are increasingly being used in a wide range of biomedical applications , from cell 
 biology to medical diagnostics . they have a core diameter of 210  nm and 
 significantly larger hydrodynamic diameter , making them suitable as large yet 
 relatively biocompatible markers , and have remarkable photophysical properties related 
 to quantum confinement effects . their superior brightness , higher photostability , and narrower 
 spectral emission compared to conventional organic fluorophores have 
 progressively lead biophysicists to adopt them as a new tool for 
 single - molecule imaging , in vitro 
 and in vivo . ncs have become an 
 alternative for organic 
 fluorophores and complementary tool of fluorescent proteins in single - molecule 
 fluorescence and whole - cell labelling assays . in this review , we first 
 discuss their unique optical properties , we then introduce some considerations 
 on their surface chemistry and we explore in the following sections the different 
 possible strategies to deliver nc inside the cell and to specifically target 
 them to a protein of interest . finally , we report on recent applications of ncs 
 in whole animal imaging in vivo 
 and address the risk of potential cytotoxicity . ncs are inorganic particles 
 of 200 to 1000 atoms . nc cores are commonly synthesized from group ii - vi ( e.g. , 
 cdse , cds , znse , and cdte ) and iii - v ( e.g. , inas , inp , and pbs ) semiconductor 
 materials . for any energy exceeding the band gap , which depends on the core 
 diameter , absorption of a photon generates an electron - hole pair , which on 
 recombination results in the emission of a less - energetic photon . due to their 
 broad absorption spectra , ncs variations in the particle composition and size result in 
 different band - gap energies and hence ncs different photoluminescent ( pl ) 
 emission , ranging from the near uv to the ir ( 4001350  nm ) . ncs have narrow and symmetric photoluminescence ( pl ) emission 
 peaks with typical full widths at half maximum ( fwhm ) of 2535  nm   that facilitate multicolour imaging by allowing efficient 
 single - colour excitation whilst minimizing emission cross - talk , see   for a critical discussion . unlike with organic dyes , the pl 
 emission arises from the radiative recombination of an exciton . for ncs , 
 relaxation to the ground state takes 10 nanoseconds , about one order of 
 magnitude longer than singlet - singlet electronic transitions in organic 
 fluorophores . the slow pl decay makes ncs attractive sources for time - gated 
 imaging , which can be used to reduce the relative contribution of cellular 
 autofluorescence to the total collected signal . figure 1 graphs the 
 evolution of the collected fraction of long - lived nc emission , relative to that 
 of the short - lived autofluorescence for different time gates t at a fixed lifetime ratio of 1:10 . larger gates are required to 
 attain the same suppression of background for increasing levels of 
 autofluorescence . for intensity - based detection ncs benefit from their large 
 brightness (   ) which results from a 10-to-100 time larger molar extinction coefficients ( 105106  mcm ) than organic dyes [ 7 , 8 ] at 
 comparable quantum yield . finally , due to their significantly higher photostability than 
 organic fluorophores , the 
 resistance to photobleaching results from the deposition of an additional 
 semiconductor shell ( e.g. , zns or cdse ) having a larger band gap than the core . the photophysical properties 
 facilitate lpo at the single - nc level , a particularly interesting property in single - particle 
 tracking ( spt ) applications , 
 tracing cell lineage , and 
 live animal imaging , that all combine the demand for imaging small numbers of 
 fluorophores over extended observation periods . beyond their established 
 function as molecular markers , ncs are increasingly being used for fret - based 
 biosensing ( see   for review ) . ncs are both a scaffold and central donor for exciting 
 multiple organic acceptor fluorophores in these inorganic / organic hybrid fret 
 sensors [ 1316 ] . also , ncs are attractive fret donors because , through selecting the 
 appropriate size , they can be dialed into almost arbitrary acceptors . the large 
 overlap integrals between donor emission and acceptor absorbance allow for 
 larger fret efficiencies or transfer over larger donor / acceptor distances . due 
 to the broad absorption bands and narrow - band emission , one can chose 
 excitation wavelengths minimizing direct acceptor excitation and minimal 
