the interactions of semiconductor quantum dots ( qds ) with living cells remain poorly understood . qds of different materials ( e.g. , cdse and cdte ) , sizes , colors , and surface 
 coatings demonstrate very different toxic effects to cells in culture [ 1 , 2 ] . much of the toxicity differences associated with a given type of 
 nanoparticle are attributable to whether the particles are able to enter the 
 cell , escape from endosomes , and enter the nucleus or mitochondria . no satisfactory explanation exists for differences 
 in these properties among batches of particles although there appear to be 
 loose correlations with particle size , particularly for nuclear entry . however , these experiments 
 were performed using thiol - capped particles , and because the small size 
 correlates with thiol coating stability , no firm conclusions can be drawn from the results . a quantitative understanding of the fate of conjugated qds in biological systems 
 is therefore critical if these particles are to be used in in vitro diagnostics 
 or in vivo systems . our 
 previous work demonstrated that qd - dopamine conjugates ( see figure 1 ) can be 
 used not only as static fluorescent labels , but also as sensors for 
 intracellular redox processes such as endocytosis , lysosomal processing , and 
 mitochondrial depolarization . this is due to the 
 electron - donating properties of dopamine ( da ) , which permit this molecule to 
 act as an electron shuttle between the nanoparticle and other molecules . the goal of this work is to improve the spatial and temporal resolution of the 
 qd - dopamine redox sensor by determining , in as quantitative as possible a 
 fashion , the relationship between the number of dopamine molecules on the 
 particle and two optical properties which can be measured within the cell : 
 photoenhancement and photobleaching . the eventual goal is to make an intracellular redox sensor that can yield nanometer 
 spatial resolution . the possibility of 
 several - nanometer resolution would become a reality if the photophysical 
 properties of single qds could be 
 regulated by their immediate environment in a controllable fashion . fluorescence intermittency , or blinking , is a classic example of a poorly - understood feature of qd 
 fluorescence that is often neglected or suppressed rather than exploited . under 
 continuous illumination , a number of 
 studies have been reported which look at various effects on blinking , such as 
 excitation power [ 8 , 9 , 11 , 13 , 14 ] , the shell material around the qds [ 7 , 8 , 10 ] , as well as environmental conditions such as temperature [ 8 , 15 ] and surrounding medium [ 16 , 17 ] . from these studies , two physical models have been advanced which 
 attempt to explain the inverse power law behavior of the blinking statistics . the first model assumes a fluctuating distribution of electron traps in the 
 immediate vicinity of , but external to , the qd . tunneling of the electron out of the qd results in a charged particle , quenching any emission and , thus , resulting in an off state . neutralization 
 of the qd by recapture of the electron recovers the emission , resulting in an 
 on state . the second model does not assume external traps , but rather posits 
 internal hole traps , presumably at surface states or crystal imperfection sites . energetic diffusion of the electronic states results in a 
 time - dependent resonance condition in which auger - assisted trapping of the hole 
 results in an off state . given the variability of the possible mechanisms , it is not possible to predict the effects 
 of conjugation of a redox - active molecule such as dopamine . in this work , we evaluate the effects of 
 dopamine of blinking and evaluate the possibility of the use of intermittency 
 as a tool for the ultrasensitive detection of subcellular environments and 
 biochemical processing of qd - bioconjugates . in this study , we used red - emitting cdse / zns qds ( qd605 , emission peak 605  20 nm ) for 
 photoenhancement and blinking studies , and green - emitting qds ( qd560 , emission 
 peak 560  20 nm ) for cellular uptake studies . qds were conjugated to the 
 neurotransmitter dopamine via the primary amine located on the opposite end of 
 the molecule from the redox - active catechol ( see figure 1 ) . the number of bound ligands was quantified in edc - coupling reactions containing varying concentrations of dopamine and/or of 1-ethyl-3-(3-dimethylaminopropyl ) 
 carbodiimide hydrochloride ( edc ) , using the o - phthaldialdehyde ( opa ) assay as 
 described previously . a strong dependence was observed of the 
 number of edcs per qd on the number of dopamine molecules that bound . the number of bound ligands increased 
 linearly with the number of edc molecules until a certain breakpoint and a 
