Cooperative Blinking from Dye Ensemble Activated by Energy

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Cooperative Blinking from Dye Ensemble Activated by Energy Transfer for Super-resolution Cellular Imaging Zhihe Liu, Jie Liu, Zezhou Sun, Zhe Zhang, Ye Yuan, Xiaofeng Fang, Fei Wang, Weiping Qin, and Changfeng Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00279 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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Analytical Chemistry

Cooperative Blinking from Dye Ensemble Activated by Energy Transfer for Super-resolution Cellular Imaging Zhihe Liu,a,ξ Jie Liu,b,ξ Zezhou Sun,a Zhe Zhang,a Ye Yuan,a Xiaofeng Fang,b Fei Wang,b Weiping Qin,a and Changfeng Wu*,b State Key Laboratory of Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012, China b Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 510855, China ξ These authors contributed equally. a

*Corresponding author: [email protected]

ABSTRACT: Photoblinking is a fundamental process that occurs exclusively in single fluorophores such as organic dyes, fluorescent proteins, and quantum dots. Here, we describe a strategy to achieve pronounced, high on/off ratio, and cooperative blinking in donoracceptor multi-fluorophore systems. An ensemble of dye molecules doped in semiconducting polymer dots (Pdots) exhibit robust photoblinking, while the pristine Pdots and the dye in optically-inert polymer matrices fluoresce continuously. Energy transfer from Pdots to dye acceptors produces photoblinking via a cooperative process, in which the bright state originates from the dye ensemble and the dark state is due to quenching of semiconducting polymer by hole polarons. Using the blinking Pdots in subcellular structures labeling, we demonstrated approximately 3.6-fold enhancement of imaging resolution in high-order super-resolution optical fluctuation nanoscopy as compared to conventional microscopy. Our findings not only demonstrate the exciting possibility of transforming a non-quantized ensemble into a single-emitter-like optical source, but also provide an effective approach to generate superior photoblinking fluorescent probes for super-resolution imaging applications.

Fluorescence microscopy is an indispensable imaging technique in biological studies.1-6 The resolution of conventional optical microscopy is limited by the Abbe diffraction limit. In recent years, many new techniques have been developed to overcome the optical diffraction limit via different principles; these endeavors have led to the invention of super-resolution imaging modalities. Super-resolution imaging techniques include Stimulated Emission Depletion (STED),7-9 Reversible Saturable Optical Linear Fluorescence Transitions (RESOLFT),10-14 Photoactivated Localization Microscopy (PALM),15-17 Stochastic Optical Reconstruction Microscopy (STORM),18-21 Bayesian analysis of Bleaching and Blinking (3B),22, 23 Structured Illumination Microscopy (SIM),24-26 and Super-resolution Optical Fluctuation Imaging (SOFI).27 As an emerging super-resolution technique, SOFI was first described by T. Dertinger in 2009, and has become a mainstream super-resolution imaging method because of its balanced spatial and temporal resolution, as well as background-reducing capability and simple optical setup.28-34 The SOFI technique is based on high-order cross-cumulants statistical analysis of temporal fluctuations of fluorescent probes recorded in a time-elapsed images. A unique requirement for fluorescent probes used in SOFI is stochastic photoblinking under continuous excitation.27 Originally, photoblinking quantum dots (Qdots) were used as fluorescent probes for SOFI development.35-37 Later on, fluorescent proteins

have been used in SOFI studies.30, 38, 39 As Pdots exhibit prominent optical properties such as high brightness and photostability,40, 41 Pdots have been developed and used as biodegradable nanoprobes for bioimaging and tumor phototherapies.42-49 We have recently developed small photoblinking Pdots for single- and dual color subcellular SOFI imaging.50-52 Because the spatial resolution of SOFI nanoscopy can increase linearly with the cumulant order,29, 31, 32, 53 fluorescent probes with controllable and pronounced blinking are critical for development of high-order SOFI nanoscopy. Organic dyes are the most widely used probes in fluorescence imaging. As single dye molecules exhibit blinking owing to intersystem crossing to the dark triplet state, the usefulness of dyes in SOFI encounters several challenges54: (1) the photobleaching of dyes upon continuous illumination limits the acquisition time, but the SOFI algorithm requires a movie that the fluctuating signal is recorded; (2) the low signal-to-noise ratio of dyes constrains the image quality of SOFI; (3) typical intersystem crossing rates for organic dyes result in microsecond-timescale “on/off” blinking, whereas most movie acquisitions for SOFI are limited to millisecond timescales. Dye-loaded latex or silica nanoparticles exhibited improved brightness and photostability as compared to single-dye molecules.55 However, fluorescence from dye-loaded nanoparticles originates from an ensemble of dye molecules,

