ARTICLE pubs.acs.org/JPCC
Ensemble and Single-Particle Fluorescence Photomodulation in Diarylethene-Doped Conjugated Polymer Nanoparticles Christina M. Davis, Elizabeth S. Childress, and Elizabeth J. Harbron* Department of Chemistry, The College of William and Mary, Williamsburg, Virginia 23187-8795, United States ABSTRACT: Fluorescent systems that can undergo intensity photomodulation in aqueous environments are finding increasing applications, particularly in high-resolution imaging of biological samples. We seek to develop conjugated polymer nanoparticles (CPNs) with bright fluorescence that can be modulated with a light signal. Here, we present CPNs, doped with a photochromic diarylethene dye, that exhibit efficient fluorescence photomodulation that is thermally irreversible. In their UVabsorbing open form, the diarylethenes have no effect on the fluorescence properties of the bright CPNs. A brief period of UV irradiation converts the dyes to their visible-absorbing closed form, which is an efficient fluorescence quencher for the CPNs, likely via a fluorescence resonance energy transfer mechanism. Aqueous suspensions of dye-doped CPNs prepared from the homopolymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) or a polyfluorene-phenylenevinylene copolymer (PFPV) exhibit thermally stable bright and dark levels. The dye-doped MEH-PPV CPNs also exhibit photomodulation in single-nanoparticle imaging experiments, which reveal that nearly all CPNs retain a small amount of residual emission in the dark state. Their PFPV counterparts undergo irreversible fluorescence photobleaching rather than photomodulation in singlenanoparticle studies. The photostability of the CPNs under the UV irradiation conditions required for photochromic conversion is investigated on the single-particle level, and PFPV CPNs are found to be particularly susceptible to photobleaching upon 254 nm irradiation. These results will guide the selection of polymers and photochromes for CPNs intended for single-particle photomodulation.
’ INTRODUCTION The ability to modulate the emission properties of fluorescent molecules and materials has enabled an increasing number of applications in chemistry, materials science, and biology. Photomodulation, where emission intensity modulation is induced by a light signal, is the basis for ultrahigh-resolution microscopy techniques that can image features much smaller than diffraction-limited spot sizes.1 Many such “nanoscopy” techniques use temporal switching of fluorophore intensity to achieve this ultrahigh resolution: a given fluorophore can be localized with high precision if neighboring fluorophores are switched to a dark or “off” state at the time of imaging. Many fluorophores for these imaging techniques, as well as other photomodulation applications, rely on a photochromic moiety to induce the photomodulation.2,3 Here, we report fluorescence photomodulation of highly fluorescent conjugated polymer nanoparticles doped with a photochromic diarylethene derivative. Photochromic molecules have two different molecular forms that, by definition, have different absorbance properties, i.e., color.46 The most common photochromic molecules are colorless until ultraviolet (UV) irradiation induces the photochromic conversion to their photogenerated form, which is typically colored. Most photochromic molecules are not appreciably fluorescent on their own, but strategies have emerged to harness their photoswitching capability for fluorescence photomodulation. Li and co-workers synthesized polymeric nanoparticles in which photochromic spiropyrans are part of a hydrophobic core r 2011 American Chemical Society
surrounded by a hydrophilic shell that enables stable suspension of the particles in water.7 The nanoparticles are nonfluorescent when the spiropyrans are in their colorless, UVabsorbing form but become highly fluorescent when the photochromes are switched to their colored, visible-absorbing merocyanine form. The rigid, hydrophobic environment of the nanoparticle interior restricts nonradiative decay pathways such that the normally nonfluorescent merocyanine becomes highly fluorescent. The use of these nanoparticles in nanoscopic imaging has been demonstrated.8,9 Another strategy for fluorescence modulation with photochromic molecules is to combine the photochromic unit, covalently or noncovalently, with a separate fluorophore. In this method the photochrome acts as a switch for fluorescence quenching: it has no effect on fluorescence intensity in its UVabsorbing form but becomes an efficient fluorescence quencher when switched to its visible-absorbing form. The quenching mechanism may be fluorescence resonance energy transfer (FRET), photoinduced electron transfer (PET), or a combination of mechanisms. Irie and co-workers recently demonstrated a nondestructive readout of the fluorescence signal of a photochrome-dye dyad at the single-molecule level.10 In this system the fluorescence of a perylenebisimide dye is quenched by PET Received: July 7, 2011 Revised: August 24, 2011 Published: August 30, 2011 19065
dx.doi.org/10.1021/jp206438p | J. Phys. Chem. C 2011, 115, 19065–19073
