the use of fluorescent 
 nanoparticles as imaging agents is an increasingly 
 important topic in the field of bioimaging . the utility of fluorescence spectroscopy as a detection method for 
 cellular imaging arises from the sensitivity of the technique , as 
 well as the ability to discriminate based on both intensity and wavelength 
 of emission . fluorescent nanoparticles provide additional advantages 
 over molecular organic fluorophores , including a reduction in fluorophore 
 aggregation , reduced cytotoxicity , improved microenvironment inertness , 
 better stability , and increased brightness . nanoparticles 
 derived from silica and gold , as well as quantum dots and carbon dots , 
 have all been utilized as fluorescent imaging agents . however , polymer nanoparticles perhaps provide the greatest 
 scope for versatility in particle properties and composition , such 
 as hydrophobicity / hydrophilicity , surface chemistry , and analyte / cargo 
 transport . additionally , polymer nanoparticles 
 can be designed to respond to a range of external stimuli , including 
 temperature , ph , oxidation / reduction , biomolecules , and light . it is particularly desirable , in the case of fluorescent particles , 
 if this response can be coupled to a change in emission . for example both hydrophobic 
 and hydrophilic dyes can be used to detect morphology changes in block 
 copolymer ( bcp ) solution state self - assemblies . however , the covalent attachment , rather than physical 
 absorption , of dye molecules to polymer nanoparticles has the advantage 
 of greater efficiency , decreased dye leaching from the nanoparticles 
 and eliminates uncertainties regarding the fluorophore location . covalent labeling can be applied to a range of 
 synthetic methodologies , such as nanoprecipitation and bcp self - assembly , and can also be applied to the synthesis of polymer nanogels , conjugated polymer nanoparticles , and dendrimers . synthetic diversity is also increased by the potential for dye incorporation 
 using fluorescent monomers and/or initiators during polymer synthesis or by subsequent particle modification . covalent attachment of fluorophores to 
 bcps has long been exploited 
 to provide a wealth of information about the bcp self - assembled state 
 in model systems , for example via excimer emission , fret measurements , 
 and fluorescence lifetimes . more recently , this self - assembly information has 
 also been collected in vitro and in vivo . for example , the aggregation of dye 
 labeled polymers can cause quenching processes to be enhanced or inhibited , 
 leading upon micellization to decreased or increased emission , respectively . the degradation of polymer micelles derived from intrinsically fluorescent 
 copolymers has also been observed by detecting a decrease in emission , while the loss of mobility upon bcp micelle 
 gelation has allowed for the glass transition temperature and critical 
 micelle temperature to be measured by changes in emission from a covalently 
 attached fluorophore . changes in the 
 morphology of bcp assemblies can also be observed by measuring emission 
 from fluorescent labels . for example , the swelling of micelle coronas 
 in response to temperature and ph can be detected due to the effect 
 on fluorophore quenching or excimer formation caused by changes in 
 coronal hydration . the controlled assembly 
 and disassembly of bcp nanoparticles in 
 response to a stimulus can also be detected by measuring the emission 
 of covalently attached fluorophores . for example , gao et al . have developed a series of  ultra - ph - sensitive bcp 
 nanoparticles , where the core block is labeled with a self - quenching 
 fluorophore . the core block comprises of ph - responsive poly(aminomethacrylates ) , 
 and protonation of this block causes a transition from hydrophobic 
 to hydrophilic , leading to micelle disassembly . micelle disassembly can therefore 
 be detected by increased emission , while the ph range for response 
 can be tuned from ph 47.4 by tailoring the poly(aminomethacrylate ) 
 allowing in vitro and in vivo detection 
 of disassembly in the early or late endosome , for example . this approach 
 of detecting ph triggered bcp disassembly with a self - quenching dye 
 can also be coupled with the use of a ph - responsive fluorophore in 
 the hydrophilic block . in this example 
 the ph - responsive dye emitted at a longer wavelength and was less 
 emissive once protonated ( which coincides with core block protonation 
 and micelle disassembly ) , so that an enhanced signal was achieved 
 by taking the ratio of emission at the two different wavelengths . in addition to ph , response of bcp micelles to temperature and the 
 presence of metal ions has also been detected by fluorescence spectroscopy , 
 using either dyes that respond to changes in aggregation or dyes whose 
 emission changes upon binding to the metal ions . recent work in our group has highlighted the 
 utility of simple 
 fluorophores based on substituted maleimides . these dithiomaleimide ( dtm ) fluorophores were easily incorporated 
 into superbright nanoparticles via a one - pot emulsion polymerization and were also incorporated into bcp micelles 
 whereby a change in emission enabled the detection of a micelle - to - vesicle 
 morphology transition . fluorescence lifetime 
 imaging microscopy ( flim ) was also utilized to allow in vitro detection of micelle - to - unimer disassembly , as fluorophore protection 
 from solvent collisional quenching in the assembled micelles led to 
 longer fluorescence lifetimes , whereas the limited protection afforded 
 to the polymer unimers resulted in a drastic reduction in fluorescence 
 lifetime . for these self - reporting bcp 
 micelles , the dtm fluorophore was located at the interface between 
 the core and coronal blocks , which required the use of a dtm - labeled 
 asymmetric dual - functional initiator for ring - opening and reversible 
 addition  fragmentation chain - transfer ( raft ) polymerization . 