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and does not exhibit photoblinking because the fluctuations of different dye molecules may compensate each other. Because of these difficulties, the application of fluorescent dyes in highorder SOFI nanoscopy remains very challenging. Here, we describe a strategy based on Förster resonance energy transfer (FRET) to activate photoblinking from an ensemble of dye molecules. Previous reports showed that large pristine Pdots (>15 nm) do not exhibit blinking.50 We show that the fluorescence from an ensemble of dye acceptors doped in the Pdots can be effectively modulated by the FRET process, yielding discrete, single-emitter-like photoblinking feature. Furthermore, we use the blinking Pdot probes in single-particle and subcellular-specific labeling for high-order SOFI analysis. We obtained a 3.6-fold enhancement in imaging resolution as compared with conventional wide-field microscopy. The new blinking mechanism provides fluorescent probes with tunable on/off intensity and time ratios for SOFI nanoscopy. EXPERIMENTAL SECTION Preparation and characterization of Pdots. Cooperative blinking Pdots were prepared by optimizing nanoreprecipitation method reported in previous studies.41, 51, 56 Poly (9, 9-dioctylfluorenyl-2, 7-diyl), (PFO: Mw: ~147000; polydispersity: 3.0), poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1cyanovinylene-1,4-phenylene)] (CN-PPV, MW: ~15,000, polydispersity, 5.9) were purchased from ADS Dyes. Coumarin 6 (3-(2-Benzothiazolyl)-N, N-diethylumbelliferylamine) (MW: 350.43), NIR695 (2,9,16,23-Tetra-tert-butyl-29H,31Hphthalocyanine) (MW:738.96), amphiphilic functional polymer poly (styrene-co-maleic anhydride) (PSMA, cumene terminated, MW: ~1,700) and tetrahydrofuran (THF, anhydrous, 99.9%) were purchased from Sigma-Aldrich. For preparation of green emission (PFO-Coumarin 6) Pdots, 1 mL THF solution containing PFO (100 μg), Coumarin 6 (1 μg) and PSMA (20 μg) was injected into 10 mL Milli-Q water under ultrasonic concussion. The THF was scavenged by nitrogen gas flow on a 90oC hot plate. A 0.22 μm polyamides filter (Millipore) was used to filtrate large aggregates. For nearinfrared emission Pdots (CNPPV-NIR695), the procedure was consistent with green emission Pdots (PFO-Coumarin 6) expect for a lower concentration of NIR695 (0.5 μg/mL). Size distributions of cooperative blinking Pdots dispersed in Milli-Q water were measured with a dynamic light scattering (DLS) instrument (Malvern NANO ZS). UV-Vis absorption spectra of cooperative blinking Pdots were measured with an ultraviolet-visible spectrophotometer (Agilent Cary 60) using 1cm quartz cuvettes. A Horiba FluoroMax-4 fluorescence spectrophotometer was used for measuring fluorescence spectra. Bioconjugation and specific subcellular labeling. Bioconjugation of cooperative blinking Pdots was catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Sigma). 1 mL of Pdots solution (50 μg/mL) was added 20 μL of PEG (5 wt%), 20 μL of concentrated HEPES buffer (1 μM), 60 μL of Streptavidin (1 mg/mL) and 20 μL of EDC solution (5 mg/mL in water). These reagents were mixed with rotation for 4 hours at room