 bleed - through of donor fluorescence into the fret detection channel . at the single - nc level , the 
 radiative recombination of the exciton can temporarily be prevented despite 
 ongoing excitation , resulting in intermittent pl emission , known as  blinking  . blinking results from the stabilization of the exciton at the nc 
 surface and is associated with surface defects . dark states reduce the duty 
 cycle , complicate the interpretation of intensity - based measurements , and prompt the elaboration of 
 specific algorithms for quantitative spt . however , blinking can be 
 turned to an advantage in as much as it allows the identification of single ncs 
 and the detection of single - pair fret ( spfret , figure 2(a ) ) , as shown on panel ( b ) between a qd565stv nc donor and an alexabiotin organic fluorophore acceptor ( yakovlev , luccardini , and others ' personal observations ) . blinking of neighboring 
 ncs can also be used for ultrahigh resolution studies beyond the classical 
 resolution limit   and allows the emission of single particle to be 
 isolated from the crowd . their electron density and 
 crystal structure provide sufficient contrast in transmission electron 
 microscopy ( em ) [ 9 , 20 ] . their use in em is an additional advantage over labelling samples with 
 conventional dyes that need to be photoconverted or require the addition of electron - dense 
 material to generate contrast on em images . however , the contrast obtained with 
 ncs is lower than when using au nanoparticles for immunolabelling . successful cell biological 
 applications of semiconductor ncs had to await the development of reliable 
 protocols for synthesizing water - soluble and colloidally stable nanoparticles . 
 to be of use in cellular imaging , ncs need to be first rendered water - soluble 
 and nonaggregating and then functionalized to be specifically targeted to a 
 molecule of interest . they should also be stable and ideally have a long shelf life 
 as well as to allow 
 for experiment series under reproducible conditions . the time needed to develop 
 potent solubilization and functionalization strategies justifies the time 
 elapsed after the first proposition of ncs as biological probes [ 3 , 22 ] and 
 their wider use by the biological community which is only beginning . ncs are 
 synthesised in organic solvents and are subsequently coated with a hydrophobic 
 shell of surfactant trioctyl phospine oxide ( topo ) to maintain the particles 
 monodispersed in organic solvents . their water solubility is obtained by 
 capping the nc surface with an additional hydrophilic coating layer . among the 
 many solubilization strategies that have been designed the most efficient , in 
 terms of colloidal stability and biocompatibility , is at present the 
 amphiphilic polymer coating [ 2325 ] . particle aggregation can further be reduced through the addition of a polyethylene 
 glycol ( peg ) layer , which also minimizes nonspecific interactions [ 20 , 26 , 27 ] . 
 taken together , the improvements in understanding nc surface chemistry and 
 hence controlling their colloidal properties have prompted an ever increasing 
 number of studies using colloidal semiconductor ncs as pl markers in cell 
 biological applications ( see , e.g. , [ 28 , 29 ] for review ) . the easier accessibility of 
 extracellular 
 epitopes of cellular membrane 
 antigens readily motivates the increasing number of studies using ncs instead 
 of organic - fluorophore conjugated antibodies as extracellular markers in immunofluorescence [ 9 , 30 , 31 ] . different linkers have been used for functionalizing ncs , including 
 streptavidin [ 3234 ] , 
 receptor ligands [ 35 , 36 ] , peptides 
 , as 
 well as secondary   or primary antibodies . the popularity of ncs for studying molecular migration comes , at 
 least in part , from the fact that ncs often offer a viable compromise between 
 the desired stability and the tolerable degree of invasiveness . on the one 
 hand , they are clearly more stable than small organic fluorophores that in turn 
 exert less influence on the bound ligand . on the other hand , over tags offering 
 a comparable long - term stability , such as the much bigger ( and hence invasive ) 
 fluorescent nanobeads or light - scattering gold particles , through their smaller size , so that ncs are less prone to 
 reduce ligand mobility and access to the binding site . despite their obvious 
 advantage for extracellular labelling , four main difficulties are encountered 