The Journal of Physical Chemistry C upon photocyclization of a covalently attached diarylethene moiety. The wavelengths required for photochromic switching are all shorter than those required for fluorescence readout, thus decoupling the switching and fluorescence readout. The use of photochromic molecules as FRET or PET gates for fluorescence photomodulation has been reviewed.2,1115 Conjugated polymer nanoparticles possess physical and photophysical properties that make them extremely desirable for use as the fluorophore component of photochromefluorophore systems. Popularized by McNeill and co-workers,16,17 conjugated polymer nanoparticles18,19 (CPNs) prepared via reprecipitation are small, spherical, surfactant-free particles that are stable in aqueous suspension. CPNs are easily prepared by injecting a small portion of a dilute conjugated polymer solution in a water-miscible organic solvent into water, and their size (5 nm and up) can be controlled through the concentration of the polymer solution. Their key features are bright fluorescence and good photostability: the total number of photons emitted by CPNs prior to photobleaching is generally 34 orders of magnitude larger than that of typical small molecule fluorophores.16 These features originate from the parent conjugated polymers, which are multichromophoric chains known for their high absorption cross sections and radiative rates.16 As nanoparticles, the conjugated polymers gain additional advantages associated with organic nanoparticles including a dramatic reduction in the blinking and photobleaching that can plague traditional fluorophores. The combination of outstanding brightness to volume ratio with excellent photostability has enabled the use of CPNs in live cell imaging. 16 These features also make CPNs promising fluorophores for photomodulation. We previously demonstrated efficient photomodulation in CPNs prepared from the conjugated polymer poly[2-methoxy5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) and doped with a photochromic spirooxazine dye.20 In its thermally stable UV-absorbing form, the spirooxazine has no effect on CPN fluorescence spectral intensity, position, or shape. A brief 365 nm irradiation converts the spirooxazine to its merocyanine form, the absorbance of which overlaps the CPN fluorescence. The merocyanine acts as an efficient fluorescence quencher, likely through a FRET mechanism, and CPN fluorescence is reduced to 5% of initial intensity. The merocyanine form of the photochrome is not thermally stable and reverts to the nonquenching spirooxazine within seconds, restoring CPN fluorescence to its initial level. The quenching and recovery can be cycled numerous times by repeated application of UV irradiation and dark recovery periods. The MEH-PPV/spirooxazine CPNs demonstrated that CPNs are appropriate fluorophores for photomodulation and that the photochromic conversion occurs in this system in spite of environmental constraints imposed by the particles. In the MEH-PPV/spirooxazine CPNs, the spirooxazine’s lack of thermal stability facilitated easy study of fluorescence intensity cycling. Whether thermal recovery of the photochrome’s photogenerated form is desirable depends on the intended application. In addition to the spirooxazine, several of the common photochromic families (azobenzenes, spiropyrans) have photogenerated forms that are not thermally stable in many environments. In contrast, fulgides21 and diarylethenes22 are prized for their thermal irreversibility, meaning that the photogenerated form of the photochrome is stable until irradiation with the appropriate (usually visible) wavelength returns it
ARTICLE
Scheme 1
Scheme 2
to its initial form. The photochromic conversion of a diarylethene derivative is depicted in Scheme 1. The UV-absorbing open form (1o) is converted to the visible-absorbing closed form (1c) via conrotory photochemical cyclization. The 1cf1o cycloreversion reaction can occur photochemically in a conrotory fashion or thermally in a disrotory fashion, but the activation energy barrier for the thermal reaction is so large that it precludes the thermal cycloreversion of 1c.23 Their thermal irreversibility has made diarylethenes the photochrome of choice for many applications,22,24,25 and their use in fluorescence photomodulation has been reviewed.26,27 The many diarylethene-containing systems developed for fluorescence photomodulation include examples in which the photochromic dye is covalently attached to the side chain of an organic conjugated polymer.2831 Our goal is to develop photochromic CPNs for thermally irreversible fluorescence photomodulation. We present herein CPNs doped with diarylethene derivative 1, which has no effect on CPN fluorescence in its 1o form and acts as an efficient fluorescence quencher in the photogenerated 1c form. Photochrome 1 is paired with two different conjugated polymers, the copolymer PFPV and homopolymer MEH-PPV (Scheme 2), to produce two sets of dye-doped CPNs. With thermally stable “on” and “off” fluorescence intensity levels, these CPNs can be used in contexts not possible with our previous spirooxazinedoped particles. The results of single-nanoparticle fluorescence imaging experiments will be presented in addition to ensemble data. The PFPV and MEH-PPV CPNs are found to have similar photomodulation capabilities when studied in aqueous suspension. For single-nanoparticle imaging, however, the photochromic MEH-PPV CPNs are much more photostable than their PFPV counterparts. Control experiments reveal that photobleaching induced by the UV irradiation required for the photochromic conversion is significant for the PFPV CPNs when they are deposited on slides. These results will guide the selection of polymers and photochromes for CPNs intended for single-particle photomodulation. 19066
dx.doi.org/10.1021/jp206438p |J. Phys. Chem. C 2011, 115, 19065–19073
The Journal of Physical Chemistry C
