Fluorescence Behavior of Single Guest Molecules in Nonpolar Oil

Article pubs.acs.org/JPCC

Fluorescence Behavior of Single Guest Molecules in Nonpolar Oil Droplets Covered with Stabilizing Surfactants Syoji Ito,* Atsushi Iida, Masakazu Yasuda, and Hiroshi Miyasaka* Division of Frontier Materials Science, Graduate School of Engineering Science and Center for Quantum Materials Science under Extreme Conditions, Osaka University, Toyonaka, Osaka 560-8531, Japan ABSTRACT: By using confocal and wide-field microscopic techniques we have investigated fluorescence behaviors of a single perylenediimide derivative, N,N′dipropyl-1,6,7,12-tetrakis(4-tert-butylphenoxy)-3,4,9,10-perylenetetracarboxydiimide (BP-PDI), in ca. 200−300 nm sized droplets of n-octane covered with surfactant molecules. The guest dye did not show distinguishable blinking in the droplets prepared with a nonionic surfactant, Triton X-100, while frequent blinking was observed in the droplets with a cationic surfactant, trimethylstearylammonium chloride (STAC). The survival time of the guest dye before the permanent photodegradation was much longer in the droplets with Triton X-100 than those with STAC. On the basis of the analysis with a model assuming the transition from the S1 state to the dark state (DS) and the permanent photodegradation during the dark state, it was deduced that the probability of the transition from the S1 state to the DS in the droplet with STAC, 7.2 × 10−6, was significantly larger than that with Triton X-100, 1.1 × 10−6. On the other hand, the probability of the photodegradation during the dark state was comparable between these two systems. These results indicate that the probability of transition from the S1 state to the DS essentially dominates the survival time of the guest dyes. Fluorescence polarization measurements also revealed that guest dyes frequently stay in the vicinity of polar interface in the droplets prepared with STAC.

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INTRODUCTION Since the pioneering works by Moerner et al.1 and by Oritt et al.,2 optical single-molecule detection (SMD) techniques have been developed and applied to various investigations as powerful tools to provide detailed information inaccessible by traditional bulk measurements.3−11 The SMD methods have been contributing to the elucidation of essential issues in a wide range of research fields such as spectral diffusion,3 Rabi oscillation,4 and photon-antibunching5 in the quantum theory, elementary photophysical processes such as energy transfer and migration6 in photochemistry, and conformational rearrangements of biopolymers7−16 in bioscience. In single-molecule studies the efficient detection of the limited number of fluorescence photons from individual molecules is crucial for reliable measurement, for which chromophores with high fluorescence quantum yields have been synthesized and single-photon detectors with high detection efficiencies have been developed. In spite of these efforts to improve detection efficiency, the interconversion between emissive (ON) and nonemissive (OFF) states in single molecules, so-called blinking, sometimes inhibits the effective measurements. Hence the reduction of blinking is also a key of reliable measurement. On the other hand, in recent years the fluorescence intermittency has been positively used in super-resolution fluorescence microscopy on the basis of the localization technique,17−19 for which highly frequent blinking is rather preferable. There have been several approaches to manipulate the dark state by photoinduced phenomena20 and/ or by chemical reagents.21,22 © 2014 American Chemical Society

The dark state (DS) observed in single-molecule studies has been attributed to several processes:23,24 triplet state formation, occasional quenching by the oxygen, and photoionization that is probably induced by the stepwise two-photon absorption via the long-lived intermediate species such as the triplet state.24,25 Among these processes, photoionization through two-photon absorption, probably due to stepwise two-photon absorption via the triplet state, has been considered to contribute to longtime DS formation at room temperature. Conventional ensemble measurements using laser multiphoton excitation26−31 revealed that the charge recombination between a cation−electron pair produced via two-photon ionization takes place in the time range from milliseconds to several hours in polymer matrices28 and on the surface of porous materials29 even at room temperature. On the other hand, the recombination time of the cation−electron pair produced via multiphoton ionization is at most several tens of picoseconds in nonpolar alkane solutions26,27,30,31 because of the large mobility of the electron and/or the intrinsic fast charge recombination.30−32 We can therefore expect that the blinking through photoionization is suppressed in the case where fluorescent molecules are confined in small droplets of nonpolar solvents. Along this line, we investigated the dynamic behavior of individual organic molecules encapsulated in micro/nanodroplets of octane solution by means of single-molecule Received: November 15, 2013 Revised: March 9, 2014 Published: May 1, 2014 10348

dx.doi.org/10.1021/jp4112266 | J. Phys. Chem. C 2014, 118, 10348−10357

The Journal of Physical Chemistry C

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detection methods,33 leading to the observation of continuous emission with little blinking. The measurement demonstrated that the importance of the photoionization as a mechanism of the blinking and that single droplets of nonpolar solutions of the fluorescent dye serve single-photon emitting systems with little blinking at room temperature. It is, however, well-known that the effect of liquid−liquid interface is much more pronounced in ultrasmall droplets because of the large specific surface. In actuality, we observed the adsorption and desorption of individual molecules at the interfaces of the octane droplets in the previous study,33 suggesting that the adsorption is related to the formation of the dark state of the emission. In order to more precisely elucidate the effect of the interface on the blinking behavior and the permanent photodegradation, we have investigated the fluorescence dynamics of single dye molecules in ultrasmall octane droplets covered with surfactants that modify the interfacial property. On the basis of the analysis of experimental results on the blinking and photodegradation processes, we will quantitatively discuss key processes leading to the permanent photobleaching of guest dyes in the droplets.