 in the present work we aim to simplify the synthetic route to obtain 
 self - reporting fluorescent dtm - labeled bcp micelles by utilizing a 
 dtm - labeled acrylate monomer to allow bcp synthesis by sequential 
 raft polymerizations . the greater versatility of this synthetic approach 
 also allowed the position of the fluorophore to be varied , and we 
 therefore also investigated the effect of locating the fluorophore 
 in the micelle core or corona . this approach has enabled the simplified 
 fabrication of highly emissive fluorescent bcp micelles , whose fluorescent 
 lifetime self - reports on the supramolecular assembled state , while 
 the emission from the micelles can also report on the presence and 
 location of an encapsulated organic dye . tert - butyl acrylate ( tba ) 
 was vacuum distilled over cah2 prior to use and stored 
 at 4 c . 2,2-azobis(2-methylpropionitrile ) ( aibn ) 
 was recrystallized twice from methanol and stored at 4 c in 
 the dark . triethylene glycol monomethyl ether acrylate ( tega ) , and dithiomaleimide acrylate ( dtma ) , were synthesized as previously reported . the 
 raft agent cyanomethyldodecyl trithiocarbonate ( cmdt ) , nile 
 red ( nr ) , and rhodamine b ( rhb ) were purchased from aldrich and used 
 as received . 1,4-dioxane for polymerizations ( fisher , reagent grade ) 
 was passed through a column of basic alumina immediately prior to 
 the reaction . 1,4-dioxane for fret experiments ( aldrich , spectroscopy 
 grade ) was used as received . solvents for size exclusion chromatography 
 ( fisher , hplc grade ) were used as received . water for self - assembly 
 and spectroscopy was purified to a resistivity of 18.2 mcm 
 using a millipore simplicity ultrapure water system . h and c nmr spectra were recorded on a bruker dpx-400 
 spectrometer in cdcl3 unless otherwise stated . size exclusion chromatography ( sec ) measurements were conducted using 
 a varian 390-lc - multi detector suite fitted with differential refractive 
 index ( dri ) , uv  vis , and photodiode array ( pda ) detectors . 
 a guard column ( varian polymer laboratories plgel 5 
 m , 50 mm  7.5 mm ) and two mixed d columns 
 ( varian polymer laboratories plgel 5 m , 300 mm  7.5 mm ) the mobile phase was tetrahydrofuran with 2% triethylamine 
 or dimethylformamide with nh4bf4 ( 5 mm ) eluent 
 at a flow rate of 1.0 ml / min . data were analyzed using cirrus v3.3 
 with calibration curves produced using varian polymer laboratories 
 easi - vials linear poly(styrene ) standards ( 162 g mol240 kg mol ) or linear poly(methyl methacrylate ) 
 standards ( 690 g mol790 kg mol ) . transmission electron microscopy ( tem ) imaging was performed on 
 a jeol 2011 200 kv lab6 instrument fitted with a gatan 
 ultrascan 1000 camera , using agar graphene oxide support film grids . static light scattering ( sls ) and 
 dynamic light scattering ( dls ) measurements were performed on an alv 
 cgs3 goniometer operating at  = 632.8 nm . the temperature of 
 the toluene bath was regulated using a julabo f32-me refrigerated 
 and heating circulator set to 20 c . intensity autocorrelation 
 functions ( g2(q , t ) ) were fitted with the repes routine using gendist software , which performs an inverse laplace transformation 
 to produce a distribution of relaxation times a( ) . an error of 10% was applied to light scattering data , in accordance 
 with previous reports . refractive index 
 increment ( dn / dc ) was measured by 
 injecting samples of a known concentration into a shodex ri-101 refractive 
 index detector . an aggregation number ( nagg ) for the particles can be calculated according to eq 1 , where mw , polymer can be approximated by mn ( calculated 
 by h nmr spectroscopy end - group analysis ) multiplied by m ( calculated by sec).1assuming that the micelle core is completely 
 dehydrated , it is then possible to approximate the radius of the core 
 ( rcore ) from nagg according to eq 2 . this equation simply relates the volume of a 
 sphere with radius rcore to the mass of 
 the polymer core of the micelle ( mw , core = mn , core(nmr )  m , core(sec ) ) , whose density is approximated by the bulk 
 density of the core - forming polymer (  = 1.00  10 g m for ptba).2core volume ( vcore ) can subsequently be calculated from rcore , while shell volume ( vshell ) is calculated 
 as the difference between total micelle volume ( from rh ) and vcore . the approximate 
 local concentration of the fluorophore ( [ dtm ] ) in the slms and clms 
 can then be calculated according to eqs 3 and 4 , respectively.34 all steady state emission , 
 excitation , and anisotropy spectra were obtained with a horiba fluoromax4 
 with automatic polarizers and analyzed in fluoressence ( horiba ) and 
 originpro 8.6 ( origin laboratories ) . a long - pass emission filter (  
 = 360 nm ) was used to eliminate the detection of first- and second - order 
 rayleigh scattering . for the emission intensity measurements the full 
 emission spectra was integrated using the integrate function in originpro 
 and normalized by dividing by the concentration of polymer . time - correlated 
 single photon counting ( tcspc ) was employed to obtain all fluorescence 
 lifetime spectra . this was done with a fluorotime 100 fluorometer 
 and 405 nm solid state picosecond diode laser source ( picoquant ) in 