temperature. An ultrafiltration centrifugation (100KD Amicon® Ultra-4) with 3000 rpm (Eppendorf 5804R centrifuge) was used to separate free streptavidin. Purified Pdots-streptavidin bioconjugates were stored at 4 ºC for use. Specific subcellular labeling was performed according to previous reports.51, 52 Optical setup. Single-particle spectrum was performed on an Olympus IX71 inverted microscope with an Andor Spectrograph (Shamrock SR-500i-D2, UK). A 395 nm or 490 nm optical fiber coupled LED (Thorlabs, M395F3, M490F3) collimated by a collimator (Thorlabs, F240SMA-532) illuminated on the back pupil of objective (Olympus, UPLSAPO 40X, NA: 0.9). For PFO-Coumarin 6 Pdots, a single-edge standard epi-fluorescence dichroic beam splitter (Semrock, 409 nm edge BrightLine®, FF409-Di03-25x36) was used for reflecting the excitation light and transmitting emission light. Subsequently, the emission light passed through 409 nm long-pass emission filter (Semrock, FF02-409/LP-25) was introduced into spectrograph and projected on an EMCCD (Andor, Newton 920, UK). For CNPPV-NIR695 Pdots, 490 nm LED, 495 nm dichroic beam splitter (Semrock, FF495-Di0325x36), 515 nm long-pass emission filter (Semrock, FF01515/LP-25) were used. To obtain high SNR single-particle spectra, EMCCD was operated at -55 oC with 200 EM gain. Single-particle and subcellular cytoskeleton imaging were performed on a Nikon N-STORM microscope equipped with an 100x TIRF (total internal reflection fluorescence) oil objective (Nikon, NA: 1.49, Japan). 405 nm laser (Coherent, OBIS, 405 nm, 200 mW, USA) and 488 nm sapphire laser (Coherent, Sapphire, 488 nm, 300 mW, USA) were aligned in an optical fiber coupled TIRF illuminator (Nikon, TIRF-E, Japan). Multiband excitation filter (Chroma, ZET405/488/561/640xv2) and dichroic filter (Chroma, ZT405/488/561/640rpcv2) aligned in filter cube were used. Band-pass emission filters (Chroma, ET525/30m and Semrock, FF01-711/25-25) were manually configured for PFO-Coumarin 6 and CNPPV-NIR695 Pdots, respectively. Perfect focus system (PFS) was used for locking the focus. Another 1.5x magnification was performed to yield a 106 nm per pixel imaging on EMCCD (Andor, DU897, UK). Data analysis methods. All single-particle and subcellular cytoskeleton imaging stacks were corrected by a home-written code in Matlab 2017b (Mathworks Inc., USA) based on a subpixels drift correction algorithm.57 Single-particle blinking and off-time interval data were extracted by a home-written Matlab code. Cross-cumulants SOFI analyzing of singleparticle and subcellular cytoskeleton imaging stacks were based on balanced SOFI algorithm.35 RESULTS AND DISCUSSION Preparation and characterization of cooperative blinking Pdots. In previous studies, we found that Pdots with a particle size larger than 15 nm exhibited steady fluorescence kinetics with no obvious photoblinking, while photoblinking was often observed for small Pdots when their size decreased to ~10 nm.5052, 58 Although the small photoblinking Pdots were used for SOFI,51, 52 their low brightness, limited by the small particle size, is a severe drawback. A variety of organic dyes can be doped in Pdots, yielding improved brightness, photostability, and tunable emission colors.52, 59, 60 We were inspired to