the APDs were sent to a PC-controlled photon-counting module (PicoHarp300, PicoQuant) with time-tagged timeresolved (TTTR) option, and time trajectories of fluorescence intensity, fluorescence lifetimes, and photon correlation histograms were obtained. A mode-locked Nd3+:YAG laser (DPM-1000&SBR-5080FAP, Coherent, 1064 nm, 30 ps fwhm) was used as an excitation light source. The repetition rate was reduced into 8 MHz with an electro-optic modulator (model 360-80, Conoptics). The second harmonic (532 nm) was focused onto the samples by the oil-immersion objective. The typical excitation intensity at the sample plane was estimated to be 1.1 MW·cm−2 from the input laser power and the spot size of the excitation laser in a scanning confocal image of single molecules embedded in a polymer (PMMA) film. The instrumental response function (IRF) of the system was estimated by the deconvolution analysis of a fluorescence decay curve of erythrosine B in water, of which fluorescence lifetime was reported35 to be 85 ps. The fwhm of IRF thus estimated was typically 600 ps. n-Octane droplets containing the fluorescence dyes were selected from confocal fluorescence images of the sample obtained by stage-scanning with a piezoelectric device (Figure

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EXPERIMENTAL SECTION 1. Sample Preparation. A nonionic surfactant, Triton X100 (Sigma-Aldrich), and a cationic one, trimethylstearylammonium chloride (SATC, Tokyo Chemical Industry), were used to stabilize nanometer-sized droplets of n-octane (GR grade, Wako). N,N′-Dipropyl-1,6,7,12-tetrakis(4-tert-butylphenoxy)-3,4,9,10-perylenetetracarboxydiimide (BP-PDI, Yamada Chemical) was employed as a fluorescence guest molecule. These chemical compounds were used as received. Water used for sample preparation was purified with an ultrapure water system (Direct-Q, Millipore). n-Octane solution of BP-PDI (typically 2.0 × 10−10 M) was mixed with an aqueous solution of the surfactants in a volume ratio of 1:50. By sonicating the mixture solution with an ultrasonic homogenizer (Cell Disruptor185, Branson), ultrasmall droplets of the n-octane covered with the surfactants were prepared. Hydrodynamic diameter and its distribution of droplets thus prepared in water were evaluated by a dynamic light-scattering (DLS) particle-size analyzer (LB-500, Horiba). This oil−water emulsion was added to an aqueous solution of agarose (LM-200, Dojindo, 2.0 wt %) at 343 K in a volume ratio of 1:1. A small amount of this mixed solution (20 μL) was sandwiched with two well-cleaned coverslips to make liquid thin film of the solution, and it was kept in a refrigerator at 283 K for several hours. The mixed agarose solution turned to solid (gel) from liquid by this cooling process, resulting in the immobilization of the droplets in the gel. 2. Single-Molecule Detection with a Confocal Microscope. A confocal microscopic system was mainly used for single-molecule detection. This system consists of an inverted optical microscope (IX-71, Olympus), a pinhole with a 50 μm diameter, and two avalanche photodiodes (APDs, SPCM-AQR14, PerkinElmer). A Hanbury-Brown and Twiss type photon correlation setup34 was employed as the detection configuration. Fluorescence photons from single molecules were collected by an oil-immersion objective with high NA (Olympus, ×100, NA = 1.3) and focused to the center of the pinhole. After passing through the pinhole, the fluorescence light was equally divided into two orthogonal propagation directions by a cube half beam splitter, and respectively guided into each of the two APDs. A polarizing cube beam splitter was employed to measure fluorescence polarization. The outputs of

Figure 1. (a) Size distribution of octane droplets prepared with Triton X-100 in water; their sizes were measured by DLS. (b) Size distribution of octane droplets after immobilization in the agarose gel; the size of the droplets was individually estimated from optical transmission micrographs. (c) Size distribution of octane droplets prepared with STAC in water before the immobilization in agarose gel; their sizes were measured by DLS. (d) Size distribution of octane droplets prepared with STAC after immobilization in agarose gel; individual sizes of the droplets were estimated from optical transmission micrographs.