 matched quartz 0.7 ml cells ( starna cell ) . instrument response functions 
 ( irf ) were determined from scatter signal solution of ludox hs-40 
 colloidal silica ( 1% particles in water w / w ) . fluorescence lifetime imaging was performed 
 using a flim lsm upgrade kit for the fv1000 ( picoquant ) mounted on 
 a fv1000 ( olympus ) confocal microscope on a ix-81 inverted base ( olympus ) . a planapo n 60 oil lens ( na 1.42 , olympus ) was used for all 
 imaging . the fv1000 system was driven with the fv10-asw v3.1a software 
 platform ( olympus ) with scan rates of 4 s / pixel at 256  
 256 pixels . flim images and spectra were collected using bins of 16 
 ps with a 405 nm laser ( ldh - p - c-405b , picoquant ) driven at 2.5 mhz . the fwhm for the 405 nm laser head was 60 ps , and the maximum power 
 was 0.21 mw ( attenuated by variable neutral density filters to prevent 
 count pileup and maintain counting rates below 1% bin occupancy ) . symphotime 64 ( picoquant ) software was used for collection and analysis 
 of flim images and spectra . all irf deconvolved exponential fits were 
 performed with the 3 or 4 exponents selected for completeness of fit 
 as determined by boot - strap  analysis in fluorofit . quantum yield experiments were performed on an edinburgh instruments 
 fls920 steady - state spectrometer fitted with an integrating sphere 
 and a r928 ( visible ) hamamatsu photomultiplier tube detection system . experiments 
 were carried out in solution using 1 cm path length quartz cuvettes 
 with four transparent polished faces . a solution 
 of cmdt ( 0.282 g , 887 mol ) , tba ( 5.00 g , 39.0 
 mmol ) , and aibn ( 14.6 mg , 88.7 mol ) in 1,4-dioxane ( 5.66 ml ) 
 was added to a polymerization ampule . the solution was degassed by 
 three freeze  pump  thaw cycles and sealed under n2 . the reaction was stirred at 65 c for 2 h and then 
 quenched by rapid cooling and exposure to air . the product was purified 
 by repeated precipitation into ice - cold methanol / h2o ( 9/1 , 
 v / v ) and isolated as a yellow glassy solid . dptba(nmr ) = 44 , mn(nmr ) = 6.0 kg mol , and m(sec ) = 1.08 . a solution of cmdt ( 40.0 
 mg , 126 mol ) , tba ( 0.807 g , 6.30 mmol ) , dtma 
 ( 81.2 mg , 189 mol ) , 
 and aibn ( 2.07 mg , 12.6 mol ) in 1,4-dioxane ( 0.914 ml ) was 
 added to a polymerization ampule . the solution was degassed by three 
 freeze  pump  thaw cycles and sealed under n2 . the reaction was stirred at 65 c for 5 h and then quenched 
 by rapid cooling and exposure to air . the product was purified by repeated precipitation into ice - cold methanol / h2o ( 9/1 , v / v ) and isolated as a fluorescent yellow glassy solid . dptba(nmr ) = 36 , dpdtma(nmr ) 
 = 1.1 , mn(nmr ) = 5.4 kg 
 mol , and m(sec ) = 1.13 . a solution of 1 ( 0.150 g , 25.2 
 mol ) , tega ( 0.878 g , 4.02 mmol ) , and aibn ( 0.41 mg , 2.5 mol ) in 1,4-dioxane ( 2.37 
 ml ) was added 
 to a polymerization ampule . the solution was degassed by three freeze  pump  thaw 
 cycles and sealed under n2 . the reaction was stirred at 
 65 c for 4.5 h and then quenched by rapid cooling and exposure 
 to air . h2o ( 10 ml ) was added , and the solution purified 
 by exhaustive dialysis ( mwco 3.5 kg mol ) against 
 distilled water . dptega(nmr ) = 120 , mn(nmr ) = 31.3 kg mol , and m(sec ) = 1.38 . a solution 
 of 1 ( 0.150 g , 25.2 mol ) , tega ( 1.10 g , 5.03 mmol ) , 
 dtma ( 16.2 mg , 37.7 mol ) , and aibn ( 0.41 mg , 2.5 mol ) 
 in 1,4-dioxane ( 2.96 ml ) was added to a polymerization ampule . the 
 solution was degassed by three freeze  pump  thaw cycles 
 and sealed under n2 . the reaction was stirred at 65 c 
 for 5 h and then quenched by rapid cooling and exposure to air . 1,4-dioxane 
 ( 2 ml ) was added , and the solution precipitated into ice - cold hexane 
 ( 200 ml  2 ) . the crude product was redissolved in 1,4-dioxane / h2o ( 1/2 , v / v ) and purified by exhaustive dialysis ( mwco 3.5 
 kg mol ) against distilled water . dptega(nmr ) = 140 , dpdtma(nmr ) = 1.1 , mn(nmr ) = 37.7 kg mol , and m(sec ) = 1.35 . a solution of 2 ( 0.130 g , 24.3 
 mol ) , tega ( 1.06 g , 4.86 mmol ) , and 
 aibn ( 0.40 mg , 2.4 mol ) in 1,4-dioxane ( 2.86 ml ) was added 
 to a polymerization ampule . the solution was degassed by three freeze  pump  thaw 
 cycles and sealed under n2 . the reaction was stirred at 
 65 c for 3.5 h and then quenched by rapid cooling and exposure 
 to air . h2o ( 10 ml ) was added , and the solution purified 
 by exhaustive dialysis ( mwco 3.5 kg mol ) against 
 distilled water . dptega(nmr ) = 130 , mn(nmr ) = 33.1 kg mol , and m(sec ) = 1.38 . nonlabeled micelles ( nlms ) , 
 shell - labeled micelles ( slms ) , and core - labeled micelles ( clms ) were 
 assembled by direct dissolution of 3 , 4 , 
 and 5 , respectively , in water ( 18.2 mcm ) 
 at a concentration of 1 g / l . in order to fully disperse the particles the solutions were stirred at 60 c for 3 h and then sonicated 
 until completely transparent . for the composition of solutions for 
 fret experiments shown in figure 8 , see table s1 in the supporting information . a stock solution of nr in 1,4-dioxane 
 was prepared at a concentration of 0.1 mm . a 1 g / l solution of clms 
 ( 82.8 l ) was diluted with water ( 2417 l ) to give [ dtm ] 
 = 1 m . to this micelle solution was added 2.5 l of the 
 nr stock solution to give a final [ nr ] = 0.1 m . the solution 