’ EXPERIMENTAL METHODS The photochromic dye 1,2-bis(2,4-dimethyl-5-phenyl-3thienyl)-3,3,4,4,5,5-hexafluoro-1-cyclopentene (1) and 4,40 -dimethyltriphenylamine were obtained from TCI America and used as received. Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4phenylenevinylene] (MEH-PPV) with an average molecular weight of 260 000 and a polydispersity of 4.3 and poly[(9,9hexyloxy)-1,4-phenylene}] (PFPV) with an average molecular weight of 49 000 and a polydispersity of 5.1 were obtained from American Dye Source (Quebec, Canada) and used as received. Dye-doped conjugated polymer nanoparticles were prepared according to a literature procedure.32 A 1 mg/mL solution of the conjugated polymer in tetrahydrofuran (THF) was stirred for at least 1 h under argon before filtering through a 0.7 μm filter to remove any aggregates. A portion of a 0.1 mg/mL solution of diarylethene 1 in THF was added to the polymer solution in the desired proportion, and the dyepolymer solution was diluted with THF to achieve a polymer concentration of 0.01 (PFPV) or 0.02 mg/mL (MEH-PPV). The resulting solution was sonicated for 30 s before a 1 mL portion of solution was injected into 8 mL of water under sonication. After 2 min of additional sonication, the THF was removed by rotary evaporation. The resulting aqueous nanoparticle suspension was then filtered through a 0.22 μm filter. Undoped control nanoparticles were prepared by the same procedure with the omission of dye. The dye-to-polymer ratio for 1-doped MEHPPV CPNs was 28 wt %, which equates to 133 dyes per polymer chain given the average chain length. The MEH-PPV CPNs also contained 4,40 -dimethyltriphenylamine at one-third the molar concentration of 1 as a tertiary amine additive to prevent quenching by hole polarons, which are formed on the polymers upon irradiation.33 The 1-doped PFPV CPNs were 40 wt %, which equates to 36 dyes per polymer chain due to the shorter chain length of this polymer. Two experiments in which 60 wt % PFPV CPNs were used (53 dyes/chain) are explicitly noted in figure captions. Nanoparticle size distributions were measured in aqueous suspension by dynamic light scattering using a Nicomp 380 ZLS Submicron Particle Sizer (Particle Sizing Systems). Ensemble absorption and fluorescence experiments were conducted with as-prepared aqueous suspensions of nanoparticles that were degassed with argon in semi-micro septum-sealed cuvettes (Starna) except where otherwise noted. Absorption and fluorescence measurements were made with a Varian Cary 50 and a Varian Eclipse, respectively. Samples for single-nanoparticle imaging experiments were prepared by spin coating (Chemat Technology Spin-Coater KW-4A) a sonciated solution of 0.1 mL of CPN suspension, 0.2 mL of 10% (w/v) poly(vinyl alcohol), and 0.7 mL of water onto rigorously cleaned glass coverslips (Fisher). The inverted fluorescence microscope (Zeiss Axiovert 200) has been described previously.34 Briefly, the sample is raster scanned over a 488 nm laser beam (Melles Griot) by a nanopositioning stage (Mad City Labs), and fluorescence is collected by an avalanche photodiode detector (APCM-AQR-14, PerkinElmer Optoelectronics). In both ensemble and single-nanoparticle fluorescence experiments, the 1of1c photochromic conversion was induced by irradiating for 2 s with a 254 nm pencil lamp (Spectroline). The 1cf1o cycloreversion was induced by irradiating with a white light light-emitting-diode (LED) flashlight for 48 min except where room light exposure was used instead as noted in the text and figure captions. Average
ARTICLE
Figure 1. (A) Absorption spectra of photochromic dye 1 in dilute THF solution before (1o, solid line) and after (1c, dotted line) 254 nm irradiation. Inset: expansion of the visible region of the spectra. (B) Fluorescence spectra of PFPV (dotted line) and MEH-PPV (dashed line) CPNs in aqueous suspension along with the absorption spectrum of 1c (solid line) in THF.
intensities for each individual CPN in a given image were determined using ImageJ software.
’ RESULTS AND DISCUSSION The photochemical and photophysical properties of diarylethene derivative 1 are well-matched to the requirements for efficient fluorescence photomodulation. The quantum yield for cyclization of 1 in solution is 46%,22 which is close to the theoretical maximum of 50% that is imposed because only one of the two possible conformations can cyclize. A photochrome with a high cyclization quantum yield will require a shorter period of UV irradiation to reach its photostationary state than one with a lower quantum yield, thus minimizing the potentially damaging effects of UV irradiation on the fluorophore. The absorption spectra of 1o and 1c are widely separated such that CPN fluorescence spectra will overlap exclusively with the absorption of 1c and not at all with 1o. As shown in Figure 1A, 1o in THF solution absorbs only in the UV with a λmax,abs below 300 nm, while photogenerated 1c has a new absorption centered below 600 nm as well as a second peak at ca. 380 nm. The minimal absorbance of 1c in the 400500 nm region means that these wavelengths can be used for fluorescence excitation in ensemble and single-nanoparticle experiments with minimal perturbation to the 1o1c population distribution. The structures of the two conjugated polymers used to prepare 1-doped CPNs are shown in Scheme 2. MEH-PPV was selected because it exhibited efficient photomodulation in our previous work with spirooxazine-doped CPNs.20 PFPV was also employed here because PFPV CPNs have been shown to have a much higher quantum yield of fluorescence than their MEH-PPV counterparts (Φfl = 0.08 vs 0.01).16 Size distributions of 1-doped CPNs and undoped control CPNs prepared from both polymers were measured in aqueous suspension using dynamic light scattering. For all samples, light scattering data fit well to a bimodal distribution in which 99100% of the 19067
dx.doi.org/10.1021/jp206438p |J. Phys. Chem. C 2011, 115, 19065–19073
The Journal of Physical Chemistry C
Figure 2. Absorption spectra of 1-doped PFPV CPNs (A) and 1-doped MEH-PPV CPNs (B) in degassed aqueous suspension before (solid lines) and after (dotted lines) 254 nm irradiation. The insets show expansions of 30 (A) and 10 nm (B) portions of the visible region.