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Analytical Chemistry manipulate the photoblinking property by using energy transfer from the semiconducting polymer to the dye acceptors. In principle, fluorescence from the dye acceptors can be modulated when the energy transfer is switched on and off.61, 62 We choose the poly(9, 9-dioctylfluorenyl-2, 7-diyl) (PFO) polymer doped with Coumarin 6 and the poly[2-methoxy-5-(2ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CNPPV) polymer doped with 2,9,16,23-Tetra-tert-butyl29H,31H-phthalocyanine (NIR695) to construct donoracceptor Pdots. Their chemical structures are illustrated in Figure 1a. A functional polymer poly(styrene-co-maleic anhydride) (PSMA) was used for surface functionalization and conjugation with streptavidin.41, 51, 56 Because of the large spectral overlap between the polymer and the dye (see Figure S1), the Pdots show dominant fluorescence from the dye acceptors despite the low doping ratios (1 wt% coumarin 6 in PFO and 0.5 wt% NIR695 in CNPPV Pdot) (Figure S2). Dynamic light scattering (DLS) measurements show that the particle sizes of PFO-Coumarin 6 and CNPPV-NIR695 Pdots are 18 nm and 16 nm, respectively. The transmission electron microscopy (TEM) images indicated that the monodispersed particles are spherical morphology (Figure 1b, 1c). As shown in Figure 1d-e, spectra of PFO-Coumarin 6 and CNPPV-NIR695 Pdots in aqueous solutions indicated successful doping of the dyes into the Pdots and efficient FRET from the polymer donors to dye acceptors.

Figure 1. (a) Chemical structures of donors (PFO and CN-PPV), acceptors (Coumarin 6 and NIR695) and the functional polymer PSMA. Size distributions determined by dynamic light scattering for (b) PFO-Coumarin 6 and (c) CNPPV-NIR695 Pdots, inserts are corresponding transmission electron microscopy (TEM) images. Normalized absorption and emission spectrum of (d) PFOCoumarin 6 and (e) CNPPV-NIR695 Pdots.

Single particle fluorescence kinetics were used to validate the cooperative blinking activated by the energy transfer from polymer donors and dye acceptors. Non-conjugated polymer

polystyrene (PS) doped with 1 wt% coumarin 6 or 0.5 wt% NIR695 were used for comparative studies. As shown in Figure 2a and 2b, single-particle intensity trajectories of the pristine PFO and CNPPV Pdots do not exhibit photoblinking, which is consistent with our previous results that larger Pdots (>15 nm) produce relatively steady fluorescence due to a larger number of chromophores.50, 52, 63, 64 Similarly, fluorescence intensity trajectories of Coumarin 6 or NIR695 doped polystyrene (PS) nanoparticles also do not exhibit photoblinking, which is consistent with single-particle fluorescence of dye-loaded latex nanospheres.65 However, the two types of dye-doped Pdots (PFO-Coumarin 6 and CNPPV-NIR695) exhibited pronounced photoblinking by the excitation of the semiconducting polymers (Figure 2c and 2d), indicating that the blinking was cooperatively enabled by both the polymer donor and the dye acceptor. FRET efficiency of PFO-Coumarin 6 and CNPPVNIR695 Pdots was 81% and 60%, respectively. Nearly all the dye-doped Pdots studied in our experiment showed discrete blinking trajectories, despite some variations in the fluorescence-on time ratios (Figure S3 and S4). Typical Photoblinking on/off intensity ratio of PFO-Coumarin 6 and CNPPV-NIR695 Pdtos is 4.2 and 3.6, respectively (Figure S5), indicating a robust blinking property. Generally, the organic molecules can be oxidized by reactive oxygen species generated from donor polymers under laser illumination, 66, 67 as shown in figure 2c-2d and figure S3-S4, after 30s excitation (~50W/cm2 laser power density), both these types of cooperative blinking Pdots show high photostability. Notably, the fluorescence from the dye ensemble was stochastically turned “on” and “off”, strongly implying that the energy transfer was modulated by a stochastic process. Singleparticle spectroscopy showed that the fluorescence of the dyedoped Pdots at single-particle levels was dominated by the emission peak from dye acceptors (Figure 2e-f). Narrow-band emission filters corresponding to coumarin 6 or NIR695 were used in a single-particle kinetics study, indicating the fluorescence “on” state was exclusively attributable to the dye acceptors. The non-fluorescent “off” state was most probably due to the semiconducting polymer, because the dye molecules in polystyrene nanoparticles showed steady fluorescence without blinking. As described in previous reports,58, 68 hole polarons can effectively quench the fluorescence of Pdots, yielding intensity fluctuations and centroid displacement in single-particle imaging. We postulate that the generation of hole polarons in semiconducting polymers may drastically modulate the FRET efficiency, to an extent that FRET can be completely tuned off because it is sensitively dependent on the distance and spectral overlap between donor and acceptor. The above mechanism is illustrated in the Jablonski diagram (Figure 2g). The blinking of the dye-doped Pdots was unambiguously enabled by the polymer donor and an ensemble of dye molecules, which is distinctive as compared to the photoblinking systems previously reported.62 Both of the two types of dye-doped Pdots showed power-law blinking processes (Figure 2h and 2i), resembling the photon statistics of a singleemitter system. This finding highlights the exciting possibility of the use of FRET pathway to transform ensemble multiple chromophores into a quantized optical emitter.