 was mixed with a vortex mixer for 1 s , and the emission was monitored 
 by fluorescence spectroscopy . the procedure above ( clms and nr ) 
 was repeated for solutions of nlms . in this case a 1 g / l solution 
 of nlms ( 79.9 l ) was diluted with water ( 2420 l ) to 
 give   = 1 m . the procedure above ( clms and nr ) 
 was repeated for solutions of clms and rhb . in this case a stock solution 
 of rhb in water was prepared at a concentration of 0.1 mm . in order 
 to synthesize bcp 
 micelles with dtm fluorophores in the shell or core , it was necessary 
 to synthesize two different bcps . shell - labeled micelles ( slms ) require 
 a bcp with the dtm fluorophore in the hydrophilic block , while core - labeled 
 micelles require a bcp with the dtm fluorophore in the hydrophobic 
 block ( figure 1 ) . schematic 
 representation of the route to shell - labeled micelles 
 ( slms ) and core - labeled micelles ( clms ) containing the dtm fluorophore 
 and the route to nonlabeled micelles ( nlms ) . the bcps used to form the labeled micelles were based on 
 poly(triethylene 
 glycol acrylate)-b - poly(tert - butyl 
 acrylate ) , p(tega)-b - p(tba ) , with 
 an average of approximately one repeat unit per chain of dithiomaleimide 
 acrylate ( dtma ) copolymerized into either 
 the p(tega ) shell - forming block or p(tba ) core - forming 
 block , as shown in scheme 1 . a nonfunctional p(tega)-b - p(tba ) was also synthesized to allow self - assembly of nonlabeled micelles 
 ( nlms ) for comparison . the dtm fluorophore is ideally suited to this 
 variable approach to bcp labeling , as the small size and intermediate 
 polarity of the fluorophore mean that it is simply incorporated into 
 both hydrophobic and hydrophilic polymers . conditions for all 
 polymerizations : 
 aibn ( 0.1 equiv with respect to raft agent ) , 1,4-dioxane , 65 c . the hydrophobic core blocks ( 1 and 2 ) 
 were synthesized first by raft polymerization of tba , using the commercially available raft agent cyanomethyldodecyl 
 trithiocarbonate , with aibn ( 0.1 equiv with respect to raft agent ) 
 as radical initiator , as a solution in 1,4-dioxane at 65 c . the nonlabeled core block 1 ( to be used to form shell- 
 and nonlabeled micelles ) consisted of a p(tba ) homopolymer , 
 while for the labeled core block 2 ( to be used to form 
 core - labeled micelles ) a copolymer of tba with dtma 
 was synthesized . for 2 , an average dp of 1 was targeted 
 for dtma to give incorporation of a single fluorophore per chain . h nmr spectroscopy indicated that for the nonlabeled homopolymer 
 ( 1 ) dptba = 44 , while for 
 the labeled copolymer ( 2 ) dptba = 36 and dpdtma = 1.1 . for both 1 and 2 the presence of the trithiocarbonate end - group was confirmed 
 by characteristic resonances of the dodecyl chain ( both h1 and h4 
 in figures s1 and s2 ) . sec analysis of 1 and 2 indicated a good control over molecular 
 weight ( m = 1.08 and 1.13 , respectively ) , 
 with trithiocarbonate retention indicated by polymer absorption at 
 309 nm ( figure 2 and table 1 ) . additionally , sec 
 analysis of 2 using a photodiode array detector showed 
 incorporation of the dtm chromophore , with the polymer peak having 
 the characteristic dtm absorption at ca . measured by sec ( 1 , 2 : thf eluent and 
 ps calibration ; 3 , 4 , 5 : dmf 
 eluent and pmma calibration ) . molecular 
 weight distributions obtained by sec using differential 
 refractive index ( dri ) and uv ( abs = 309 or 400 
 nm ) detectors for ( a ) p(tba ) ( 1 ) and 
 p(tega)-b - p(tba ) ( 3 ) , ( b ) p(tba ) ( 1 ) and p(tega - co - dtma)-b - p(tba ) ( 4 ) , and ( c ) p(tba - co - dtma ) 
 ( 2 ) and p(tega)-b - p(tba - co - dtma ) ( 5 ) . bcps were produced by the chain extension of the macro - raft 
 agents 1 and 2 according to scheme 1 . chain extension of 1 with 
 tega resulted in the nonlabeled bcp 3 , the precursor 
 to the nonlabeled micelles , while chain extension of 1 with tega and dtma ( targeting an average dp of 1 for dtma to give 
 incorporation of a single fluorophore per chain ) resulted in 4 , the precursor to shell - labeled micelles containing the 
 dtm fluorophore in the corona forming tega block . h nmr 
 spectroscopy indicated that 3 had dptega = 
 120 , while 4 had dptega = 140 and dpdtma = 1.1 ( figures s4 and s5 ) , giving hydrophobic 
 weight fractions ( fc ) of 18% and 15% for 3 and 4 , respectively , which would likely favor 
 the formation of star - like spherical micelles upon aqueous self - assembly . chain extension of 2 with tega 
 resulted in bcp 5 with a labeled core forming block ( the 
 precursor to core - labeled micelles ) . h nmr spectroscopy 
 indicated that 5 had dptega = 130 ( figure s6 ) , corresponding to a hydrophobic weight 
 fraction ( fc ) of 16% . in all cases sec 
 indicated good blocking efficiency , with molecular weight distributions 
 obtained from both differential refractive index and uv ( abs = 309 nm ) detectors showing consumption of the macro - raft 
 agents 1 and 2 , with a reasonable control 
 over molecular weight ( m = 1.351.38 
 for 35 ) . by monitoring absorption 
 at 400 nm ( absorption due to the dtm chromophore ) , incorporation of 
 dtma into the corona forming block of 4 the amphiphilic bcps 35 were assembled by direct dissolution 
 in water ( 18.2 mcm ) at 