2c). Fluorescence photons from individual emissive droplets were recorded as a function of time from the beginning of the measurement using the photon-counting module. Typical duration of the measurement was 120 s. The size of the droplets immobilized in the agarose gel was estimated from optical transmission images obtained by using a CCD camera attached to the microscope.36,37 In order to avoid fluctuation of the fluorescence intensity due to the translational diffusion of the dye going into/out of the confocal volume, droplets with diameters of ca. 200−300 nm were selected for the measurements of the fluorescence dynamics of single 10349



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fluorescence image, one can find two small bright spots with a diffraction-limited size near the droplet marked by C. These two spots are attributable to nanometer-sized droplets containing BP-PDIs that are not detected in the optical transmission image in Figure 2a. Figure 2c shows a typical scanning-confocal image of octane droplets immobilized in the agarose gel. As in the wide-field fluorescence image shown in Figure 2b, several bright spots in the confocal image correspond to octane droplets including the guest dye. As already noted in the Experimental Section, we measured single-molecule fluorescence dynamics in droplets with diameters from 200 to 300 nm in order to avoid fluorescence fluctuations originating from the lateral motion of the guest dyes. To confirm the measurement of droplets with diameters in the target size range, we also checked the size of droplets in scanning-confocal images. 2. Fluorescence Intensity Trajectories of BP-PDIs in Small Octane Droplets. Figure 3a shows a fluorescence

Figure 2. (a) An optical transmission micrograph of octane droplets immobilized in the agarose gel and (b) the corresponding wide-field fluorescence image in panel a. The length of scale bars in panels a and b is 4 μm. (c) A scanning-confocal image of octane droplets prepared with STAC. These droplets were immobilized in the agarose gel in the manner described in the text.

molecules. To obtain the information on the adsorption and desorption of the guest dyes at the interface, the time profile of the fluorescence polarization was measured by guiding the fluorescence photon into the polarizing beam splitter and detecting two orthogonally polarized signals with the two APDs. The degree of polarization, P, was calculated using eq 1. p=

IAPD1 − GIAPD2 IAPD1 − GIAPD2

(1)

Here, IAPD1 and IAPD2 are respectively numbers of photons detected by the APD1 and APD2. G is a factor that corrects the difference of detection efficiency between the two detectors. All the measurements were performed at 22 ± 1 °C.

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RESULTS AND DISCUSSION 1. Size Distribution of Droplets. Figures 1a and 1c show typical size distributions of octane droplets in water prepared with Triton X-100 and STAC before being mixed with the agarose aqueous solution. The sizes of the emulsions were measured by DLS in the range from 50 nm to 1 μm. Figures 1b and 1d show typical size distributions of the octane droplets with Triton X-100 and STAC after immobilization in the agarose gel. Because the individual sizes of the immobilized droplets were estimated from their optical transmission images, only the droplets larger than the diffraction limit of visible light (∼200 nm) are involved in the histograms. The diameters of the droplets after the immobilization are, however, still in the range of sub-micrometers, indicating that the immobilization in the agarose gel did not seriously affect the sizes of the octane droplets. Figures 2a and 2b respectively show optical transmission and fluorescence micrographs of octane droplets at the same position in agarose gel. Although four droplets (A−D) are observed in the optical transmission image, one of the four droplets marked by A disappears in the corresponding fluorescence micrograph. That is, the droplet contains no fluorescent dye inside. Because the sizes of the droplets marked by B, C, and D are somewhat larger (>1 μm), the lateral Brownian motion of the guest dyes along the optical axis caused fluorescence intensity alteration in a video frame, which also indicates that the number of guest dyes inside the droplets is quite small. In addition to the three bright droplets in the

Figure 3. (a) A typical fluorescence intensity trajectory of a BP-PDI in an octane droplet prepared with Triton X-100. (b) Photon correlation histogram for the result in panel a. (c) A typical fluorescence intensity trajectory showing blinking of a BP-PDI in an octane droplet prepared with Triton X-100.

intensity trajectory of BP-PDI in an octane droplet prepared with Triton X-100. The BP-PDI shows a continuous fluorescence emission from the time origin until the sudden drop in fluorescence intensity at 35 s. This discrete decrease in fluorescence intensity is characteristic of single-molecule emission behavior. Among 85 droplets successfully measured, 84.7% of them (72 droplets) showed such a one-step decrease of fluorescence intensity, indicating that single molecules are encapsulated in individual emissive droplets. To further confirm the encapsulation of one molecule in one droplet, we obtained a second-order photon correlation histogram, g(2)(t), by analyzing time profiles of the fluorescence intensity. As shown in Figure 3b, the g(2)(t) value around the time origin (t = 0) is almost zero, while peaks appear at around the time interval of laser pulses, 125 ns. This result directly indicates that the present system can be regarded as a singlephoton emitter.5,9,38 Because the steady-state ensemble emission spectrum indicated no apparent aggregation of BPPDIs in the droplets, the constant fluorescence intensity before its one-step decrease is safely ascribed to the single-molecule emission in the single droplets. In addition, the one-step 10350



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Table 1. Comparison of τON_med, τOFF_1/e, τsur_med, p, q, and s between Nanodroplets Prepared with Triton X-100 and Those Prepared with STACa surfactant

τON_med/s

Triton X-100

22 71 (L) 12 (S) 1.5 2.1 (L) 1.4 (S)