 when using ncs for intracellular labelling of cytoplasmic constituents in live cells . first , to deliver ncs into 
 the cell , the plasma membrane has to be made transiently permeable for these nanoscale 
 ( but in a cellular context yet relatively large ) objects , while maintaining the 
 cell intact and viable . second , as ncs are also unspecifically taken up , probably by a 
 process similar to pinocytosis , any specific uptake has to dominate over these 
 nonspecific uptake mechanisms to ensure a specific labelling . pinocytosis 
 occurs in all types 
 of cells , leading to pinosomes which can be bigger than 1  m 
 ( macropinocytosis ) . because their size , macropinosomes provide an efficient 
 route for nonselective endocytosis of solute macromolecules , and hence ncs in 
 solution . third , once the ncs have penetrated 
 the cell , they must stay monodispersed and reach their molecular target through 
 diffusion or transport . however , nanometric hard particles are frequently 
 recognized as exogenous objects and are engulfed in endo-/lysosomal 
 compartments . finally , even in the case of a successful cytoplasmic loading , 
 the main obstacle remains the difficulty in addressing ncs to their specific 
 target sites and in removing the unbound nc fraction from the cytoplasm . whole - cell labelling has 
 been demonstrated with biocompatible , but nonfunctionalized ( bare ) ncs . the addition of ncs to the extracellular medium leads to their spontaneous uptake [ 28 , 42 ] . not only specialized 
 macrophages and fibroblasts but also many cells internalize both extracellular 
 particles and fluid via phagocytosis and pinocytosis , respectively . this uptake leads 
 to endodomes that are much bigger than the ncs itself ( macropynosomes > 1  m , 
 clathrin coated pits 120  nm , caveolae 60  nm , and clathrin- and 
 caveolin - independent endocytosi 90  nm ) . however , these tracks often lead to aggregations of 
 ncs crowded in intracellular compartments ( recognized by the absence of blinking ) . thus , additional and more specific loading techniques are required for specific 
 nc loading . microinjection is a simple 
 tool for loading monodispersed ncs into the cytoplasm [ 10 , 36 ] . dubertret and coworkers injected ncs into xenopus laevis oocytes and traced the cell lineage 
 throughout embryonic development . single - cell electroporation   potentially is another technique for loading charged ncs into individual 
 cells , but its efficiency critically depends on the size and charge of ncs ( luccardini 
 and yakovlev unpublished observations ) . however , similar to patch clamping or microinjection , 
 it is time - consuming techniques ; 
 and more efficient techniques are desirable when the loading of larger cell 
 populations is required . bulk electroporation of cell suspensions allows the 
 parallel delivery of ncs into thousands of cells , but has been reported to go 
 along with nc aggregation [ 36 , 45 ] . this technique probably traps ncs on the plasma membrane where they are 
 endocytoted during the time that is required for the cells to settle on the 
 cover glass before imaging ( luccardini and yakovlev , personal observations ) . thus , the osmotic lysis of pinosomes ( figure 3 , upper panel ) 
 provides a simple and convenient method to efficiently load monodispersed ncs 
 into many cells simultaneously , under identical 
 conditions . during loading , the cell morphology did not change 
 and plasma membrane integrity and cell viability were not affected through the 
 osmotic shock and inclusion of ncs ( figure 3 , lower panel ) . this 
 technique enabled , for example , the loading of ncs to track single kinesin 
 motors in live cells . chemical methods to deliver ncs to the cytoplasm include the use of 
 cationic polymers [ 36 , 45 , 47 ] and cationic 
 lipids [ 10 , 48 ] . after liposome formation , ncs penetrate the plasma 
 membrane , but accumulation in endosomal compartments is frequently observed [ 36 , 39 , 49 ] . also , 
 liposome - loaded ncs have been found in late endosomes / lysosomes 
 , and in keeping with this observation , tend to concentrate in regions 
 close to the nucleus . overcoming nc 
 sequestration , encapsulation of ncs in a peg - grafted polyethylenimine coat another possibility for ncs delivery into the cytosol is their conjugation to specific 
 peptide sequences [ 52 , 53 ] , similar to 
 what has been used for the delivery of magnetic nanoparticles . although this 
 is a particularly interesting and active area of research , and nc translocation 