 a concentration of 1 g / l . in order to fully disperse the particles , 
 the solutions were stirred at 60 c for 3 h and then sonicated 
 until completely transparent . self - assembled solutions of 35 were analyzed by multiangle laser light scattering 
 using a goniometer allowing simultaneous dynamic and static light 
 scattering ( dls and sls ) measurements ( see table 2 and figure s7 ) . particle hydrodynamic radius ( rh ) 
 was obtained directly from dls measurements and in all cases was approximately 
 equivalent with rh = 3436 nm ( figure 3 ) . measurement of 
 particle mw by sls allowed for the calculation 
 of aggregation number ( nagg ) , which was 
 found to vary between the systems ( table 2 ) . the trend of increasing nagg with fc could be explained 
 by considering that polymer unimers with higher fc ( greater hydrophobic character ) are less stable in aqueous 
 solution and therefore have a lower energy barrier for insertion . 
 despite this variation in nagg , the structural 
 similarity of the dtm - labeled micelles ( prepared from 4 and 5 ) to the nonlabeled micelles ( prepared from 3 ) indicates that incorporation of the dtm label has not had 
 a detrimental effect on the bcp self - assembly . from rh and nagg it is also possible 
 to estimate the micelle core and shell volumes ( vcore and vshell ) , and hence the local concentration of dtm fluorophores within the 
 micelles ( [ dtm ] ) could be calculated ( see experimental 
 section for details ) . these calculations revealed that despite 
 using the same ratio of dye for labeling the bcps 4 and 5 ( ca . 1 equiv per chain ) , two very different 
 local environments can be created : a ca . 400-fold 
 decrease in local concentration is obtained by locating the dtm in 
 the shell ( slms ) compared to locating the dtm in the core ( clms ) . ( a ) size 
 distribution obtained by dls ( detection angle of 90 ) 
 for a solution of nlms , slms , and clms at 1 ( b ) slms imaged by tem on a graphene 
 oxide support . scale bar = 100 nm . micelle solutions were 
 imaged by dry state transmission electron 
 microscopy ( tem ) using graphene oxide support tem grids in order to 
 examine micelle morphology . as shown in figure 3 , particles provided 
 a circular projection when dried to a graphene oxide surface , suggesting 
 they had a spherical morphology . in line with previous observations , only the p(tba ) micelle cores 
 provided sufficient contrast to be visualized by tem , with core diameters 
 in reasonable agreement with those obtained by light scattering . the steady state 
 emission and excitation spectra for solutions of labeled micelles were found to be very similar to that of analogous small molecule 
 dtms . a 2d excitation emission 
 spectrum for the core - labeled micelles is shown in figure 4a , with excitation maxima occurring 
 at 267 and 407 nm , with the corresponding emission maximum of 510 
 nm ( figure 4b ) . the 
 fluorescence quantum yield ( f ) for the core - labeled 
 micelles was measured using an integrating sphere to give an absolute 
 value of 17  2% . excitation and emission spectra were also recorded 
 for the shell - labeled micelles , which showed similar excitation and 
 emission . however , a red - shift in the emission maximum ( em , max ) to 520 nm was observed with a drastic reduction in 
 f to < 1% , as compared to the core - labeled micelles . the drastic reduction of f and bathochromic shift 
 of emission indicates the different environment of the chromophore , 
 which is consistent with collisional ( solvent ) quenching in the more 
 polar environment of the solvated micelle shell . these results are 
 in agreement with previous work using small molecule dtm fluorophores 
 which show both bathochromic shifts and reductions in f upon increasing solvent polarity ; for example , dithiobutanemaleimide 
 has em , max = 486 nm and f = 28% 
 in cyclohexane , whereas in methanol em , max = 546 
 nm and f < 1% . while 
 the possibility of ordered , coherent effects can not be overtly discounted , 
 we have seen nothing to indicate aggregation - induced emission , a process which is typically reserved for discussions 
 of neat or chromophore - rich , highly ordered systems with j - type emission 
 or h - type systems that interconvert to j - type emission . emission 
 spectra with a 5 nm step for 
 an aqueous solution of core - labeled micelles . ( b ) excitation and emission 
 spectra of aqueous solutions of core- and shell - labeled micelles . emission intensity was measured 
 over a range of concentrations 
 for aqueous solutions of the polymers 4 and 5 , whereby the integrated emission was calculated for the whole spectrum 
 and these values normalized by the concentration of polymer chains 
 in solution ( figure 5 ) . for both polymers a relatively flat emission intensity over 3 
 orders of magnitude in concentration was observed , corresponding to 
 the micellar state ( shell - labeled micelles for 4 and 
 core - labeled micelles for 5 ) . deviation from the flat 
 emission intensity occurred at c  1  
 10 m for 4 and c  5  10 m for 5 and 
 was assigned to a transition from micelles to solvated polymer unimers 
 upon decreasing concentration . for polymer 4 the dtm fluorophore is already solvated by water in the 
 micelle shell , so the transition from micelles to unimers leads to 