STAC

τOFF_1/e/s 0.7 0.8 0.5 0.6 0.4 0.6

τsur_med/s

p

q

savg

80

1.1 × 10−6

0.23 (eq 10) 0.60 (Ns=0/Nall)

4.5 (eq 10) 2.0 (count)

6.3

7.2 × 10−6

0.29 (eq 10) 0.47 (Ns=0/Nall)

2.1 (eq 10) 2.0 (count)

(L) (S) (L) (S)

a Here, τON_med, τOFF_1/e, and τsur_med are respectively medians of the ON time, OFF time, and survival time, p and q are respectively probabilities of transitions from the S1 state to the DS and the DS to the PPB, and savg is the averaged number of transitions from the DS to the S0 state before PPB. The letters in parentheses, L and S, respectively show the droplet size from 200 to 300 nm and that ≤200 nm. The values of q and savg with “(eq 10)” were derived from the analysis by using eq 10, while those with “(Ns=0/Nall)” and “(count)” were respectively derived by directly counting the number of molecules and the number of OFF times. Here, Nall is the total number of molecules, and Ns=0 is the number of molecules that did not show blinking before PPB.

excitation to the permanent photobleaching (PPB). The medians of τON, τOFF, and τsur obtained by the analysis are summarized in Table 1. With an aim to investigate the size dependence, the 72 droplets were classified into two groups based on their size: small (≤200 nm) and large (200−300 nm). The medians of τON and τOFF for each group are also listed in Table 1. The median ON time of the large droplets (71 s) is somewhat larger than that of small droplets (12 s), indicating that the oil−water interface affects the ON-time duration, i.e., the dark-state formation. On the other hand, the median OFF times showed no marked difference. Figures 4a and 4b respectively show normalized histograms of τON and τOFF for the 72 molecules. The insets of the figures are double logarithmic plots of the histograms. It should be noted that 20 molecules did not show OFF time in the time

decrease is ascribable to the photodegradation of the guest dye in the droplet. In the fluorescence-intensity trajectory of single molecules, the blinking of fluorescence has been observed in many systems. No marked blinking was however observed for the guest dye in the small droplet covered with Triton X-100 until permanent photobleaching as shown in Figure 3a. Among several mechanisms which were suggested to account for the blinking behaviors, photoionization via successive two-photon absorption through intermediate species, such as triplet state, has been considered as a main process leading to the tentative formation of the nonemissive (OFF) state.24 In the case where the photoionization is responsible for the OFF state formation, the recombination between the ejected electron and its parent cation is necessary for the recovery from the dark (OFF) state to the emissive (ON) state. Accordingly, the OFF-time distribution is related to the recombination time of electron− cation pairs. In amorphous solids such as polymer matrices, electrons ejected through the successive multiphoton absorption are trapped in stable trap sites in the matrix, leading to the long OFF time; the recombination time is typically from microseconds to a few hours even at room temperature.28,29 Owing to this long recombination time, ionized states can be detected as OFF times in fluorescence intensity trajectories. On the other hand, the electron−cation pair in nonpolar alkane solutions undergoes the geminate recombination very quickly owing to the small stabilization energy of charged species and the large mobility of electrons. The recombination time is usually less than a few picoseconds.26,27,30,31 This rapid charge recombination does not lead to detectable OFF times in the fluorescence intensity trajectory of single molecules with a time bin of milliseconds. In actuality, we reported small probability of blinking in the fluorescence behavior of single molecules in droplets of octane prepared without surfactants.33 The present experimental result could be well interpreted as the short recombination time of the photoionized state as observed in the octane droplet without surfactant. Although similar behaviors were observed for most emissive droplets with Triton X-100, it should be noted that some trajectories showed blinking as shown in Figure 3c. To statistically characterize the blinking behavior, we analyzed 72 molecules in octane droplets covered with Triton X-100 for τON, τOFF, and τsur. Here, τON and τOFF are respectively durations of the emissive and the dark states in the fluorescence intensity trajectory. The survival time, τsur, is defined as the time interval from the beginning of photo-

Figure 4. Histograms of (a) τON, (b) τOFF, and (c) τsur of single BPPDIs in the octane droplets prepared with Triton X-100 and immobilized in the agarose gel. A black solid curve in panel a is the result of a curve fitting using eq 4. A black solid curve in panel c is the result of a curve fitting using eq 10. The number of molecules with τsur ≥120 s was 25, and these molecules are not included in the figure. The insets in panels a and b are the double logarithmic plots of the ONtime and OFF-time histograms. 10351