Figure 2. Typical fluorescence trajectories of single nanoparticles and scheme of cooperative blinking. (a) Pure PFO Pdots and Coumarin 6-doped polystyrene nanoparticles, (b) pure CNPPV Pdots and NIR695-doped polystyrene nanoparticles, (c) PFO-Coumarin 6 Pdots, (d) CNPPV-NIR695 Pdots. Single-particle spectra of (e) PFO-Coumarin 6 and (f) CNPPV-NIR695 Pdots. (g) Schematic illustration of cooperative blinking in dye-doped Pdots. Statistics of the off-time interval for (h) PFO-Coumarin 6 Pdots (n = 50) and (i) CNPPV-NIR695 Pdots (n = 49). (g) Schematic illustration of cooperative blinking in dye-doped Pdots

Single-particle SOFI nanoscopy with cooperative blinking Pdots. Cooperative blinking is not only fundamentally interesting, but also practically useful for subcellular imaging and SOFI nanoscopy. To evaluate the SOFI performance of the Pdots, 1000 frames of single-particle images were analyzed by calculating 4th-order cross-cumulants. Conventional wide-field, and 4th-order SOFI reconstructed imaging of PFO-Coumarin 6 Pdots was shown in Figure 3a-b and 3c-d, respectively. Two optical-diffraction-limited blurry points marked with white arrows in the conventional wide-field image can obviously be resolved by 4th-order SOFI analysis (Figure 3c-d). The spatial resolution is quantified by plotting the intensity profiles of the point spread function (PSF). As shown in Figure 3e, approximately 5.3-fold enhancement in imaging resolution for PFO-Coumarin 6 Pdots were obtained. The statistical full width at half maximum (FWHM) of a dozen single particles in widefield images (Figure 3f) was 287 ± 11 nm, while the FWHMs after 4th-order SOFI analysis was determined to be 75 ± 9 nm, which indicates an approximately 3.8-fold enhancement. Similarly, 4th-order SOFI analysis was performed for CNPPVNIR695 Pdots (Figure 3g-h and 3i-j) and the spatial resolution was also found to be dramatically enhanced about 4.0-fold (Figure 3k). Statistical FWHMs of 4th-order SOFI images show 85 ± 6 nm and indicating that about 3.9-fold compared with conventional wide field images (333 ± 12 nm) (Figure 3l). These results provide a general approach for tuning photon statistics of Pdots for super-resolution imaging applications. Subcellular structures SOFI nanoscopy with cooperative blinking Pdots. Finally, we demonstrate the application of the dye-doped Pdots for blinking-based super-resolution nanoscopy. Microtubules (α-tubulin) of COS-7 cells were labelled with streptavidin-conjugated Pdots. Both the two types of Pdots show excellent subcellular labeling with high signal-to-noise ratios (Figure 4). Next, we performed SOFI

Figure 3. Single-particle SOFI nanoscopy of PFO-Coumarin 6 and CNPPV-NIR695 Pdots. (a) Conventional wide-field image and (b) 4th-order SOFI image of PFO-Coumarin 6 Pdots. Magnified views of (c) wide field and (d) 4th-order SOFI images from the area indicated by the white box labelled “I” in panel (a-b). (e) Intensity profiles of PFO-Coumarin 6 Pdots for the white arrows shown in panels (c)-(d). (f) Statistical FWHM of individual PFO-Coumarin6 Pdots in wide field and SOFI images. (g) Conventional wide-field image and (h) 4th-order SOFI image of CNPPV-NIR695 Pdots. Magnified views of (i) wide field and (j) 4th-order SOFI images from the area indicated by the white box labelled “II” in panel (i-j). (k) Intensity profiles of CNPPV-NIR695 Pdots for the white arrows shown in panels (i)-(j). (l) Statistical FWHM of individual CNPPVNIR695 Pdots in wide field and SOFI images.