 plateau was reached , which was considered as the saturation point for the qds ( see 
 figure 2 ) . indirectly , we can interpret 
 the saturation point as an indicator of the number of functional groups 
 available on the surface of the qds . this appears to be slightly smaller for green qds than for red , as 
 expected due to the smaller size of these particles , although it is the same 
 within error at the maximum edc concentration . manipulating the 
 extent of ligand coverage of the qd surface can be an effective way to modulate 
 fluorescent properties if the ligands can act as energy or electron donors or 
 acceptors to or from the qds . in our 
 previous work , we have shown that dopamine can be used to modulate the emission 
 characteristics of qds by a mechanism of electron transfer . in figure 3(a ) , we demonstrate the effect of 
 the number of bound dopamine ligands and the subsequent reduction in the 
 emission intensity of the qds . to 
 distinguish this type of quenching from the stern - volmer collisional quenching , 
 we purified the conjugates from excess unbound ligand . our results showed a 
 large decrease in intensity when a relatively small number of ligands were 
 bound to the surface , owing to the electron transfer from dopamine to the qds . oxidation of these quenched qds , 
 either by photoexposure or chemical means , led to a restoration of fluorescence 
 from the qd . in addition , oxidized 
 dopamine emitted fluorescence in the blue regime ( peak emission 460  nm ) . thus , there were three parameters that could 
 be used to indicate the number of dopamine conjugates on the particle surface : 
 ( a ) blue fluorescence from oxidized dopamine ; ( b ) brightness of qd fluorescence 
 without uv pre - exposure ; and ( c ) time course of qd fluorescence under 
 photooxidation . this 
 latter quantity must be determined for each light source ; broad hg - lamp excitation ( through a qd or dapi filter , see figure 3(b ) ) yielded very different results than 488  nm laser - line excitation , showing essentially 
 all - or - nothing dependence on illumination power ( see figure 3(c ) ) . similarly , lifetime measurements with 
 time - correlated single photon counting ( tcspc ) and 400 nm laser illumination 
 revealed extreme sensitivity to illumination power . a full study with laser powers varying from 
 tens of microwatts to several milliwatts is forthcoming . little or no binding was seen to cells without dopamine receptors , and 
 the qd conjugates were readily washed away . we investigated the effect 
 of dopamine ligand coverage and corresponding uptake in da - receptor - expressing 
 pc12 cells . as expected , cells that were 
 treated with qds alone did not show any detectable qd association . for the qd560-da conjugates , particles with fewer than 
 100 da / particle were not taken up in significant amounts after 1530 minutes of 
 incubation ( not shown ) . the 
 variation of photoenhancement properties with position in the cell was then 
 used to semiquantitatively identify regions where oxidation had occurred . the location 
 and appearance of various organelles in cells were determined by 
 labeling with specific dyes such as lysotracker and mitotracker ( see figure 4 ) . studies of unquenching time - courses of the 
 simultaneously loaded qd - dopamine were then performed under 488  nm laser 
 illumination . the results showed a 
 consistent and reproducible pattern with three distinctive qd behaviors . in 
 fixed cells , with depolarized mitochondria , qds did not show overlap with the 
 mitochondrial - targeting dye mitotracker . qd fluorescence brightened slightly under light exposure ( see figures 4(a ) , 
 4(b ) , and 4(g ) ) . in live cells , however , the 
 mitochondrial region brightened quickly and intensely , with qd - mitotracker 
 overlap becoming apparent ( see figures 4(c ) , 
 4(d ) , and 4(g ) ) . in both live and 
 fixed cells , a good deal of qd fluorescence was seen colocalized with 
 lysosomes . qd fluorescence within 
 lysosomes shows only bleaching with time ( see figures 4(e ) , 
 4(f ) , and 4(g ) ) . qds 
 that were outside the cell , in aggregates outside the membrane , exhibited no 
 brightening but only photobleaching over time ( see figures 4(e ) , 
 4(f ) , and 4(g ) ) . observation of the blue qd - dopamine fluorescence confirmed 
 what was suggested by the time - course spectra . qds outside the cells showed no blue emission , confirming the absence of 
 dopamine , whereas those in the cytoplasm and mitochondria showed visible 460  nm 
 emission ( see figure 5 ) . blinking 
 is conveniently studied by taking an image series with time of a number of 
 immobilized qds [ 911 ] . details of the analysis may be found in the methods section and in previous publications [ 9 , 10 ] . the result of the analysis is that the fluorescence time trace for each identified qd is 