number-weighted distribution was 11 nm or smaller. Larger aggregates comprised the remaining 1% or less of total CPNs in all samples. These results are consistent with our previous work, which showed an average CPN size of 8 nm by atomic force microscopy (AFM) and no size difference between doped and undoped CPNs.20 The average number of polymer chains per CPN was calculated by two methods, one that assumes a typical organic density20,35 and the other that makes reference to the hydrodynamic diameters of polystyrene standards.36,37 Both methods yielded the same result, with MEH-PPV CPNs containing a single polymer chain and CPNs prepared from the lower molecular weight PFPV containing 7 chains/CPN. Fluorescence spectra of 1-doped CPNs prepared from both polymers are shown in Figure 1B. In addition to different quantum yields, the two polymers also have very different fluorescence spectra in aqueous suspension with λmax,fl values of 507 and 590 nm for PFPV CPNs and MEH-PPV CPNs, respectively. The fluorescence spectra of both sets of CPNs are red-shifted from those of their respective parent conjugated polymers in organic solution. This shift is standard for CPNs and is attributed to energy transfer to and subsequent emission from low-energy, aggregated sites within the tightly packed nanoparticles.17 The CPN fluorescence spectra in Figure 1B are consistent with those from the literature for the same polymers16,38 and exhibit no change in intensity, shape, or position upon doping. Despite their widely separated fluorescence spectra, both types of CPNs have substantial overlap with the broad visible absorbance of 1c (Figure 1B). Thus, 1c should be able to quench the fluorescence of both CPNs via FRET if it can be photogenerated in the doped nanoparticles. Absorbance studies reveal that 1c can indeed be photogenerated in both types of CPNs. Figure 2 shows absorption spectra of PFPV (A) and MEH-PPV (B) CPNs before and after 2 s of 254 nm UV irradiation. At full scale, the spectra appear to be unchanged, indicating that irradiation did not damage the polymer structures to a detectable extent. Closer inspection of the 500650 nm region of the spectra reveals a small but highly
ARTICLE
Figure 3. (A) Fluorescence spectra of 1-doped PFPV CPNs in degassed aqueous suspension before (solid line) and after (dotted line) 254 nm irradiation and after visible irradiation (dashed line). (B) Peak fluorescence intensity of 1-doped PFPV (black squares) and MEH-PPV (black circles) CPNs and undoped control PFPV (open squares) and MEHPPV (open circles) CPNs before irradiation, after 254 nm irradiation, and after visible irradiation via white LED (PFPV) or room light (MEHPPV) exposure. MEH-PPV intensities were recorded 3 min after cessation of irradiation to eliminate transient effects due to hole polarons. (C) Peak fluorescence intensity of 1-doped PFPV CPNs during irradiations and dark periods as described in the text. (D) Peak fluorescence intensity of 1-doped MEH-PPV CPNs during irradiations and dark periods as described in the text.
reproducible increase in the absorbance upon UV irradiation. Magnified in the Figure 2A inset for PFPV CPNs, the UVgenerated absorbance in this region matches the 1c absorbance spectrum shown in Figure 1A. Both absorbance spectra show their greatest intensity between 530 and 620 nm with a λmax,abs of 578 nm. The 1c absorbance is more difficult to detect in the MEH-PPV CPNs due to the much greater overlap between the absorption spectra of the CPNs and 1c in the region of interest but can still be seen in the magnified Figure 2B inset. The average ΔA upon UV irradiation in the 576580 nm region corresponding to the 1c λmax,abs is 0.0011 for both samples depicted in Figure 2. The concentration of 1c can be calculated from this absorbance and corresponds to 11% of total doped 1 for PFPV CPNs and 8% for MEH-PPV CPNs. These numbers represent higher photochromic conversions than we observed previously for spirooxazine-doped MEH-PPV CPNs (1.55%). This result might be due to the much smaller amount of free volume required for the cyclization of 1 as compared to the ring opening of a spirooxazine, the increased quantum yield for photochromic conversion of 1 over the spirooxazine,22,39 or a combination of these factors. The fluorescence of both PFPV and MEH-PPV CPNs is significantly quenched by the photogenerated 1c dyes. The fluorescence of the PFPV CPNs from Figure 2A is shown in Figure 3A before irradiation, after UV irradiation to induce 1of1c cyclization, and after visible irradiation to induce the 1cf1o cycloreversion. The peak fluorescence intensity is quenched to 7% of its initial value when the dyes are in their 1c form and recovers nearly all of its intensity when the dyes are returned to their 1o state. We quantify the magnitude of fluorescence photomodulation and recovery with the modulation efficiency 19068
dx.doi.org/10.1021/jp206438p |J. Phys. Chem. C 2011, 115, 19065–19073
The Journal of Physical Chemistry C
ARTICLE
(Emod, eq 1) and modulation recovery (Erec, eq 2) Emod ¼ 1 Erec ¼
Ipss I0
Irec I0
ð1Þ ð2Þ
where I0, Ipss, and Irec are the fluorescence intensities before irradiation, after UV irradiation to produce the photostationary state (pss) concentration of 1c, and after visible irradiation to return the photochrome to its 1o form, respectively. Both the Emod and Erec for the PFPV CPNs in Figure 2A have values of 93%. The relative peak intensity values from these PFPV spectra as well as for the MEH-PPV sample from Figure 2B are displayed in Figure 3B. The MEH-PPV CPNs exhibit slightly poorer metrics than the PFPV sample, with an Emod value of 83% and an Erec value of 80%. However, the poorer recovery is to be expected due to the fact that it was induced with room light exposure for the MEH-PPV sample versus more intense LED white light illumination for the PFPV sample. For both types of CPNs, Emod values generally range from 80 to 95% and Erec values from 80 to 93% depending on the doping level and the individual sample. Undoped MEH-PPV and PFPV CPNs were subjected to the same UV and white light irradiation conditions as a control to demonstrate that the intensity modulation is a result of the photochromic conversion. Relative peak fluorescence intensities shown in Figure 3B demonstrate that neither control sample exhibits any modulation: MEH-PPV CPNs actually become slightly brighter upon light exposure while PFPV CPNs exhibit minor photobleaching. As exemplified by the Figure 3B data, the PFPV CPNs generally exhibit somewhat higher Emod values than their MEH-PPV counterparts when samples with comparable concentrations of photogenerated 1c quenchers are compared. Although the quenching mechanism has not been verified, these CPNs were designed to undergo efficient FRET. The spectral overlap of donor fluorescence and acceptor absorbance required for FRET is quantified by the overlap integral, J(λ), which depends on the corrected fluorescence intensity of the donor (FD) and the extinction coefficient of the acceptor (εa) as shown in40 Z ∞ JðλÞ ¼ FD ðλÞ εa ðλÞλ4 dλ ð3Þ 0
J(λ) for 1c-doped PFPV CPNs is 63% of that for the MEH-PPV CPNs. However, the FRET efficiency also depends on a number of other factors in addition to J(λ), including the quantum yield of the fluorescence donor.40 Given that the quantum yield is much higher for PFPV CPNs than MEH-PPV CPNs (Φfl = 0.08 vs 0.01),16 the FRET efficiency for PFPV CPNs is predicted to be on the order of 1.2 times that of the MEH-PPV CPNs, which is in qualitative agreement with the results presented here. The significant fluorescence quenching of PFPV and MEHPPV CPNs observed upon photochromic conversion is induced by a relatively small number of 1c dyes. Using the photogenerated 1c absorbance and the polymer concentration, the number of 1c quenchers per CPN can be estimated to be 1025 dyes/ nanoparticle. Conjugated polymers are multichromophoric, and we previously estimated that a typical MEH-PPV CPN contains roughly 100 chromophores.20 Therefore, each 1c dye quenches multiple conjugated polymer chromophores. This amplified
Figure 4. (A) Peak fluorescence intensity of 1-doped PFPV CPNs in degassed aqueous suspension over multiple cycles of 254 nm irradiation and visible irradiation. (B) Peak fluorescence intensity (squares) and absorbance integrated over 524624 nm (circles) for 60 wt % 1-doped PFPV CPNs in non-degassed aqueous suspension over two UV/visible irradiation cycles.
