ys al 98
11-13 nm 750 eV pho on ene gy. Labelling cell wi h 1.8 nm
AuNPs 93
SAXS X- ay sca e ing depending on he elec on densi y
o he NPs. I equi es in ense, monoch oma ic X-
ays o low di e gence.
I on oxide co e-polyme shell NPs 140. 100-200 nm PMMA NPs
using synch o on adia ion (pho on ene gies om 6 o 10 keV) 141
GISAXS
AuNPs size (10-30 nm)128,142. Wa eleng h ange below 1 nm
co esponds o a pho on ene gy o 1.24 keV, eaching 10 keV using
monoch oma ized synch o on adia ion
Spec oscopic Techniques
DLS Sca e ed ligh due o he in e ac ion be ween he
NPs and he sou ce o ligh is ela ed o he
hyd odynamic NPs diame e
0.15 nm Ag 113 AuNPs 114, polyme ic NPs 278, I on oxide (7-8
nm) silica coa ed NPs 115,116
SLS 5 nm Cellulose nanoc ys als 109
SANS NPs sca e ed beam o neu ons as a unc ion o
sca e ing angle. 0.01 nm
Spa ial dis ibu ions o magne ic momen s o Fe3O4
NPs (9 nm).,magne ic shells 1.0 o 1.5 nm 146.
Dodecano hiol-AgNPs in-si u ligand sol a ion
measu emen s147 and Z O2 NPs 148 (SANS/SAXS).
LIBD Pulsed and ocused lase exci es he sample which
a omically emi s.
< 10 nm-
1000 nm
T aces o hexa alen u anium (U(VI)) colloidal
pa icles 119 e.g., pulsed Nd:YAG lase and pulse
epe i ion a e = 20-100 Hz
UV- is Plasmon peak is ela ed wi h he shape and size o
he NPs
AuNPs o di e en shapes and sizes, including nanosphe es,
nanocubes, nanob anches, nano ods, and nanobipy amids 279
SERS
Inelas ic sca e ing o pho ons om he inciden
lase ligh due o an elec omagne ic and a
chemical enhancemen
SERS due o di e en shapes o Au o Ag NPs 122,123
Gold nano ods as SERS subs a es o de ec ion o sc ambled p ions
127
NMR
1H NMR in eg al alues o he inne mos p o ons
a e modi ied by he NPs
1H NMR expe imen can be used o eadily dis inguish be ween
dend ime s capsula ed NPs 138
a Fo abb e ia ions see ex . A omic dis ances: ~ 0.1 nm

49
Table 5. Hyphena ed echniques in elec on mic oscopy and X- ay de ec o s. In o ma ion
abou s uc u e and elemen al composi ion o NPs (only selec ed examples a e desc ibed)
Technique Main cha ac e is ic
measu ed phenomenon Combined commen s Applica ions in ma e ials science
X-Ray Spec oscopy
EDS, EDX,
XEDS o
EDAX
High-ene gy beam o cha ged pa icles, such as
elec ons o p o ons o a beam o X- ays, is ocused
in o he sample, egis e ing he X- ay spec a.
Elemen al composi ion ela ed o gene a ed X- ays o
an imaged NPs o hei agg ega es can be ob ained
HRTEM NPs composi ion
280
FE-SEM
UHV STEM P @Fe2O3 co e-shell NPs 194
TEM
An ibac e ial e ec s Ag(I) by in e ac ing wi h hiol
g oups in p o eins and inac i a ing he enzyme ac i i y
due he p esence o sulphu 195
ETEM
Liquid-Cell: P NPs g ow h54. By Mic ochip-TEM
TNPs51. Specimen holde allows high empe a u e and
gaseous a mosphe es, P o e Al2O3 66
We SEM Au, TiO2, ZnO and Fe2O3 NPs 6
ESEM Ni , Co, TiO2,SiO2, and PVC 64 in a muscle
STEM
HAADF, mo phology and composi ion o AgPd@P
NPs in ca alysis196 and Au concen a ion in
luo escen AuNPs197
WDS,
WDXRF
Displays X- ay o a single wa eleng h a ime wi hou
gi ing a b oad spec um o wa eleng hs o ene gies
simul aneously as EDX.