 an increase in emission intensity due to increased protection from 
 solvent interactions with the presence of the hydrophobic core block 
 in the unimer coil . however , for polymer 5 the dtm fluorophore 
 is protected from the surrounding solvent due to its location in the 
 micelle core . therefore , upon transition to the polymer unimer state 
 an increase in solvation occurs , leading to dye  solvent quenching 
 and a corresponding decrease in emission intensity . in both cases , the emission intensity self - reports on the supramolecular state of 
 the polymer allowing a convenient way to determine the critical micelle 
 concentrations ( cmcs ) , which correspond to 3.8 and 1.7 mg / l for shell- 
 and core - labeled micelles , respectively . the higher cmc of the shell - labeled 
 micelles relative to the core - labeled micelles is in agreement with 
 the shell - labeled micelles possessing a lower nagg  both phenomena being explained by a greater solubility 
 of unimers of polymer 4 relative to 5 due 
 to 4 having a lower fc . within 
 the micellar region emission anisotropy ( r ) for both 4 and 5 was found to be 0.29  0.01 and 
 0.19  0.01 , respectively , further confirming that the emissive 
 dtm fluorophore was incorporated into a macromolecular structure , 
 as analogous small molecule dtm dyes have rca . 0 in solution . it is valuable to observe 
 that the total increase in emission intensity for polymer 4 is not as severe as the decrease in the emission intensity observed 
 in polymer 5 on transition to the unimer state from the 
 micellar state . additionally , it is interesting to note that the higher 
 dye density ( [ dtm ] ) within the core block of the core - labeled micelles 
 does not result in overt quenching . this is important in terms of 
 application , where total change in intensity for a given species will 
 be critical and where the initial species ( micelle ) should be as bright 
 as possible , and points to a core - labeled system being more viable 
 than a corona - labeled one . emission intensity ( normalized to polymer chain 
 concentration ) 
 with respect to concentration for polymers 4 and 5 . fluorescence lifetime was measured for 
 aqueous solutions of polymers 4 and 5 using 
 time - correlated single photon counting . samples were excited with 
 a pulsed 405 nm diode laser ( 60 ps full width at half - maximum ) , and 
 the resultant emission decays were modeled as a sum of exponential 
 decays after deconvolution with the instrument response function . decay spectra are shown in figure 6 , with the average lifetimes and lifetime components 
 listed in table 3 . for both 4 and 5 spectra were recorded for 
 an aqueous solution at 5  10 m corresponding 
 to the micellar regime ( shell- and core - labeled micelles ) and an aqueous 
 solution at 5  10 m corresponding to polymer 
 unimers ( below the cmc ) . a dehydrated thin film was also prepared 
 by drying a drop of micelle solution to a glass slide , with the spectra 
 collected by fluorescence lifetime imaging microscopy , where the intensity 
 decay was calculated by summation of the decays for each pixel in 
 the image ( figure s8 ) . fluorescence lifetime 
 decay spectra ( points ) , with fitting ( lines ) , 
 residuals ( bottom ) , and instrument response function ( irf ) , for aqueous 
 solutions of ( a ) 4 and ( b ) 5 . the fluorescence lifetime decay spectra clearly exhibit two important 
 features . the first is that the shell - labeled micelles formed from 4 have a significantly faster decay than the core - labeled 
 micelles formed from 5 , with intensity - averaged lifetimes 
 of the excited state ( av , i ) of 7.0  0.1 and 
 18.8  0.3 ns , respectively . this is as a result of a near ultrafast 
 lifetime component with significant amplitude for shell - labeled micelles 
 ( 1 = 0.40  0.06 ns , a1 = 0.71 ) , which is assigned to excited state annihilation 
 by solvent collision and can be interpreted as the result of poor 
 fluorophore protection . in contrast , the major lifetime component 
 for the core - labeled micelles is 2 = 17.5  
 0.1 ns , with amplitude a2 = 0.96 . for 
 the core - labeled micelles the dye is located within the dehydrated 
 core and is therefore encapsulated within the supramolecular structure , 
 whereas for the shell - labeled micelles location of the dye within 
 the solvated corona provides poor protection to the dtm fluorophore 
 from solvent quenching . this interpretation is supported by the decay 
 spectrum of unimers of 4 , which also have av , i = 7.0  0.1 ns ( near ultrafast lifetime component 
 1 = 0.32  0.06 ns , a1 = 0.72 ) , indicating that shell - labeled micelle formation 
 does not change the local environment for the dtm , whereas an increase 
 in av , i to 14.8  0.3 ns for the dehydrated 
 film of 4 gives a closer representation to the intrinsic 
 lifetime for polymer 4 . these results are in agreement 
 with the observation of a lower f for the shell - labeled 
 micelles compared to the core - labeled micelles and further emphasize 
 that the optimum location for the dtm dye to obtain the greatest emission 
 is within the micelle core . the second important feature that 
 the decay spectra highlight is 
 the ability to discriminate the micellar state of 5 from 
 measurements of fluorescence lifetime . a relatively long lifetime 