quenching is possible because of conjugated polymers’ intrinsic energy migration processes that can funnel multiple excitons to a single low-energy quencher and is the basis for sensing applications that harness this amplified response.41 The key feature of the 1-doped CPNs is their thermal stability in both on and off states, and parts C and D of Figure 3 demonstrate the stability of the dark level for PFPV and MEHPPV CPNs, respectively. Figure 3C shows the drop of the peak fluorescence intensity during the UV irradiation period that produces 1c and then confirms that the dark level (Emod = 88%) remains constant a half-hour later. Visible irradiation then returns the sample to its bright state with an Erec value of 85%. Likewise, the peak fluorescence intensity of the MEH-PPV sample in Figure 3D drops during UV irradiation with an Emod value of 92%, which remained stable for the 1.72 days it was studied. The MEH-PPV sample was not exposed to a strong visible light source for 1cf1o conversion; however, upon opening of the fluorimeter lid to expose the sample to weak ambient room light, the fluorescence intensity recovered with an Erec value of 44%. Both samples demonstrate that the famed thermal irreversibility of 1 enables stable dark levels in the fluorescence photomodulation of photochromic CPNs. The fluorescence photomodulation demonstrated above for individual cycles can be repeated over multiple cycles by alternating UV and visible irradiation periods to switch between the 1o and 1c forms of the photochrome. Figure 4A demonstrates cycling for PFPV CPNs with average Emod and Erec values of 84 and 83%, respectively. Although some intensity is not recovered after the first UV irradiation, the Erec value remains fairly consistent after that first cycle, and stable cycling is observed. This repeated cycling requires photostability of both the CPN fluorophore and the photochrome 1. With 1 known to be fatigue-resistant over hundreds of cycles,22 the most likely culprit for the Erec values of less than 100% is the fluorophore. CPNs are known for their excellent photostability under normal ensemble and singleparticle fluorescence conditions.16 However, these normal conditions seldom include higher energy UV irradiation such as that applied here to induce the photochromic conversion. To investigate CPN photostability under our experimental conditions, we monitored the visible absorbance of 1c and the peak fluorescence intensity of 60 wt % 1-doped PFPV CPNs over two cycles of UV and visible irradiation (Figure 4B). A higher doping level was employed here so that easily detectable 1c absorbances would be generated. Unlike the samples depicted in Figure 3, this sample was not degassed so that the full effect of 19069
dx.doi.org/10.1021/jp206438p |J. Phys. Chem. C 2011, 115, 19065–19073
The Journal of Physical Chemistry C
ARTICLE
Figure 5. Single-nanoparticle fluorescence images (10 μm2, left) and expansions (1.5 μm2, right) of 60 wt % 1-doped PFPV CPNs embedded in poly(vinyl alcohol) before irradiation (A, B), after 254 nm irradiation (C, D), and after visible irradiation (D, E). Scale bar is 1 μm.
Figure 6. Single-nanoparticle fluorescence images (10 μm2, left) and expansions (1.5 μm2, right) of 1-doped MEH-PPV CPNs embedded in poly(vinyl alcohol) before irradiation (A, B), after 254 nm irradiation (C, D), and after visible irradiation (D, E). Scale bar is 1 μm.