analysis of the 4th-order cross-cumulants of the microtubule structures labeled by PFO-Coumarin 6 Pdots. Compared with the wide-field images, the resolution of the 4th-order SOFI images was apparently enhanced (Figure 4a-d). The FWHMs of the intensity profiles of the fibrils highlighted by the white arrows in Figure 4e, f, are shown in Figure 4i; these indicate that the resolution of 4th-order SOFI (90 nm) increased by a


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Analytical Chemistry factor of 3.6 compared with the figure for averaged wide-field images (325 nm). Furthermore, after 4th-order SOFI analysis, two adjacent filaments (separated by 155 nm) were clearly distinguished (Figure 4g, h, j), whereas the averaged widefield image showed a broad intensity profile due to the optical diffraction limit. Subsequently, we obtained a 4th-order SOFI image of microtubules stained with CNPPV-NIR695 Pdots in a COS-7 cell (Figure 4l, n), which were reconstructed from

wide field images (Figure 4k, m). Intensity profiles of the microtubule filaments marked by white arrows (Figure 4m, n) are shown in Figure 4o, indicating a 3.2-fold enhancement in the spatial resolution. These results indicate that FRETactivated blinking Pdots hold promise for super-resolution imaging of subcellular structures.

Figure 4. SOFI nanoscopy of microtubules labeled with cooperative blinking Pdots. (a, c) Conventional wide-field images of PFO-Coumarin 6-labelled microtubules in COS-7 cells. (b, d) 4th-order SOFI nanoscopy images of the views shown in panels (a) and (c), respectively. (e, f) Magnified views from the images of areas indicated by the white boxes labelled “I” in panels (a) and (b), respectively. (g, h) Magnified views of areas of the images indicated by the white boxes labelled “II” in panels (c) and (d), respectively. (i, j) Intensity profiles of the microtubules indicated by white arrows shown in panels (e, f) and (g, h), respectively. (k) Conventional wide-field view of CNPPV-NIR695labelled microtubules in a COS-7 cell. (l) 4th-order SOFI nanoscopy image of the view shown in panel (k). (m, n) Magnified views of the image area bounded by the white boxes labelled “III” in panels (k) and (l), respectively. (o) Intensity profiles from wide-field and 4th-order SOFI images of the features indicated by the white arrows shown in panels (m) and (n).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

CONCLUSION In conclusion, we have developed a unique strategy to achieve pronounced photoblinking in a nanoscale multichromophore system. While pristine Pdots (15-20 nm) and dye ensemble in polystyrene nanoparticles show steady fluorescence, the dye molecules doped into Pdots exhibit cooperative photoblinking as a single optical emitter. We demonstrate that the photoblinking is activated by FRET, where the bright state is due to the dye ensemble and the dark state is attributable to the hole polarons of the semiconducting polymer. By conjugation with streptavidin, we performed subcellular labeling using these photoblinking probes. As compared with conventional wide-field microscopy, an approximately 3.6-fold enhancement in imaging resolution (~90 nm) was obtained by 4th-order SOFI analysis of single particles and subcellular structures. In this study, we successfully transformed a non-quantized ensemble system into a single-emitter-like optical probe. This study highlights the effective approach to manipulate photophysical properties of fluorescent dyes for super-resolution optical imaging.

Supplementary Figures 1-4 (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ORCID Zhihe Liu: 0000-0002-5134-4116 Changfeng Wu: 0000-0001-6797-9784

Author Contributions ξ These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

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This work was supported by the grants from the National Natural Science Foundation of China (Grant No. 61335001; Grant No. 81771930), the National Key R&D Plan of China (Grant No. 2018YFB04007200), and the Shenzhen Science and Technology Innovation Commission (Grant No. JCYJ20170307110157501).

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Cooperative Blinking from Dye Ensemble Activated by Energy

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