STEM MoNPs (25keV)
200
SP
Bi composi ion as esidual syn hesis componen o
magne ic me al oxide nanoc ys als 201. esidual B – and
Cl– capping agen s on Pd nanowi es198
XPS
Kine ic ene gy o pho oemi ed elec ons when
i adia ed a sample wi h ocused X- ays wi h
pho on ene gy ange o 8–11 keV Synch o on 101
SP
Au-CuNPs e olu ion 202. Con i ma ion o Au(I) on
Au@Au(I) 203., chemical s a es o Ag in o silica
nanocomposi es 204 Cu2S nanocage composi ion wi h
encapsula ed AuNPs 101
XRD
Impinging X- ays o e he sample sa is ies he
B agg’s equa ion, cons uc i e in e e ence occu s,
sample o a es in he pa h o he collima ed X- ay
beam a an angle θ while he de ec o o a es 2θ
SP
Magne i e 115 and maghemi e 116 co e composi ion in o
luo escence silica shell NPs Ag nanocubes pa e ns
205, I on oxides composi ion encapsula ed in polyme
140
XRF
Sho X- ay o gamma - ay wi h ene gy highe han
he ioniza ion po en ial o a oms in he sample induces
emission o cha ac e is ic luo escence.
SP Composi ion o Ru-P bime allic NPs 207 o pu i y o
ZnO nanopla es 208
XAS
Inciden pho on in e ac s wi h he elec ons bound in
an a om. The ene gy o he inciden pho ons is
su icien o cause exci a ion o a co e elec on o he
abso bing a om o a con inuum s a e o p oduce a
pho oelec on.
1s: k-edge; 2s, 2p:L-edge; 3s,3p,3d: M-edge
Synch o on
P e-edge; XANES; NEXAFS; EXAFS.
La ex pa icles and mic oballoons dispe sed in wa e
211. S uc u al de e mina ion o Ru-P NPs 207, QD
su ace passi a ion 210
O he s echniques coupled o EM, elemen al in o ma ion
EELS Measu e o he ene gy-loss o inciden elec on when
passing h ough he sample
TEM
Elec onic s uc u e o he oxide shell on he su ace o
FNPs 214. Di e ences in me allic o semiconduc ing
CNTs 215
SEM Ca bon ilm a 30 keV beam ene gy 216
STEM Image SERS ho spo 217
EFTEM
Disc imina ion in a composi e he di e en plasmon
exci a ion ene gies o he nylon (~22 eV) and he
nano ube (~27 eV)
CEND Di ac ion in o ma ion abou s uc u e and
mo phology STEM <3 nm in diame e AgNPs 38
SAED Di ac ion pa e ns o he impinging elec ons due o
he a oms o he sample ac as a di ac ion g a ing TEM
A omic laye deposi ion is applied o coa ca bon
nanocoils wi h magne ic Fe3O4 o Ni 219, SAED pa e n
o Ag nanocubes pa e ns 205
a Fo abb e ia ions see ex . A omic dis ances: ~ 0.1 nm. SP: Spec ome e
50
Table 6. X- ay mic oscopy, spec oscopic echniques and Auge , Mössbaue and BET
echniques. The mos impo an issues ela ed o some NPs applica ions a e desc ibed o each
echnique
Technique Main cha ac e is ic measu ed phenomenon Applica ions in ma e ials science
X-RAY Mic oscopy
STXM Use adia ion in he ene gy ange 250 o 3000 eV, ocused by
F esnel Lens, being hen he sample scanned pe pendicula o he
op ical axis, while he in ensi y o he ansmi ed X- ays is
de ec ed a he same ime.
ZnO NPs in cells 220, down o ~20 nm in ca bon
nano ubes 222, Polyme NPs composi ional maps 103.
Chemical s a e in o ma ion o Fe and ac inide NPs
(100 o 1,000 nm)108.
TXM 3D elec on densi y mapping o Au/Ag nanoboxes ~
100 nm 92
O he echniques
AES
Su ace-sensi i e echnique ela ed o he inelas ic sca e ing ha
occu s o elec ons; Mos o he emi ed Auge elec ons a e
p oduced wi hin a e y sho dis ance om he sample su ace,
ypically 0.3–3 nm.
Ca alys laye s and he CNT sys ems 223
P ussian-blue analogues (PBA) NPs ( om 6 o 25 nm)
o e silicon su aces 224.
Mössbaue
spec oscopy
Resonan abso p ion and emission o gamma ays o ene gy le el
ansi ions gi es in o ma ion abou he a om's local en i onmen Fe3O4 NPs pa hway o ma ion 227
BET Physical adso p ion o gas molecules on a solid su ace.
Speci ic su ace a ea and po e size dis ibu ion o
SWCNTs 231 and on FNPs (gas adso p ion 3 m2·g-1
wi h a diame e (Ф) be ween 38-45 nm) 221
Spec oscopic echniques o de e mine NPs concen a ion
LIBS The s ong hea ing o he sample leads o a ionized ligh (plasma)
ela ed o he chemical elemen s Quan i ica ion o pa icle numbe densi y 24.