UV light and oxygen on the sample could be measured. Oxygen is known to play a critical role in the photobleaching of conjugated polymers in general.42 Therefore, a greater loss of fluorescence intensity should be observed in this sample if fluorophore photobleaching is indeed occurring. The Emod values for the sample in Figure 4B are high and consistent, 97 and 98%, while Erec seems acceptable after the first cycle at 81% but then plummets to 47% after the second cycle. The absorbance of 1c over the same two cycles is completely consistent, demonstrating that the same concentration of photochrome is achieved with each irradiation. Taken together, the data indicate that CPN photobleaching and not photochrome fatigue is responsible for Erec values of less than 100%. The UV irradiation required to induce the photochromic conversion is most likely responsible for the photobleaching of the CPN fluorescence. One of our goals in preparing thermally irreversibly photochromic CPNs was to utilize them in single-particle fluorescence imaging experiments to probe why all photochromic CPN samples retain a small amount of residual emission in their dark states, i.e., have Emod values of less than 100%. The residual emission could be due to weak emission from all CPNs or bright emission from a small number of unquenched CPNs or aggregates in a sample composed primarily of completely quenched CPNs. Ensemble spectroscopy cannot distinguish between these two scenarios, but single-particle imaging experiments can. McNeill and co-workers recently demonstrated single-particle photomodulation for spirooxazine-doped CPNs prepared from the conjugated polymer poly[(9,9-dioctylfluorenyl-2,7-diyl)co-(1,4-benzo{2,10,3}thiadiazole)].33 Slides for single-particle imaging for 1-doped CPNs were prepared by spin coating onto glass a diluted aqueous suspension of nanoparticles with a small
amount of poly(vinyl alcohol) added. The slides were then imaged under ambient conditions by raster scanning over 488 nm laser excitation. Figures 5 and 6 show representative sets of 10 μm2 images of 1-doped PFPV CPNs (Figure 5) and MEH-PPV CPNs (Figure 6). For each type of CPN, images are shown before irradiation (A), after UV irradiation to induce 1of1c (C), and after visible irradiation to return the photochromes to 1o (E). For each image, a representative 1.5 μm2 expansion is also shown (B, D, F). At first glance, the 1-doped PFPV CPNs appear to show decent photomodulation upon UV irradiation to produce the dark state. The average Emod value calculated from individual CPN intensities in images A and C of Figure 5 is 60%. Upon visible irradiation, however, the individual nanoparticles not only fail to recover intensity but become significantly dimmer. The spots shown in the expansions (B, D, F) exemplify this result, losing nearly all of their intensity after visible irradiation. The individual PFPV CPNs appear to have suffered irreversible photobleaching that prevents their photomodulation. The CPNs in Figure 5 had the highest doping level studied (60 wt % dye:polymer), and the same result was observed at all doping levels. The PFPV CPNs thus cannot be used to study the source of residual emission in CPNs. The 1-doped MEH-PPV CPN images in Figure 6 convey a completely different story than their PFPV counterparts. The majority of individual nanoparticles undergo a dramatic reduction in intensity upon UV irradiation and then recover nearly to their original intensities upon visible irradiation. Particle analysis reveals that the average Emod for this sample is 64%, which is lower than what was typically observed in ensemble experiments in aqueous suspension. Matrix effects on the photochromic conversion could be the source of this difference. While Emod is 19070
dx.doi.org/10.1021/jp206438p |J. Phys. Chem. C 2011, 115, 19065–19073
The Journal of Physical Chemistry C
ARTICLE
Table 1. Single Nanoparticle Image Analysis polymer
a
Figure 7. Histogram of ratio values calculated from the images in Figures 5 and 6 as described in the text for 1-doped PFPV (white) and MEH-PPV (gray) CPNs.
lower for this sample than in the ensemble experiments, the recovered intensity in the images is consistent with the ensemble results, with an Erec value of 91%. A number of CPNs even became brighter upon recovery than they were initially. The two representative molecules shown in the expanded images in Figure 5 have Emod and Erec values of 84 and 94% (left) and 79 and 53% (right). The successful fluorescence photomodulation of single particles will enable us to explain residual emission observed when 1c quenchers are present, as discussed further below. The CPNs prepared from PFPV and MEH-PPV clearly exhibit very different behavior in the single-particle imaging experiments. The different photomodulation ability of the two samples can be quantified by calculating the ratio of the intensity recovered upon visible irradiation (Irec Ipss) to the intensity lost upon UV irradiation (I0 Ipss). By this measure, a CPN that lost intensity and then recovered all of it will have a ratio of 1 while a particle that lost intensity upon UV irradiation and then lost additional intensity upon visible irradiation will have a negative ratio. Figure 7 shows a histogram of these ratio values for the CPNs in the images in Figures 5 and 6. The PFPV sample clearly experienced photobleaching almost exclusively as the vast majority of molecules (88%) have negative ratios, and the average ratio value is 0.26 ( 0.41. Only 6% of PFPV CPNs showed any intensity recovery upon visible irradiation, and the magnitude of the recovery was extremely small in all of these cases. The remainder of PFPV CPNs (6%) actually became brighter upon UV irradiation and then exhibited photobleaching upon visible irradiation, leading to larger negative ratios. In contrast, MEH-PPV shows a broad distribution of mostly positive ratio values with an average ratio of 0.85 ( 0.47. The vast majority (94%) of molecules have positive ratio values, indicating that they successfully underwent photomodulation and recovery. Some of these molecules (36%) have ratios greater than 1, which means that they became brighter upon visible irradiation than they were initially. This behavior is explored in more detail below. Only a small fraction (6%) of MEH-PPV CPNs exhibited the sort of photobleaching observed for PFPV and had negative ratio values. These results indicate that MEH-PPV CPNs are appropriate for photomodulation experiments on the single-particle level while the PFPV CPNs are clearly unsuitable for these experiments. We attribute the very different responses of PFPV and MEHPPV CPNs in the single-particle experiments to polymer-structuredependent photobleaching caused by the irradiations required
Emod a
Erec b
I254/I0
I365/I0
MEH-PPV
0.64 ( 0.17
0.91 ( 0.28
1.10 ( 0.35
1.04 ( 0.31
PFPV
0.60 ( 0.29
0.25 ( 0.19
0.50 ( 0.20
0.92 ( 0.24
Calculated according to eq 1. b Calculated according to eq 2.