 extracted from the image series , and the durations of on and off times ( time durations for 
 which the signal is above and below the threshold level , resp . ) are 
 extracted . a typical image and extracted fluorescence trace of a single , 
 immobile qd are shown in figure 6 . we 
 studied the effect of conjugating dopamine to qds on their blinking statistics . the probability distributions of on times , p(on ) , and off times , p(off ) , for qds with and without conjugated 
 dopamine are shown in figures 7(a ) and 7(b ) , respectively , as measured by 
 extracting fluorescence time traces , such as shown in figure 6(b ) , setting a 
 threshold and calculating on and off times . approximately 500 qds were analyzed 
 from 5 sets of movies , taken at 50-millisecond resolution for 100 seconds . except 
 for the dopamine , the qds and the experimental conditions were identical . clearly , under these conditions , the conjugation of dopamine to the qd reduced 
 the on times and increased 
 the off times . we previously showed   that qd - dopamine conjugates 
 show altered fluorescence in living cells in accordance with the cellular and 
 subcellular redox potential with brighter fluorescence corresponding to more 
 oxidizing conditions . first , different colors , batches of qds , and 
 conjugate preparations showed widely varying degrees of uptake . second , the mechanism of brightening in 
 response to oxidation was not elucidated although it was presumed to be related 
 to cap decay . in this work , we 
 explain the differing levels of uptake by showing that using standard msa - edc 
 coupling techniques , approximately 100 dopamines per particle are required to 
 obtain efficient uptake in our stably transfected dopamine - receptor cell 
 lines . future work will explore variations on solubilizing - agent chain 
 length and the addition of spacers 
 to improve the presentation of the dopamine to its receptor , as this high 
 requirement probably reflects biological inactivity of most of the 
 dopamines on these particles . of general 
 interest is the observation that controlling the average number of dopamine 
 molecules bound to qds affects the photoenhancement of ensembles of particles 
 in a measurable fashion . this makes 
 these conjugates a more useful tool than one based upon quenching alone , as the 
 presence of fully - quenched particles can obviously not be detected under 
 fluorescence microscopy . on slides , more 
 dopamines per particle correspond 
 to slower photoenhancement ( see figure 3(b ) ) ; qds without dopamine show 
 bleaching without enhancement . confocal 
 laser illumination shows a quite different pattern from hg - lamp illumination ; 
 this could be due to several factors . the illumination is at a single wavelength ; the most commonly used line 
 ( 488 nm ) will not excite the dopamine quinone , eliminating issues of signal 
 confusion as well as eliminating the possibility that excitation of the quinone 
 affects the qd enhancement or bleaching . 
 finally , laser illumination is intermittent due to scanning , perhaps permitting 
 qds to recover in - between pulses . in cells , a minimum number of dopamines , corresponding to slow enhancement , is necessary 
 for uptake . however , as the particles 
 travel through the cell , particularly to oxidizing regions , enhancement becomes 
 more rapid suggesting that the cap decay mechanism is in fact correct . these 
 data could be used for a semiquantitative model of qd processing in cells 
 ( schematic in figure 8) . it is immediately obvious from figure 7 that the addition of dopamine to the qd surface affects blinking by reducing 
 on times and increasing off times . many groups have found that the distribution 
 of off times fits to a power law function [ 810 , 12 , 18 , 23 ] , whereas the on times distribution 
 is the source of some debate . some have found that the on times fit to a power 
 law function [ 18 , 24 ] ; whereas others have found that they 
 fit better to a power law function convoluted with an exponential function at 
 long on times [ 8 , 10 , 19 ] . the effect of coating the cdse with 
 a zns shell on the blinking has also been studied by several groups . nirmal et al . found that a thick zns shell results in the observation of longer 
 on and off events . subsequently , a more thorough 
 statistical analysis revealed that the power law slopes for both the on times 
 and the off times distributions are not affected by zns capping , but that the 
 exponential cutoff time in the on times slightly increases by zns capping . heyes et al . found that , within experimental error , there was no 
 effect in both the power law slope and the exponential cutoff upon increasing 
 the zns capping thickness . this lack of dependence was explained as the 
 physical origin of blinking lying in hole - trap states at the surface or 
