A general perspective of the characterization and quantification of nanoparticles: Imaging, spectroscopic, and separation techniques

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. 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