for the photochromic conversion. To test the response of the polymers to UV irradiation, we prepared samples of undoped control CPNs in poly(vinyl alcohol) on slides using the same procedure as that for the doped CPNs. For each polymer, a sample slide was imaged before and after 2 s of 254 nm irradiation to match the conditions used to induce 1of1c in the 1-doped CPNs. The experiment was then repeated using a fresh sample of each type of CPN but irradiating this time with 2 s of 365 nm light, which matches the conditions we used in our previous study of spirooxazine-doped CPNs. Intensities of individual CPNs were then recorded, and the ratio of each particle’s postirradiation intensity to its initial intensity was calculated and presented in Table 1. Individual PFPV CPNs lost an average of 50% of their initial fluorescence intensities in response to the 254 nm irradiation. This substantial fluorescence photobleaching is most likely due to a combination of influences including oxygen, UV light, and the sample matrix. Fluorophore photobleaching of this magnitude was not observed in degassed aqueous suspensions as can be seen in the undoped PFPV CPNs in Figure 3B. Even in the non-degassed aqueous suspension studied for Figure 4B, the fluorescence intensity reached similar photobleaching levels only after two full cycles of UV and visible light irradiations. The single-particle photobleaching experiments also demonstrate that lower energy 365 nm UV irradiation is significantly less damaging than 254 nm irradiation. Under these irradiation conditions, PFPV CPNs retain an average of 92% of their initial intensity. As expected given the successful photomodulation of individual MEH-PPV CPNs, the photobleaching experiments reveal that undoped MEH-PPV CPNs do not lose fluorescence intensity upon UV exposure. Indeed, the individual MEH-PPV CPNs actually become slightly brighter under both sets of irradiation conditions, with postirradiation intensities averaging 110 and 104% of initial intensities for 254 and 365 nm irradiation, respectively. A similar effect was observed for an ensemble of undoped MEH-PPV CPNs in degassed aqueous suspension as shown in Figure 3B. The increased brightness could indicate a photoprocess that slightly increases the quantum yield of the CPNs. For example, the quantum yield of MEH-PPV has been shown to increase as the extent of conjugation is reduced by introduction of saturated defects in the polymer structure.43 A small number of photoinduced breaks in conjugation would thus explain the observed results. However, such an effect does not prevent photomodulation of individual CPNs. The successful fluorescence photomodulation of individual MEH-PPV CPNs enables us to consider the source of residual emission observed in all CPN samples after UV irradiation to produce the 1c quenchers. While 15% of particles in the dark state image (Figure 6C) have emission intensities indistinguishable from background, the majority of CPNs, including those in the Figure 6D expansion, retain a small amount of emission in the dark state. This result implies that it is a lack of complete quenching in nearly all particles, rather than strong emission from a few unquenched CPNs or aggregates, that is responsible 19071
dx.doi.org/10.1021/jp206438p |J. Phys. Chem. C 2011, 115, 19065–19073
The Journal of Physical Chemistry C for the residual emission observed in ensemble experiments on aqueous suspensions of CPNs. This conclusion is supported by consideration of CPN size, previous results, and FRET theory. Dynamic light scattering measurements showed that the particles studied herein are less than 11 nm in diameter, which is consistent with our previous AFM measurement of 8 nm for spirooxazine-doped MEH-PPV CPNs. Our work on the spirooxazine-doped CPNs also indicated that only those dyes on the particle surface were able to undergo photochromic conversion in response to UV irradiation.20 If the diarylethene dyes studied here behave analogously, then surface-based 1c quenchers would be unable to quench emission originating in or near the center of a CPN. The F€orster radius, which is the distance at which energy transfer is 50% efficient, is 2.24 nm for the 1c/MEH-PPV FRET pair. Energy transfer efficiency falls off dramatically at distances beyond the F€orster radius40 and would be just 3% at 4 nm for this system. Thus, residual emission observed in the majority of particles may come from chromophores within the CPNs that are too far from the 1c quenchers for efficient FRET. More efficient quenching would require even smaller CPNs, dyes that are able to undergo photochromic conversion both within a CPN and on the surface, and/or a dyepolymer FRET pair with a larger F€orster radius.