 to the cytoplasm [ 55 , 56 ] and specific 
 labelling of intracellular organelles such as mitochondria [ 36 , 57 ] or the nucleus [ 36 , 45 ] have been published , the true impact of these studies can only be 
 evaluated with a careful study of the three - dimensional ( 3-d ) intracellular 
 localization of the ncs , for example , combining specific immunostaining and 
 quantitative 3d imaging [ 35 , 58 ] , and careful 
 colocalization analysis . finally , the conjugation of ncs to membrane - permeable toxins like 
 botulinum toxin should represent an attractive strategy to deliver nc into the 
 cytoplasm , although further work needs to confirm these initial observations . in summary , while many different strategies of nc 
 loading have been explored and some of them to produce a monodispersed 
 cytoplasmic labelling at least in the cell types studied , the absence of 
 rigorous criteria for successful cytoplasmic loading and the lack of 
 appropriate controls along with the often uncritical and overoptimistic 
 interpretation of intracellular fluorescent puncta make it hard to be directly extrapolated from the 
 published literature on the own experiment . in principle , if ncs are localized 
 in the cytoplasm rather than sequestered in some intracellular compartment , 
 they should be evenly distributed in and randomly diffused throughout the accessible volume ; in 
 contrast , many images rather show localized distributions and heterogeneous 
 clusters of different sizes 
 and brightnesses . blinking and consistent diffusion coefficients will clarify if particles are monodispersed and trapped 
 or they can diffuse 
 freely . as yet , it seems safe to say that the uptake and internalization of 
 nanoscale particles into cells has not been completely understood and probably 
 varies both from cell type to cell type . also , it depends on the surface 
 chemistry of the nanoparticles . additionally , purification steps could play a 
 crucial role ; for example , in determining the concentration of excess ligands in 
 solution . site - specific labelling of 
 intracellular proteins is far more difficult than extracellular target recognition , 
 since the cytoplasm constitutes a crowded molecular environment , containing a 
 plethora of proteins , nucleic acids , and other molecules . so as to achieve 
 specificity in intracellular targeting , tagging strategies rely on specific 
 target recognition ( reviewed in [ 12 , 59 ] ) . another 
 requirement for lpo imaging is that the chemical bond linking the cytoplasmic 
 target and the label chosen for its detection is stable over the experiment 
 time . it is in response to this 
 need that the tsien laboratory ( university 
  of california , calif , usa ) introduced genetically encoded 
 fluorescent proteins in cell biology ( reviewed in ) . the introduction of a 
 small protein tag or of a unique combination of amino acids on the target 
 protein allows their interaction with a specific fluorophore - bearing substrate , 
 here an nc . examples of self - labelling protein tags are biarsenical compounds [ 61 , 62 ] , snap 
 tag , and halo tag . these approaches are helpful for developing new nc 
 functionalization strategies for specific intracellular targeting . compared with applications 
 to subcellular imaging in cell biology , nc - based whole - animal imaging has 
 developed very fast . due to their long - wavelength emission , brightness , and long - term 
 photostability , ncs are ideal probes for sensitive in vivoimaging in 
 deep tissues of small animals or imaging superficial tissue layers of larger 
 species . the possibility of synthesizing ncs emitting in the infrared 
 wavelengths minimizes scattering , optimizes depth penetration and allows discrimination 
 against collagen autofluorescence and thus should permit ultradeep imaging of 
  optically thick  tissue [ 66 , 67 ] , 
 provided that cytotoxicity is not an issue ( see section 7 ) . one of the first live - animal 
 applications of ncs was the selective labelling of tumor vasculature in mice by 
 using pegylated ncs coated with specific peptidic sequences against vascular 
 markers . in 2002 kerman et al .   showed in histological staining that after intravenous nc injection , 
 functionalized ncs can be addressed to specific blood vessels . a high level of 
 peg substitution on top of the functionalization of the ncs reduced their 
 uptake into the endothelial reticulum . were able 
 to image by multiphoton microscopy ncs through the skin of live mice , in 