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window of the measurement (120 s), of which data points are not plotted in Figure 4a. As clearly shown in this figure, many molecules showed ON times longer than 10 s. On the other hand, the OFF time distribution (Figure 4b) shows a very short duration of the dark state; more than 52% of τOFF were shorter than 1 s. The double logarithmic plot of the OFF-time histogram (the inset of Figure 4b) shows a linear shape, indicating that the OFF-time distribution obeys the power law. On the other hand, the ON-time distribution shows a curved shape, which is ascribed to the exponential distribution of the ON time. This is consistent with the results that the ON-time distributions of the two systems are well analyzed by the model of eq 4 with the single transition probability as will be discussed later. Figure 4c shows the normalized histogram of survival time, τsur, of the 72 molecules. In the present case, an OFF time longer than 60 s was regarded as the PPB, because the OFF time followed by the recovery to the emissive state was quite short as shown in Figure 4b. The number of molecules with τsur longer than 120 s was 25; 20 molecules showed no blinking as already mentioned in the previous paragraph, and the remaining 5 molecules showed several interconversions between ON and OFF states as shown in Figure 3c. Taking into account this number of molecules, the number of guest molecules with τsur longer than 60 s was almost 50%. As discussed in the previous section, the short recombination time of the ionized state in the nonpolar solution well accounts for the small probability of the OFF state formation and consequent long durations of τON. In addition, the rapid charge recombination in the octane droplet may also reduce the probability of photoinduced irreversible bleaching because it shortens the duration of the ionized state that generally has higher reactivity resulting in irreversible chemical reactions into nonfluorescent species. Compared to the result of the droplets with Triton X-100 (Figure 3a), single BP-PDIs in droplets prepared with STAC exhibited more frequent blinking before the one-step photobleaching as shown in Figure 5. In addition, survival times of BP-PDIs in the droplets covered with STAC were much shorter than those in the droplets prepared with Triton X-100.

Figure 6. Histograms of (a) τON, (b) τOFF, and (c) τsur of single BPPDIs in octane droplets prepared with STAC and immobilized in the agarose gel. A black solid curve in panel a is the result of a curve fitting using eq 4. A black solid curve in panel c is the result of a curve fitting using eq 10; the data of two BP-PDI molecules with τON ≥ 120 s is not included in the analysis. The insets in panels a and b are the double logarithmic plots of the ON-time and OFF-time histograms.

(1.6 s) is slightly larger than that of small droplets (0.6 s), Figure 6a clearly shows that the ON time is much shorter in the droplets covered with STAC than those covered with Triton X100 (Figure 4a), while the histogram of τOFF in Figure 6b shows no distinguishable difference from that with Triton X-100 in Figure 4b. The double logarithmic plots in the insets of Figure 6 overall show a similar trend with those exhibited in the insets of Figure 4. The OFF-time distribution with a linear shape obeys the power law, while the ON-time distribution rather obeys the exponential although the median ON time in the droplets with STAC is much shorter than in those with Triton X-100. The τsur of the droplets with STAC is mainly distributed in the time range up to 20 s, and the number of molecules with τsur > 120 s was only 2, while τsur for the Triton X-100 system is much longer as shown in Figure 4c. These results indicate that the guest dyes more easily undergo irreversible photobleaching in droplets with STAC and suggest that the permanent photobleaching closely relates to the formation of the OFF state. To elucidate the origin of the difference in the two systems, we analyzed the histograms of τON, τOFF, and τsur on the basis a simple model shown in Scheme 1. In this model, the guest dye undergoes the transition from the S1 state to the dark state (DS) with a probability of p or the deactivation into the S0 state mainly via a radiative transition with a probability of 1 − p. In addition, it is assumed that the PPB occurs from the dark state with a probability of q. It should be mentioned that the intermediate state such as the triplet state plays an important role in the formation of DS in the actual case. The present bin time of 25 ms is, however, much longer than the lifetime of the triplet state at room temperature. Hence, we simplified the model as shown in Scheme 1. Equation 2 gives the probability

Figure 5. A typical fluorescence intensity trajectory of a BP-PDI in an octane droplet prepared with STAC.

To quantitatively compare the fluorescence behaviors in these two systems, we plotted normalized histograms of τON, τOFF, and τsur of the guest dyes for the STAC system in Figure 6, where the results obtained by the analysis of 63 molecules are exhibited. The insets of Figures 6a and 6b show double logarithmic plots of the histograms of τON and τOFF. The 63 droplets with STAC were also classified into two groups based on their size in the same manner as for the droplets with Triton X-100. The medians of τON and τOFF for each group are listed in Table 1. Although the median ON time of the large droplets 10352



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involved in this estimation; the ratio of dyes with τON > 120 s was 27.8%. In other words, the actual value should be much longer than the median ON time (22 s) shown in Table 1. On the other hand, Figures 4b and 6b show the distributions of τOFF. Contrary to the large difference of τON in Figures 4a and 6a and in Table 1, there is no marked difference in the distribution of τ OFF between these two systems. The distribution of OFF time is related to the lifetime of DS, from which the nonradiative recovery of the S0 state or photodegradation takes place. Histograms of OFF times accumulated over many single-molecule measurements give a decay time constant of DS. To estimate the lifetime of the DS of the two systems, we tentatively employed an exponential process for the decay of DS although the typical geminate recombination of electron−cation pairs produced by photoionization is not monophasic.28,29 The histograms of the OFF times indeed show the power law as shown in the insets of Figures 4b and 6b. Lifetimes of the DS, τOFF_1/e, thus estimated are listed in Table 1, showing no remarkable difference in apparent time constant between these two droplet systems. The result indicates that the summation of the rate constants of the two pathways from the DS, i.e., the recovery of the S0 and the PPB, is almost the same in the two systems. For further elucidating the reaction profile from the DS, we analyzed the distribution of τsur in the two droplet systems. As shown in Scheme 1, it was presumed that the guest dye undergoes PPB from the DS with a probability q or returns to the S0 state via a nonradiative pathway with a probability 1 − q. Equations 6 and 7 represent the probability of PPB at the Nth excitation, PPPB(N;s=i) (i = 0, 1, 2, ...).