LIF Exci ed NPs by a lase and he luo escence spec um is analyzed S uc u al species cha ac e iza ion 13
EPR De ec ion o unpai ed elec on in o ela ed o NPs. Impu i ies on CdSe QDs240
Induced OH- species by i on oxide NPs 241
UV- is
spec oscopy
Posi ion o he UV- is peaks a e ela ed wi h size, shape and NPs
composi ion
Ag/Au nanocages composi ion 248
Hollow me al nanos uc u es 281
FTIR Chemical bonds in a molecule esul s in abso p ion peaks S uc u al p ope ies o e.g., Ag nanocomposi es
204. I on oxide co e-shell silica NPs115
SERS Enhancemen o a Raman in ensi y peak h ough changing he
composi ion and he mo phology o he NPs
Ligand con o ma ion on AuNPs 255
Au nanos uc u es modi ied wi h sil e 256
a Fo abb e ia ions see ex . A omic dis ances: ~ 0.1 nm
51
FIGURE LEGENDS
Figu e 1. (a) and (b) c yo-TEM o silica NPs. (b) I shows an sel -assembly o nanopa icles
in highe -o de and de ined s uc u es in 2D. (c) TEM o magne i e (Fe3O4) NPs. (d)
C yo-TEM o co e-shell NPs (magne ic co e@silica shells). (e) TEM o Ca bon
Do s. ( ) FIB-SEM o Au NPs whe e i can be seen he lase size e ec , in ol ing an
impo an limi a ion o obse e he in e io o he NPs. (g) TSEM o hyb id NPs
(gold@polyme ), (h) TEM image o gold NPs (Φ =30 nm) (i) HRTEM o seed and
g own AuNPs (Φ =100 nm). (j) and (k) a e TEM o Au nano ods and b anched
mo phologies.
Figu e 2. TSEM o polyme ic e mo esponsi e NPs (a) and (b) TEM image o a dilu ion o
NPs imaged by he pic u e "a". (c) Va ia ion o he hyd odynamic diame e o
s imuli- esponsi e polyme ic NPs, wi h empe a u e. Closed and open symbols
deno e he cooling and he hea ing cycles, espec i ely. Con inuous (Cooling) and
dashed (Hea ing) lines co espond o he Bol zmann i o he expe imen al alues.
The inse is a ypical iew o he size dis ibu ion o he NPs by DLS.
Figu e 3. No mal ope a ion mode in FFF. (Rep in ed om publica ion e . 174, Copy igh
(2005), wi h pe mission om Else ie .)
Figu e 4. In o ma ion ob ained by a EDX coupled o a TEM. (a)TEM image o alloy Au/Ag
15:85 a io NPs, (b) co esponding EDX spec um. Peak a (8.04 keV) a e om he
coppe o he suppo g id.
Figu e 5. Schema ic diag am o elec on mic oscopy (EM) and scanning ansmission X- ay
mic oscopes (STXM) oge he wi h gene a ed signals o high- esolu ion images,
nanodi ac ion pa e ns o spec oscopic ela ed o NPs cha ac e is ics.
T ansmission elec on mic oscopy (TEM), scanning elec on mic oscopy (SEM),
ocused ion beam SEM (FIB-SEM), scanning ansmission elec on mic oscopy
(STEM), ene gy dispe si e X- ay spec oscopy (EDX), Auge elec on spec oscopy
(AES) and scanning Auge mic oscopy (SAM), seconda y elec on mic oscopy
(SEM), wa eleng h dispe si e X- ay spec ome y (WDS) elec on ene gy-loss
spec oscopy (EELS), cohe en elec on nano-di ac ion (CEND) and selec ed a ea
elec on di ac ion (SAED). B igh ield (BF) and la ge angle BF (LABF), da k-
ield (DF), annula da k- ield (ADF) and high-angle annula da k- ield (HAADF);
In o STXM: Lensless p ojec ion imaging mic oscopes (PIMs) o di ac ion imaging
mic oscopes (DIMs)
Figu e 6. TEM images o (a) AgNPs ( size a ound 40-60 nm), (b) AuNPs (size a ound 20-30
nm) (c) . U -Vis- spec a o AgNPs (416 nm), AuNPs (521 nm) and alloys NPs
made o di e en Au:Ag a ios, being 15, 25 and 50% con en o Au placed a 426,
442 and 463 nm, espec i ely (sizes 40-60 nm). (d) Obse ed isual ange o Au/
Ag alloy NPs.
52
Figu e 1
53
Figu e 2

54
Figu e 3
55
Figu e 4
56
Cohe en X-Rays
F esnel Zone
Pla es
Fluo escence Pho ons
Pho oelec ons
STXM
X-Ray de ec o
SEM
TEM
Elec on beam
123
FIB-SEM
STEM
BF/LABF
EELS
ADF ADF
HAADF HAADF
CEND
EDX
AEM
SAM
SEM
SAED
WDS
PIM and DIM
Wi hou Zone Pla es
Figu e 5
57
Figu e 6
64
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