 capillaries embedded 100  m in tissue . ballou 
 et al . demonstrated the importance of long - chain peg ( 5  kda ) coating for 
 increasing the duration of ncs circulating in the blood flow of mice . they were able to detect ncs by noninvasive whole body fluorescent 
 imaging , upto four months after injection . the same report also showed that ncs 
 deposit in liver , skin , and bone marrow in a surface - coating dependent manner 
 and that polymer- and peg - coated ( upto 3,400  da mw ) ncs are cleared from the 
 blood after injection . functionalized 
 with a monoclonal antibody directed against prostate cancer cells as a 
 cell - specific marker . after nc injection in mice , transplanted with human cancer 
 prostate cells , they succeeded in specifically detecting and imaging the tumor 
 site . however , as their ncs emitted in the visible spectrum , the authors used 
 spectral unmixing algorithms to detect the nc signal in the presence of 
 autofluorescence . along these lines , kim et al . intradermally injected 
 near - infrared - emitting ncs in mice and pigs and imaged sentinel lymph nodes 
 ( snl ) one cm deep in tissue . this work enables for the first time snl mapping 
 and cancer surgery under image guidance . metastatic tumor cell extravasations 
 were monitored in mice by intravenous injection of cells labelled with nc , 
 which were examined by fluorescence emission spectroscopy . more recently , stroh et al . combined ncs and multiphoton 
 intravital microscopy to distinguish in mice tumor vessels from perivascular 
 cells and extracellular matrix . with this approach , they also investigated the ability of nc - loaded 
 silica beads ( 100500  nm diameter ) to access the tumor and monitored the 
 trafficking of the precursor cells , a promising technique for cancer prevention 
 and treatment . designed recently 
  self - illuminating  nc conjugates permitting in vivo imaging without an external light source ; instead , 
 luciferase on the nc surface transfers its excitation to the nc core in a bioluminescence 
 resonance energy transfer ( bret ) assay . intramuscular or subcutaneous injection in mice of 5 pmol of 
 polymer - coated ncs conjugated to the renilla 
 reniformis luciferase was enough to image a bret emanating from 3  mm depth 
 tissue , after coelenterazine injection for activation . we note that this study 
 is one of the few applications that used ncs as acceptors rather than donors . as ncs are increasingly being used as biological 
 photoluminescent probes , in both acute cell assays and chronic , in the entire animal , in 
 vivo , it is important to evaluate if they represent a specific risk of 
 toxicity for the organism under study . although probably not classically termed cytotoxicity in 
 a strict sense , one obvious problem resulting from the nanoscopic size of 
 nanoparticles is that ncs can directly affect the biological system under study 
 by impairing the mobility , interaction , binding , or other biological action of the 
 ligand molecule to which they are attached . hence , any study using 
 nc - conjugated biomolecules must exclude the inhibition of the enzyme , receptor , 
 motor , or other by the nc . concerns against the use of semiconductor ncs for cell 
 biological applications go well beyond arguments of steric hindrance . it is 
 well known that cd can be released from the cdse core after 
 oxydative attack ( corrosion ) . bare cdse ncs are particularly harmful in this regard [ 36 , 71 ] , 
 limiting their utility for direct - injection strategies . additional shells ( zns ) 
 and capping ( silanization ) can reduce cd leakage , and further purification 
 steps can remove already released cd [ 7173 ] . in 
 our hands , a supplementary purification step prior to loading nc reduces the 
 toxic action of ncs , as measured by a resazurin or cell adhesion assay ( figure 4 ) . nevertheless , it is important to 
 bear in mind that despite sporadic claims of nanoparticles being 
 indiscriminately harmless , there is a general consensus that nc are toxic and that their 
 toxicity depends on their concentration , precise chemical composition , the 
 particle size , colloidal stability , as well as solubilization and 
 functionalization groups . also , cdse particles are generally more toxic inside 
 the cell than extracellularly , in line with the known action of cd by inhibiting protein synthesis , carbohydrate metabolism and  with 
 time by its accumulation in kidney and liver . at 
 the same time , the undisputable cytotoxic action of cd nanoparticles has not 