 core - shell interface . in the study of heyes et al . , all blinking probability distributions fit to an 
 inverse power law for off times . for on times , the functions fit to an inverse 
 power law with exponential cutoff at longer on times , in agreement with 
 previous observations and a previously published model , which did not assume 
 the presence of external trap states . the data presented here do not fit 
 to the same functions as previous observations ( power law for off times and a 
 power law convoluted with an exponential cutoff at longer times ) suggesting 
 that the underlying mechanisms of blinking differ . at the current time , we 
 can not identify the source of these differences and much more work is needed to 
 further investigate the physical origins . we have identified several possible 
 sources that may be responsible for the observed difference in blinking 
 statistics . one such source may be that the difference in chemical environment 
 of the qd is responsible . the qds used here are coated with mercaptosuccinic 
 acid ( msa ) ligands , which carry both a negative charge and a coordinating 
 sulfur group which may both affect the blinking statistics . most previous 
 studies have investigated qds that are either coated with uncharged organic 
 ligands or are coated with large amphiphilic polymers . the chemical environment may be further 
 affected by the conjugation of dopamine which may result in the presence of 
 different trap ( off ) states . another source for the difference may lie in the 
 fact that in the experiments presented here , polychromatic excitation from a 
 halogen lamp , passed though a qd filter cube , which results in excitation with 
 a range of wavelengths from 380 nm to 460 nm , was used . while this excitation 
 configuration is a common one for biological detection of qds in cellular 
 environments , both the cdse core and the zns shell are excited ( as well as 
 dopamine when it is present ) whereas 
 most previous blinking experiments have been performed using monochromatic 
 excitation at either 488 nm , 514 nm , or 532 nm [ 712 ] where only the cdse core is excited . excitions 
 formed following excitation of the qds using the present excitation 
 configuration are more easily exposed to 
 the qd surface or surrounding environment . further work is currently underway 
 in our lab to identify the effects of both the ligand properties and the 
 excitation energy on the blinking mechanism . the average intensity of the images 
 decreases faster for qd - dopamine conjugates than for qds not conjugated with 
 dopamine . however , the decrease in average intensity is relatively small 
 compared to the total intensity due to the contribution from the background 
 signal arising from the many pixels in which no qds are present . this is 
 constant throughout the experiment , which contributes to a nonzero offset in 
 the integrated intensity of the image . on the other hand , the fraction of 
 particles in the on state decreases by approximately 3 orders of magnitude for 
 the qd - dopamine conjugates , whereas qds without dopamine show a relatively 
 constant on fraction during the experiment . this reduction in the fraction of 
 emitting qds indicates that either they are trapped in a long off time or that 
 they are permanently bleached . chung and bawendi argued that there must exist a 
 saturation in the maximum off time duration in order to explain the observation 
 that , under continuous illumination , qd emission does not reach zero intensity 
 due to all qds eventually becoming trapped in a very long off state . indeed , using ensemble fluorescence 
 spectroscopy , they determined that the maximum off time is on the order of 
 thousands of seconds  a timescale that is generally not reached 
 using single molecule experiments . in order to determine if the nonfluorescent 
 particles would turn  on  again at later times ( i.e. , the particles are in an 
 extended off period ) , we turned off the excitation source for several minutes . we then imaged the same area to determine if the particles were once again 
 fluorescent . we found that almost none of the particles regained their fluorescence , 
 indicating that the photobleaching was indeed permanent rather than the 
 particles residing in an extended off period . it has been shown that qds are 
 much more photostable than other fluorophores , and are generally photostable 
 for timescales much longer than our experiments . one possible mechanism may be that 
 radicals are formed by the dopamine , which may then eventually diffuse through 
 the solution and react with the qd surface forming trap states . this may repeat 
 until a significant number of trap states are formed which results in 
 preferential nonradiative decay rather than radiative decay of any formed 