’ CONCLUSIONS PFPV and MEH-PPV CPNs doped with thermally irreversible photochrome 1 exhibit stable intensity photomodulation in aqueous suspension. “On” and “off” fluorescence intensity levels are maintained until the photochrome is switched by irradiation. In single-particle imaging experiments on CPNs embedded in poly(vinyl alcohol), 1-doped MEH-PPV CPNs exhibit reversible quenching and recovery. In a representative sample, 94% of CPNs exhibited quenching followed by recovery, indicating that these CPNs are suitable for photomodulation experiments on the single-particle level. These experiments demonstrated that nearly all CPNs retain a small amount of residual emission when the diarylethene dyes are in their quenching form. This emission may come from chromophore populations located too far from quencher dyes to undergo efficient FRET. In contrast to the successful photomodulation of single MEH-PPV CPNs, 1-doped PFPV CPNs undergo substantial, irreversible photobleaching upon exposure to the irradiation conditions required for the photochromic conversion. UV irradiation experiments on undoped control CPNs reveal that PFPV particles are extensively photobleached by the high-energy 254 nm irradiation. These results suggest alternative irradiation conditions for future photochromic CPNs. Lower energy UV irradiation can be used with many photochromes and is shown here to cause far less photobleaching in PFPV CPNs although it did not yield sufficient 1of1c conversion to be used in these experiments. Another alternative is two-photon irradiation, which replaces a single photon of UV irradiation with two near-IR photons and has been shown to be suitable for inducing the photochromic conversion under biological imaging conditions.44 The photostability of the conjugated polymer is also an important consideration in the design of CPNs for photomodulation. ’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
ARTICLE
’ ACKNOWLEDGMENT We gratefully acknowledge support of this work by NSF through CAREER award CHE-0642513. We thank Zifan Wang and Prof. Doug English of Wichita State University for performing the dynamic light scattering measurements. C.M.D. thanks the Charles Center of the College of William and Mary for honors research funding. ’ REFERENCES (1) Hell, S. W. Nat. Methods 2009, 6, 24–32. (2) Tian, Z.; Wu, W.; Li, A. D. Q. ChemPhysChem 2009, 10, 2577–2591. (3) Cusido, J.; Deniz, E.; Raymo, F. M. Curr. Phys. Chem. 2011, 1, 232–241. (4) Brown, G. H., Eds. Photochromism; Wiley: New York, 1971. (5) Crano, J. C., Guglielmetti, R. J., Eds. Organic Photochromic and Thermochromic Compounds; Kluwer: New York, 2002. (6) D€urr, H., Henri, B.-L., Eds. Photochromism: Molecules and Systems; Elsevier: Amsterdam, 2003. (7) Zhu, M.-Q.; Zhu, L.; Han, J. J.; Wu, W.; Hurst, J. K.; Li, A. D. Q. J. Am. Chem. Soc. 2006, 128, 4303–4309. (8) Hu, D.; Tian, Z.; Wu, W.; Wan, W.; Li, A. D. Q. J. Am. Chem. Soc. 2008, 130, 15279–15281. (9) Tian, Z.; Li, A. D. Q.; Hu, D. Chem. Commun. (Cambridge, U. K.) 2011, 47, 1258–1260. (10) Fukaminato, T.; Doi, T.; Tamaoki, N.; Okuno, K.; Ishibashi, Y.; Miyasaka, H.; Irie, M. J. Am. Chem. Soc. 2011, 133, 4984–4990. (11) Cusido, J.; Deniz, E.; Raymo, F. M. Eur. J. Org. Chem. 2009, 2009, 2031–2045. (12) Raymo, F. M.; Tomasulo, M. J. Phys. Chem. A 2005, 109, 7343–7352. (13) Raymo, F. M.; Tomasulo, M. Chem. Soc. Rev. 2005, 34, 327–336. (14) Gust, D.; Moore, T. A.; Moore, A. L. Chem. Commun. (Cambridge, U. K.) 2006, 1169–1178. (15) Yildiz, I.; Deniz, E.; Raymo, F. M. Chem. Soc. Rev. 2009, 38, 1859–1867. (16) Wu, C.; Bull, B.; Szymanski, C.; Christensen, K.; McNeill, J. ACS Nano 2008, 2, 2415–2423. (17) Wu, C.; Szymanski, C.; McNeill, J. Langmuir 2006, 22, 2956–2960. (18) Pecher, J.; Mecking, S. Chem. Rev. 2010, 110, 6260–6279. (19) Tuncel, D.; Demir, H. V. Nanoscale 2010, 2, 484–494. (20) Harbron, E. J.; Davis, C. M.; Campbell, J. K.; Allred, R. M.; Kovary, M. T.; Economou, N. J. J. Phys. Chem. C 2009, 113, 13707–13714. (21) Yokoyama, Y. Chem. Rev. 2000, 100, 1717–1740. (22) Irie, M. Chem. Rev. 2000, 100, 1685–1716. (23) Nakamura, S.; Irie, M. J. Org. Chem. 1988, 53, 6136–6138. (24) Tian, H.; Feng, Y. J. Mater. Chem. 2008, 18, 1617–1622. (25) Wigglesworth, T. J.; Myles, A. J.; Branda, N. R. Eur. J. Org. Chem. 2005, 1233–1238. (26) Yun, C.; You, J.; Kim, J.; Huh, J.; Kim, E. J. Photochem. Photobiol., C 2009, 10, 111–129. (27) Matsuda, K.; Irie, M. J. Photochem. Photobiol., C 2004, 5, 169–182. (28) Li, X.; Tian, H. Macromol. Chem. Phys. 2005, 206, 1769–1777. (29) Finden, J.; Kunz, T. K.; Branda, N. R.; Wolf, M. O. Adv. Mater. 2008, 20, 1998–2002. (30) Hayasaka, H.; Miyashita, T.; Tamura, K.; Akagi, K. Adv. Funct. Mater. 2010, 20, 1243–1250. (31) Hayasaka, H.; Tamura, K.; Akagi, K. Macromolecules 2008, 41, 2341–2346. (32) Wu, C.; Zheng, Y.; Szymanski, C.; McNeill, J. J. Phys. Chem. C 2008, 112, 1772–1781. (33) Tian, Z.; Yu, J.; Wu, C.; Szymanski, C.; McNeill, J. Nanoscale 2010, 2, 1999–2011. 19072
dx.doi.org/10.1021/jp206438p |J. Phys. Chem. C 2011, 115, 19065–19073
The Journal of Physical Chemistry C
ARTICLE
(34) Lewis, S. M.; Harbron, E. J. J. Phys. Chem. C 2007, 111, 4425–4430. (35) Grey, J. K.; Kim, D. Y.; Norris, B. C.; Miller, W. L.; Barbara, P. F. J. Phys. Chem. B 2006, 110, 25568–25572. (36) Maa, Y. F.; Chen, S. H. Macromolecules 1988, 21, 1176–1177. (37) Yildiz, I.; Impellizzeri, S.; Deniz, E.; McCaughan, B.; Callan, J. F.; Raymo, F. M. J. Am. Chem. Soc. 2011, 133, 871–879. (38) Clafton, S. N.; Beattie, D. A.; Mierczynska-Vasilev, A.; Acres, R. G.; Morgan, A. C.; Kee, T. W. Langmuir 2010, 26, 17785–17789. (39) Metelitsa, A. V.; Lokshin, V.; Micheau, J. C.; Samat, A.; Gugliemetti, R. J.; Minkin, V. I. Phys. Chem. Chem. Phys. 2002, 4, 4340–4345. (40) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer Academic/Plenum: New York, 1999. (41) Thomas, S. W.; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339–1386. (42) Yu, J.; Hu, D.; Barbara, P. F. Science 2000, 289, 1327–1330. (43) Padmanaban, G.; Ramakrishnan, S. J. Am. Chem. Soc. 2000, 122, 2244–2251. (44) Zhu, M.-Q.; Zhang, G.-F.; Li, C.; Aldred, M. P.; Chang, E.; Drezek, R. A.; Li, A. D. Q. J. Am. Chem. Soc. 2011, 133, 365–372.
19073
dx.doi.org/10.1021/jp206438p |J. Phys. Chem. C 2011, 115, 19065–19073