 precluded acute staining experiments of cells , because the concentration of ncs 
 can be always kept low enough to prevent immediate cytotoxic damage within the 
 experimental time window , but still high enough for enough fluorescence [ 4 , 20 , 39 , 45 , 47 , 50 ] . however , 
 because of the ligand desorption over time , a simple ligand exchange 
 functionalization is not effective to durably prevent intracellular nc 
 degradation . since as 
 much as ncs are intrinsically colloidally unstable and cytotoxic for cells , the specific kind of coating is essential for at least retarding 
 the cytotoxic effect [ 29 , 36 , 73 ] . peg 
 coating can reduce the unspecific uptake of ncs , it reduces their toxic effect 
 for extracellular application at the same initial concentration [ 29 , 71 ] . for biomedical applications as well as chronic animal 
 experiments , the major healthcare concern of nc labelling is related to the 
 leakage of cd into the organism , even at low dose . extracellular 
 application of cdse particles already presents a cytotoxic risk because cd does not only block ca ion channels ( like co as well , 
 which is released from magnetic ncs ) but also it permeates through the channel 
 and enters the cytoplasm . we note that the absence of a visible effect , often 
 based on the detection of cell morphology changes and cell viability assays does 
 not exclude a cumulative poisoning of the organism which first impairs the 
 metabolism of the cells , without being immediately noxious . interestingly , a 
 similar debate has long haunted the evaluation of nonlinear photodamage caused 
 by two - photon fluorescence excitation , where the introduction of rigorous 
 physiologically relevant criteria based on microscopic observables like the 
 kinetics of ca transients [ 77 , 78 ] has 
 ended the futile discussion . in conclusion , more work is needed to critically 
 evaluate the cytotoxicity of ncs , both upon short- and long - term exposure . to 
 better understand the deleterious action of different ncs on the organism under 
 study , standardized samples , experimental conditions , cells , and assays would 
 be a great leap forward and pave the ground for biomedical applications that 
 would additionally benefit from a tight collaboration with toxicologists . in this review , we focus on nanocrystal applications in vivo , both in cell biology and 
 medical diagnostics , and on the potential toxicity of ncs for biological 
 imaging . the advances in understanding nc colloidal properties together with 
 the ability of developing stable surface chemistries has brought about a large 
 choice of functionalization strategies which now offer to biologists a 
 versatile tool kit for many applications that rely on fluorescence and electron 
 microscopy . the main advantages of ncs over conventional organic fluorophores 
 are the possibility to detect easily single molecules , mostly derived from 
 their superior brightness and the long - term photostability ; the spectral 
 tunability and narrow - band emission ; and , going along with these , the ease of 
 nc use in multicolour fluorescence . particularly cd - based ncs are potentially cytotoxic , and the modulation of 
 their optical properties ( e.g. , their intrinsic fluorescence intermittency ) 
 through their local chemical environment ( see , e.g. , ) needs to be considered in each application .
semiconductor nanocrystals ( ncs ) are increasingly being used as photoluminescen markers in biological imaging . 
 their brightness , large stokes shift , and high photostability compared to organic fluorophores permit the exploration of biological phenomena at the single - molecule scale with superior temporal resolution and spatial   precision . 
 ncs have predominantly been used as extracellular markers for tagging and tracking membrane proteins . 
 successful internalization and intracellular labelling with ncs have been demonstrated for both fixed immunolabelled and live cells . however , the precise localization and subcellular compartment labelled are less clear . generally , live cell studies are limited by the requirement of fairly invasive protocols for loading ncs and the relatively large size of ncs compared to the cellular machinery , along with the subsequent sequestration of ncs in endosomal / lysosomal compartments . for long - period observation the potential cytotoxicity of cytoplasmically loaded ncs must be evaluated . 
 this review focuses on the challenges of intracellular uses of ncs .