 excitons . it must also be noted here that , under these excitation conditions , 
 dopamine is also directly excited . the initial increase in on fraction of qds 
 may be the result of an initial bleaching of dopamine on the qds which results 
 in more on particles . this may be then followed by reaction of radicals with 
 the qd to once again turn them off . the changes in blinking 
 statistics upon conjugation of qds to specific ligands such as dopamine suggest 
 that such an assay may be used in the future as ultrasensitive sensors for 
 chemical and biological characterization of subcellular environments and 
 biological processing pathways . however , it is necessary for the underlying 
 mechanisms contributing to the observed changes to be fully understood in order 
 to interpret these types of experimental results . conjugation of qds to an electron 
 donor such as dopamine leads to changes in optical properties beyond simply 
 quenching . these properties , such as 
 photoenhancement and blinking , may be used in biological studies as novel means 
 to probe subcellular environments . the 
 use of other electron donors , such as any of the biological catechols , should 
 show equally interesting properties when conjugated to qds , creating a general 
 principle upon which new fluorescent indicators may be created . all chemicals 
 were purchased from sigma - aldrich canada ( oakville , on , canada ) . cdse / zns 
 core - shell nanocrystals were synthesized as previously described [ 26 , 27 ] . in brief , cdse / zns qds were synthesized as 
 follows : 0.024 g cdo was added to a 
 reaction flask containing 0.44 g stearic acid and heated to 180c under inert 
 conditions , forming a colorless solution . the solution was allowed to cool , and 
 afterwards 5 g topo and 2 g octadecylamine were added to the flask . the flask was then 
 evacuated and filled with inert gas several times , and the solution was heated 
 to 200c300c ( exact 
 temperature depends on the desired size ) . 0.2 g se was then dissolved in 
 24 ml topo under inert conditions , and added to the reaction flask . finally , 0.4 ml of zn(me)2 was added to 0.07 ml ( tmsi)2 under an inert atmosphere , and added to the reaction flask . finally the 
 solution was allowed to cool , dissolved in chcl3 , and precipitated 
 with meoh . these topo - passivated nanocrystals were then dispersed in the 
 desired solvent , including toluene , chcl3 , and hexane . aqueous 
 qd solutions were diluted in h2o to an optical density ( od ) of 0.1 
 at the exciton peak . one mg edc was 
 added to 0.2 ml of qds in aqueous solution and 0.3 ml phosphate - buffered 
 saline ( pbs ) solution . the tubes were covered in foil and put on a shaker for 
 one hour . afterwards dopamine was added to a final concentration of 2 mm , 
 and pbs was added to a final volume of 1 ml . the tube was again covered in 
 foil , and agitated on a shaker for 2 hours . solutions were dialyzed against pbs 
 for 1 hour in order to remove excess dopamine . all handling of dopamine solutions and qd - dopamine was performed in a 
 glove bag under nitrogen to avoid oxidation of dopamine and further stored 
 under an inert atmosphere until ready for use . experiments 
 with cell lines were performed using pc12 cells stably transfected with human 
 d2 dopamine receptors ( gift of stuart sealfon , mount sinai school of medicine ) . cells were maintained in high 
 glucose dulbecco 's modified eagle 's medium ( dmem ) ( invitrogen canada , 
 burlington , on , canada ) supplemented with 10% fetal bovine serum , 5% horse 
 serum , 0.2 mm glutamine , 100 u / ml penicillin , and 100 g / ml streptomycin in a 
 5% co2 atmosphere at 37c . for passage , cells were rinsed 
 first with phosphate - buffered saline ( pbs ) and then with hanks balanced salt 
 solution containing 0.05% trypsin and 0.02% edta , incubated for 2 minutes at 
 room temperature , and resuspended in supplemented dmem . cells were 
 passaged onto glass bottom dishes ( mattek co. , ashland , mass , usa ) the day 
 before use at 5080% 
 confluency . just prior to 
 labeling , growth medium was removed by 2 washes in sterile pbs , and then 
 replaced with 1 ml serum - free medium without phenol red ( optimem , invitrogen canada ) . in 
 preliminary studies , incubation times were varied between 15 minutes to 2 hours , 
 and it was found that some uptake of unconjugated qds could occur at longer 
 timescales . qd - dopamine conjugates were applied directly into serum - free medium at a 
 concentration of ~510 nm particles . for colabeling with mitotracker red or 
 lysotracker orange ( invitrogen canada ) , dyes were added to cells at a 
 concentration of 1 m at least 30 minutes before qd addition . all cells were 
 washed several times with sterile pbs after labeling , and live cells were 
 imaged in pbs . fixed cells were 
 incubated in 3.7% paraformaldehyde in pbs for 30 minutes , rinsed twice in pbs , 