Scheme 1. States Diagram for the Guest Dye in the Octane Droplet

of the transition from the S1 state to the DS at the Nth photoexcitation, PDS(N). PDS(N ) = p(1 − p)N − 1

(2)

The number of photoexcitation, N, can be expressed as a function of time, t, by using the number of photons detected per unit time, Np, and the detection efficiency of the confocal microscope, α (eq 3).

N=

Np

t (3) α The ON-time distribution is, therefore, rewritten in the form as a function of time (eq 4). PDS(t ) = Ap(1 − p)N pt /α−1

(4)

Here, A is an amplitude factor to analyze an experimentally obtained histogram of τ ON by using eq 4. Under a normalization condition that PDS(0) = 1, A is expressed as A = (1 − p)/p. Solid lines in Figures 4a and 6a are curves analyzed by using eq 4 with A = (1 − p)/p. In the analysis, we used the averaged values of Np that were experimentally obtained; these were 5.4 × 103/s for the droplets with Triton X-100 and 4.6 × 103/s for the droplets with STAC. The detection efficiency of the confocal microscope, α, is estimated at ca. 7% by using eq 5.39−41 α = ΩOLTOLTOSΦAPD

PPPB(N ;s=0) = (1 − p)N − 1pq PPPB(N ;s=i) =

(6)

1 1 (1 − p)N − s − 1ps + 1 (1 − q)s q N B(s+1,N −s)

(i = 1, 2, 3, ...)

(7)

Here, B is the beta function and s is the number of transition(s) from the DS to the S0 state. Equation 6 corresponds to the case where the dye molecule undergoes PPB at the first formation of a DS after continuous photoemission. On the other hand, eq 7 represents the situation that the dye molecule undergoes PPB at the ith formation of DS. In other words, i − 1 formations of DS result in the recovery of the S0 state. Substituting eq 3 into eqs 6 and 7 gives the probabilities of PPB, PPPB(t;s=0) and PPPB(t;s=i) (i = 1, 2, ...), as a function of time.

(5)

Here, ΩOL is the photon correction efficiency of the optical microscope that can be estimated from the NA of the objective. In the present study, the guest dyes in droplets are regarded as a point light source because the rapid rotational motion of the guest molecules spatially uniformalizes the anisotropic dipole emission. The ΩOL of the objective with NA 1.30 was estimated at 24%. TOL is the transmittance of the objective that can be obtained from a technical specification sheet provided from Olympus. TOS is the total transmittance of the optical detection system consisting of the dichroic mirror, optical long-pass filters, windows and prisms in the microscope, pinhole, and focusing lens. ΦAPD is the photon-detection efficiency of the APD. The solid lines well reproduced the experimental results on distributions of τON, with p values of 1.1 × 10−6 (Figure 4a) and 7.2 × 10−6 (Figure 6a). The median of ON time, τON_med, of the dye in the droplets with Triton X-100 is much larger than that of the dyes in the droplets with STAC as shown in Table 1. This result also indicates that the probability of the transition to the OFF state is much higher in the droplets with STAC than those with Triton X-100. It should be noted that the median ON time of the dyes in the droplets with Triton X-100 is underestimated because ON times longer than 120 s are not

PPPB(t ;s=0) = (1 − p)N pt /α−1 pq PPPB(t ;s=i) = (1 − q)s q

(8)

1 1 (1 − p)N pt /α−s−1 ps + 1 Npt /α B(s+1,Npt /α−s) (i = 1, 2, 3, ...)

(9)

The experimentally obtained histograms of τsur can be analyzed by using eq 10, which is the summation of eq 8 and eq 9. PPPB(t ) = C1(1 − p)N pt /α−1 pq + C2

1 1 Npt /α B(s+1,Npt /α−s)

(1 − p)N pt /α−s−1 ps (1 − q)s q

(10)

Here, C1 and C2 are amplitude factors to analyze an experimentally obtained histogram of τsur. The ratio of C1 to C2 is expressed by C1/C2 = q/(1 − q). Under a normalization 10353