 was observed for 5 in the micellar state ( av , i = 18.8  0.3 ns ) , whereas the unimer state showed 
 a significant decrease to av , i = 9.2  0.2 
 ns , due to a near ultrafast ( solvent collision ) component to the decay 
 ( 1 = 0.56  0.06 ns , a1 = 0.60 ) . again this interpretation was supported by fluorescence 
 lifetime imaging microscopy measurements of a dehydrated film of micelles , 
 which had the same decay as the micelle solution ( av , i = 18.5  0.2 ns ) , indicating that the core of the core - labeled 
 micelles is largely solvent free . we have previously shown with a 
 related interface - labeled system that this ability to discriminate 
 between micelles and unimers simply by measuring fluorescence lifetime 
 could be translated to in vitro imaging , such that 
 micelles and unimers could be located within discrete areas of rat 
 hippocampal tissue . as the micelle - to - unimer 
 transition is widely exploited as a trigger for controlled drug delivery 
 from polymer nanoparticles , we expect 
 that this feature of the core - labeled dtm micelles would provide a 
 simple method to identify such controlled release in vitro . fret describes a phenomenon 
 whereby two fluorophores can interact when in close proximity to one 
 another . energy transfer occurs between a donor molecule in the excited 
 state and an acceptor molecule , provided there is sufficient spectral 
 overlap between donor emission and acceptor excitation and that the 
 two molecules are positioned within the necessary frster distance . the result is emission from the acceptor fluorophore upon excitation 
 of the donor fluorophore , according to their respective excitation 
 and emission wavelengths . monitoring the fret process for fluorescently 
 labeled micelles has been exploited to measure cmcs , to identify morphology response to stimuli , and to follow the uptake and release of fluorescent payloads . because of the interest surrounding the 
 use of nanoparticles as delivery agents , we sought to investigate whether the uptake of model compounds by 
 the core - labeled micelles could be identified using fret . the dtm 
 fluorophore was designated as the fret donor due to its broad excitation 
 spectra and to also ensure that all emission originated from a labeled 
 micelle . two fret acceptor molecules whose excitation spectra overlapped 
 with the dtm emission were chosen as probes for interaction with , 
 and uptake into , the core - labeled micelles : nile red ( nr ) as a hydrophobic 
 guest expected to partition to the micelle core and rhodamine b ( rhb ) 
 as a hydrophilic guest expected to partition to the aqueous solution 
 or the solvated micelle shell ( figure 7 ) . to reduce the background fluorescence ( i.e. , non - fret 
 emission ) from the probes , a 10-fold excess in total dtm concentration 
 was used relative to nile red and rhodamine b concentration , while 
 all dyes were present at concentrations corresponding to an absorbance 
 < 0.1 to negate inner filter effects . ( a  c ) schematic 
 representation of interaction between micelles 
 and fluorescent dyes nile red ( nr ) and rhodamine b ( rhb ) . ( d ) structures 
 of nile red and rhodamine b. to study uptake of the hydrophobic dye , a solution of nile 
 red 
 in 1,4-dioxane ( 2.5 l , 0.1 mm ) was added to a solution of core - labeled 
 micelles ( 2.5 ml ) with [ dtm ] = 1 m , to give a final [ nile red ] 
 = 0.1 m . emission spectra were recorded for the solution with 
 an excitation wavelength of 422 nm , corresponding to the excitation 
 maximum of the dtm donor . quenching of the dtm emission at 515 nm 
 was observed , with a corresponding enhancement of nile red emission 
 at 610 nm ( figures 7a and 8a ) . quenching and 
 enhancement occurs within 10 s ( the time of the first measurement , 
 see figure s9 ) , at which time equilibrium 
 has been reached with no further change after 60 min . these results 
 demonstrate that fret occurs between donor ( dtm ) and acceptor ( nile 
 red ) , indicating the proximity of the two fluorescent species . as 
 fret is extinguished beyond the frster distance ( typically 
 < 4 nm ) , fret between dtm and nile red corresponds to the presence 
 of nile red within the core of the core - labeled micelles . as a control , the protocol of nile red addition was repeated for a solution of nonlabeled 
 micelles where the polymer concentration was maintained with respect 
 to the core - labeled micelles ( figures 7b and 8b ) . in this case an increase 
 in emission at 610 nm was observed , as it is well - known that nile 
 red emission is quenched in water and subsequently restored upon partition 
 to a more hydrophobic environment . however , the detectable change 
 in emission that results from this  background  increase 
 in nile red brightness upon partition was 2.5 lower than the 
 combined partition and fret effect observed for the core - labeled micelles . in addition , a greater ambiguity is associated with the interpretation 
 of changes in nile red emission on its own , as these variations result 
 from any change in environment polarity . finally , the fret experiment 
 was repeated for the core - labeled 
 micelles using the hydrophilic dye rhodamine b ( figures 7c and 8c ) , which was 
 added to the solution of core - labeled micelles as a solution in water 
 ( 2.5 l , 0.1 mm ) to give a final [ rhodamine b ] = 0.1 m . 