 and imaged in pbs . absorbance and emission 
 spectra were recorded on spectramax plus and spectramax gemini readers 
 ( molecular devices , sunnyvale , calif , usa ) . for hg - lamp exposure experiments , 
 cells and qds were examined and imaged with an olympus ix-71 inverted 
 microscope and a nuance multispectral imaging system , which provides spectral 
 data from 420720  nm in 10-nm 
 steps ( cri instruments , cambridge , mass , usa ) . qd  filter cube 
 set ( excitation = 380460  nm , dichroic 
 = 475  nm , emission = 500  lp ) , a filter cube set ( excitation = 350/50 nm , 
 dichroic = 400  nm , emission = 420  lp ) , or a  tritc  filter set ( excitation = 
 540  nm , dichroic = 565  nm , emission 605  lp ) ( chroma technologies , 
 rockingham , vt , usa ) . for evaluation of photoenhancement on slides , a droplet 
 of qd solution was placed on a glass slide and illuminated at full lamp power 
 from below using the qd filter . confocal imaging and 
 time - course experiments were 
 performed on a zeiss 510  lsm with a plan apo 100 oil objective . qds were excited with an ar ion laser with output power held at 
 55% for all experiments , corresponding to 6 a of tube current to reduce laser 
 flicker , and the percent transmission of the 488 nm line was adjusted between 
 10% and 100% . lysotracker red 
 and mitotracker orange were excited with a hene laser ( 543 nm line ) . cells labeled with > 1 probe were 
 examined for channel bleed - through before imaging . for cell imaging studies , samples were prepared for blinking studies by 
 aminosilanizing a glass coverslip with aminopropyl trichlorosilane to which a 
 drop of ~100  pm qd solution was added . qds were immobilized by electrostatic forces between the positively charged amino groups of the silane and the negatively charged carboxylates on the qd . the qds were measured with the liquid still above them to reduce 
 possible effects from oxygen in the atmosphere . drying out of the solution was 
 reduced by placing a reservoir of water next to the sample , and covering the 
 sample stage , which resulted in a humid environment around the sample . blinking 
 studies of qds were performed in an epifluorescence configuration on an olympus 
 ix70 inverted microscope using a halogen - lamp source . a qd filter cube set was 
 used for excitation and emission ( excitation = 380460  nm , dichroic 
 = 475  nm , emission = 500  lp ) . the excitation light was passed though an olympus 
 planapo oil - immersion objective ( 60 , 1.45  na ) and the emission collected into 
 the same objective . the light is then focused onto a photometrics cascade 512  b em - ccd 
 camera , and a time - stack of images taken at 20  fps ( 50-millisecond exposure ) 
 for 2000 frames using the rsimage software ( roper scientific , tucson , ariz , usa ) . andrei kobitski at the university of ulm ( germany ) 
 using matlab 6.5 ( mathworks , natick , mass , usa ) , from which traces of 
 individual qds are extracted and probability distributions of on and off times 
 are determined . the details of the analysis may be found in previous 
 publications [ 9 , 10 ] . briefly , the algorithm 
 aligns each frame of the series and integrates the image stack . thus , qds that 
 are on at least once in the time series will be identified . then , the software 
 measures the integrated signal from the 3  3 pixels around the central of the 
 emission ( the 3  3 pixels is approximately equal to the point spread function 
 of the microscope , and may be manually set by the user of the software ) as a 
 function of frame number ( time ) . the local background signal is found from the 
 pixels surrounding the qd in each frame and subtracted . if another qd is too 
 close that the local background can not be determined , 
 then this qd is ignored . due to the low qd concentration , this results in a set of traces similar to that shown in figure 6(b ) . it was previously found that if the signal : noise is strong enough , the actual threshold level used ( from 24 ) does not affect the resulting 
 statistics . then the durations of times spent in the off state 
 and on state are calculated from all the extracted traces and plotted as a probability 
 histogram ( figure 7 , normalized to a total probability of 1 ) .
cdse / zns quantum dots ( qds ) conjugated to biomolecules that quench their fluorescence , particularly dopamine , have particular spectral properties that allow determination of the number of conjugates per particle , namely , photoenhancement and photobleaching . in this work , 
 we quantify these properties on a single - particle and ensemble basis in order to evaluate their usefulness as a tool for indicating qd uptake , breakdown , and processing in living cells . 
 this creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle - cell interactions .