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condition that PPPB(0) = 1, C1 and C2 are respectively expressed as C1 = (1 − p)/pq and C2 = (1 − p)(1 − q)p−1q−2. Solid lines in the normalized histograms of τsur in Figures 4c and 6c are curves calculated using eq 10 with C1 = (1 − p)/pq and C2 = (1 − p)(1 − q)p−1q−2. In the analysis we used fixed values of p that were obtained through the analysis of τON in Figures 4a and 6a. We also used the fixed values of Np, 5.4 × 103/s for the droplets with Triton X-100 and 4.6 × 103/s for the droplets with STAC, and the detection efficiency of our optical setup α, 7% in the same manner as the analysis of the ON-time histograms. The values of q and s were estimated by the curve fitting by using eq 10 and the fixed values. Results obtained by the analysis are summarized in Table 1. The q values determined by the analysis are 0.23 for the droplets with Triton X-100 and 0.29 for those with STAC. The q value derived from the analysis using eq 10 is slightly (ca. 1.3 times) larger in the droplets with STAC, indicating that the frequency of the photodegradation (PPB) is somewhat higher in this system. The q value can be directly derived also from the ratio of Ns=0/Nall. Here, Nall is the total number of molecules and Ns=0 is the number of molecules that did not show blinking before PPB. The q values obtained by this method are also shown in Table 1. The q values obtained by the analysis with eq 10 are smaller than those obtained by directly counting Ns=0 and Nall. The limited time of measurement up to 120 s may account for the difference. Transitions to the DS in longer time region were not counted owing to the limited measurement time. This uncounted number may enhance the difference. In actuality, the difference is much pronounced in the droplets with Triton X-100 because the p value of the Triton X-100 system is much smaller. The analysis with eq 10 yielded s values of 4.5 and 2.1 respectively for droplets with Triton X-100 and those with STAC, while s values determined by directly counting the number of OFF times are 2.0 in both droplet systems. In the droplets with Triton X-100, the s value obtained by the analysis with eq 10 is larger than that obtained by directly counting the number of the OFF times, which is explained by the smaller q value in the Triton X-100 system and the limited measurement time in the present study. On the other hand, most guest dyes in droplets with STAC underwent PPB within the measurement time (Figure 6c), leading to good agreement between the s values obtained by the analysis with eq 10 and by directly counting the number of OFF times. The results thus obtained by the detailed analysis of the fluorescence intensity trajectories indicate that the probability of the transition from the S1 state to the DS (the value of p) strongly affects the survival time of the guest dye. In other words, the lengths of ON time and survival time for the present two systems are mainly governed by the probability of the transition from the S1 state to the DS. 3. Time Trajectories of the Degree of Fluorescence Polarization. As shown in the previous sections, the probability of the transition to the OFF state was significantly different between the two droplet systems. Although the photoionization may play an important role in the OFF-state formation, the ionization taking place in the solution area would not lead to the OFF state formation detectable in the present experimental condition because of the very short recombination time compared to the bin time. The long recombination time could be, however, observed in the case where the ionization of the molecule adsorbed on the interface, because the electron and the cation produced via the

photoionization can be stabilized at the interfacial area. The guest dye adsorbed on the interface for the time comparable with the bin time may show polarized fluorescence, while the polarized fluorescence is not detectable for the dye in the solution phase owing to the very rapid rotational motion. Hence, we measured the time trajectory of the fluorescence polarization. Figure 7 shows a typical result of the fluorescence polarization measurement of droplets with Triton X-100.

Figure 7. (a) Typical time courses of fluorescence intensities with orthogonal polarizations (black dotted and gray lines) from a BP-PDI in a nanodroplet prepared with Triton X-100 and their sum intensity (black solid line). (b) The time course of the fluorescence polarization, P, calculated from the data shown in panel a.

Two orthogonally polarized components of the fluorescence were respectively detected by APD1 and APD2. As shown in Figures 7a and 7b, almost the same intensities from the APD1 and APD2 are observed and the degree of polarization is almost zero during the measurement. These results indicate that the guest dye did not effectively adsorb onto the interface of octane−Triton X-100 for the time period comparable with or longer than the bin time (25 ms). Most (96%) of the guest molecules showed such a behavior showing no apparent degree of the polarization in droplets with Triton X-100. On the other hand, the fluorescence-intensity trajectory of the guest dyes in the droplets with STAC frequently showed different signal intensities between the two APDs (Figure 8a) and the nonzero values of fluorescence polarization (Figure 8 b). These results are attributable to the adsorption at the oil− water interface with a time period longer than the bin time (25 ms). Hence the long adsorption period is pronounced in the droplets covered with STAC. Indeed, 34% of the guest dyes showed time trajectories of fluorescence polarization with nonzero values. During the long adsorption time, the probability of the ionization increases and, as a result, PPB takes place more frequently. It should be noted that the frequency of the approach of the guest dye to the oil−water interface may be comparable between the two droplet systems. The difference of the polarization measurement is rather ascribed to the adsorption period; the adsorption period much shorter than the bin time (25 ms) in the droplet with Triton X100 resulted in the zero value of polarization measurement. 4. Excited Triplet-State Lifetime of the Guest Dye. We have demonstrated in the previous sections that the duration of adsorption to the oil−water interface is the primal factor 10354