 in this case no change in the intensity of emission at 515 nm was 
 observed ( dtm emission was not quenched ) , while the intensity of emission 
 at 615 nm was accounted for by a summation of the emission from core - labeled 
 micelles ( t = 0 ) and a 0.1 m rhodamine b solution 
 in water ( rhodamine b emission was not enhanced ) . this experiment 
 therefore shows that fret does not occur between the dtm fluorophore 
 in core - labeled micelles and rhodamine b , indicating that rhodamine 
 b does not partition to the core of the core - labeled micelles . collectively 
 these fret experiments demonstrate that the incorporation of the dtm 
 dye in the core - labeled micelles allows the micelles to report on 
 the presence ( nile red ) or absence ( rhodamine b ) of a cargo molecule 
 within the micelle core via a simple measure of emission . furthermore , 
 although too fast in this example , measuring the rate for fret could 
 provide details of the kinetics of cargo encapsulation and release , 
 as has been shown previously for core cross - linked polymer nanoparticles . taken in conjunction with the steady state and 
 time - resolved fluorescence data , this final finding points to dtm 
 core labeling being superior to coronal labeling for all of the most 
 major considerations in nanocontrast / nanotheranostic systems : it can 
 be seen ( bright ) , it can report on the supramolecular state ( changes 
 in emissive character ) , and it can signal with regards to loading / unloading 
 ( fret ) . ( a ) emission spectra of clms at t = 0 , clms at 
 1 min ( t = 1 ) and 60 min ( t = 60 ) 
 after addition of nile red ( nr ) , and nr in water ( 0.1% 1,4-dioxane ) . ( b ) emission spectra of nlms at t = 0 , nlms at 1 
 min ( t = 1 ) and 60 min ( t = 60 ) 
 after addition of nr , and nr in water ( 0.1% 1,4-dioxane ) . ( c ) emission 
 spectra of clms at t = 0 , clms at 1 min ( t = 1 ) and 60 min ( t = 60 ) after addition 
 of rhodamine b ( rhb ) , and rhb in water . ex = 422 
 nm in all cases , and peaks at 495 nm correspond to the raman scattering 
 of water . poly(triethylene 
 glycol acrylate)-b - poly(tert - butyl 
 acrylate ) bcp micelles have been synthesized 
 with a fluorescent dtm group incorporated into the micelle core or 
 shell . the advantages of using dtm chemistry are the small size and 
 intermediate polarity of this fluorophore as well as its excellent 
 compatibility with bcp synthesis and self - assembly and its proven 
 applicability to tissue imaging . it was found locating the dtm fluorophore 
 in the micelle core resulted in greater emission ( f = 17% ) and a longer fluorescence lifetime ( av , i = 19 ns ) , when compared to locating the fluorophore in the shell 
 ( f < 1% , av , i = 7 ns ) , as a 
 result of better protection of the fluorophore in the core from solvent 
 collisional quenching . for both shell and core - labeled micelles it 
 was possible to measure the onset of aggregation ( with respect to 
 concentration ) by measuring the emission intensity . the transition 
 from micelle - to - unimer could also be detected for the core - labeled 
 micelles by fluorescence lifetime spectroscopy since the polymer unimers 
 have a significantly shorter lifetime ( av , i = 9 
 ns ) . following our previous work , we 
 believe that the core - labeled micelles ability to self - report 
 on their supramolecular state would allow in vitro discrimination between assembled and disassembled micelles using 
 fluorescence lifetime imaging microscopy . the presence of the dtm 
 label allows the encapsulation of a fluorescent hydrophobic guest 
 ( nile red ) to be monitored by measuring fret between the dtm ( donor ) 
 and nile red ( acceptor ) . uptake of the hydrophobic guest dye was found 
 to occur very quickly ( < 10 s ) , while no fret was observed with 
 a hydrophilic guest ( rhodamine b ) , indicating that this small molecule 
 is not encapsulated in the micelle core . the use of this simple dtm 
 label can therefore produce fluorescent bcp micelles that can self - report 
 on both their supramolecular structure and the presence or absence 
 of cargo molecules .
block copolymer micelles have been 
 prepared with a dithiomaleimide 
 ( dtm ) fluorophore located in either the core or shell . 
 poly(triethylene 
 glycol acrylate)-b - poly(tert - butyl 
 acrylate ) ( p(tega)-b - p(tba ) ) was 
 synthesized by raft polymerization , with a dtm - functional acrylate 
 monomer copolymerized into either the core forming p(tba ) block or the shell forming p(tega ) block . 
 self - assembly by direct 
 dissolution afforded spherical micelles with rh of ca . 
 35 nm . 
 core - labeled micelles ( clms ) 
 displayed bright emission ( f = 17% ) due to good 
 protection of the fluorophore , whereas shell - labeled micelles ( slms ) 
 had lower efficiency emission due to collisional quenching in the 
 solvated corona . the transition from micelles to polymer unimers upon 
 dilution could be detected by measuring the emission intensity of 
 the solutions . 
 for the core - labeled micelles , the fluorescence lifetime 
 was also responsive to the supramolecular state , the lifetime being 
 significantly longer for the micelles ( av , i = 19 
 ns ) than for the polymer unimers ( av , i = 9 ns ) 
 . 
 the core - labeled micelles could also self - report on the presence of 
 a fluorescent hydrophobic guest molecule ( nile red ) as a result of 
 frster resonance energy transfer ( fret ) between the dtm fluorophore 
 and the guest . 
 the sensitivity of the dtm fluorophore to its environment 
 therefore provides a simple handle to obtain detailed structural information 
 for the labeled polymer micelles . a case 
 will also be made for the 
 application superiority of core - labeled micelles over shell - labeled 
 micelles for the dtm fluorophore .