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provided ACF curves with relatively high S/N ratio as typically shown in Figure 9c. Analyzing the ACF curves by using eq 11 yielded the triplet lifetime of the guest dye. G (τ ) = 1 +

⎛ τ⎞ FT exp⎜ − ⎟ 1 − FT ⎝ τT ⎠

(11)

Here, FT and τT are respectively the fractional population and lifetime of the triplet state. The analysis of the ACF curves shown in Figures 9c and 9d provided the same triplet lifetime, 0.4 μs. Although the triplet lifetime is too short to be precisely determined, the result indicates that the triplet lifetime itself is similar between the two droplet systems. The fractional population of the triplet state, FT, showed a difference between the two systems. Analyzing the ACF curves shown in Figures 9c and 9d determined the FT values of 0.31 and 0.48 respectively. This somewhat large difference in FT is ascribed to the higher probability of the intersystem crossing from the S1 to the T1 state in the droplet with STAC. This is probably because the guest dye in the droplet with STAC experienced a polar microenvironment for longer periods than that in the droplet with Triton X-100. The difference of micropolarity resulted in the different probability of the intersystem crossing from the S1 to the T1 state.

Figure 8. (a) Typical time courses of fluorescence intensities with orthogonal polarizations (black dotted and gray lines) from a BP-PDI in a nanodroplet prepared with STAC and their sum intensity (black solid line). (b) The time course of the fluorescence polarization, P, calculated from the data shown in panel a.

associated with the probability of the transition from the S0 state to the DS, p, that mainly determines the length of the survival time. One of the most plausible pathways to the DS from the singlet excited state is the intersystem crossing to the lowest excited triplet state, T1, followed by the absorption of the second photon by which a higher excited triplet state, Tn, is produced. Although the time bin of 25 ms is too long to measure the lifetime of the T1 state at room temperature, it is possible to estimate the triplet lifetime by calculating photonby-photon correlation of the data obtained in the present study. Figure 9 shows time trajectories of fluorescence intensity of guest dyes in droplets with Triton X-100 (Figure 9a) and

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SUMMARY We have observed emissive behaviors of single guest dyes incorporated in octane droplets with ca. 200−300 nm diameters prepared with two different surfactants: Triton X100 and STAC. The guest dyes in the n-octane droplets with Triton X-100 showed continuous emission with little blinking. On the other hand, frequently blinking guest dyes were observed in the droplet covered with STAC. The histogram of the emissive (ON) time, τON, statistically showed that the typical ON time of BP-PDI in the droplets with STAC was much shorter than in those with Triton X-100. Analyzing the histograms of the ON time revealed that the transition probability from S1 to DS in the droplets with STAC was much higher than that in those with Triton X-100. By analyzing the distributions of τsur using eq 10, we have obtained the probability of transition from the DS to the PPB, q, of the guest dye. The obtained q values were almost the same between the two droplet systems. The results obtained by the statistical analysis of τON and τsur indicated that the length of survival time is mainly governed by the probability of the transition from S1 to DS; τON and τsur indeed showed a strong correlation. The fluorescence polarization measurements clearly showed different rotational velocities of guest dyes between the two droplet systems. Most (96%) of the guest dyes in the droplets with Triton X-100 showed rapid rotational motion, indicating that they were not effectively adsorbed onto the interface for a time period longer than the bin time (25 ms). On the other hand, the guest dyes in the droplets with STAC frequently showed nonzero fluorescence polarization, which is attributable to the adsorption at the oil−water interface with a time period longer than the bin time (25 ms). During the long adsorption time, the probability of the ionization increases and, as a result, PPB takes place more frequently. Analyzing the photon correlation curves yielded the triplet state lifetime, τT, of the guest dyes. The value of τT was similar between the two droplet systems. On the other hand, the fractional population of the triplet state, FT, was larger in the

Figure 9. (a, b) Time trajectories of fluorescence intensity of (a) BPPDI in a droplet with Triton X-100 and (b) that in a droplet with STAC. (c, d) The fluorescence ACF curves calculated from the data shown respectively in panels a and b.

STAC (Figure 9b) with corresponding autocorrelation function (ACF) curves of the fluorescence (Figures 9c and 9d). Though the long-time fluorescence emission of the guest dyes in the droplets with Triton X-100 enabled ACF curves to be obtained at high signal-to-noise (S/N) ratio, the short survival time of the guest dyes in the droplets with STAC reduced the S/N ratio of ACF curves. Hence a limited number of guest dyes 10355



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droplet with STAC. This was explained by the increase in the probability of the intersystem crossing from the S1 to the T1 state at the polar interfacial area of the droplet.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The present work was partly supported by Grant-in-Aid for Scientific Research (A) (23245004) to H.M. and Grant-in-Aid for Young Scientists (A) (23681023) to S.I. from the Japan Society for the Promotion of Science (JSPS). The authors sincerely thank Profs. Shiraishi and Hirai for their kind support to the measurement of the droplet diameters using DLS.

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Fluorescence Behavior of Single Guest Molecules in Nonpolar Oil

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