Multicolor Super-resolution Fluorescence ... - ACS Publications

Jul 11, 2017 - Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 510855, China. §. State Key L...
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Multicolor Super-resolution Fluorescence Microscopy with Blue and Carmine Small Photoblinking Polymer Dots Xuanze Chen,†,ξ Zhihe Liu,‡,§,ξ Rongqin Li,†,ξ Chunyan Shan,† Zhiping Zeng,∥ Boxin Xue,† Weihong Yuan,† Chi Mo,† Peng Xi,*,† Changfeng Wu,*,‡ and Yujie Sun*,† †

State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, and Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China ‡ Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 510855, China § State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012, China ∥ College of Physics and Information Engineering, Fuzhou University, Fuzhou 350116, China S Supporting Information *

ABSTRACT: Advances in the development of small photoblinking semiconducting polymer dots (Pdots) have attracted great interest for use in super-resolution microscopy. However, multicolor super-resolution imaging using conventional small photoblinking Pdots remains a challenge due to their limited color choice, broad emission spectrum, and heavy spectrum crosstalk. Here, we introduce two types of small photoblinking Pdots with different colors and relatively narrow emission spectra: blue PFO Pdots and carmine PFTBT5 Pdots for blinking-based statistical nanoscopy. Both of these probes feature ultrahigh single-particle brightness, very strong photostability, superior biocompatibility, and robust fluorescence fluctuation. In addition, these small photoblinking Pdots serve as excellent labels for dual-color superresolution optical fluctuation imaging (SOFI) of specific subcellular structures, indicating their promise for long-term multicolor SOFI nanoscopy with high spatiotemporal resolution. KEYWORDS: multicolor super-resolution imaging, super-resolution optical fluctuation imaging, blue and carmine small polymer dots, photoblinking, bioimaging ar-field fluorescence microscopy has proven to be one of the most important imaging tools for investigation in the life sciences. However, the spatial resolution of conventional fluorescence microscopy is limited by classic optical diffraction (∼300 nm), limiting the resolution and detail of subcellular structures. Fortunately, over the past two decades, a broad range of super-resolution optical microscopy (nanoscopy) techniques have been developed to break the diffraction barrier, and these have played very important roles in biological imaging.1 Such techniques include stimulated emission depletion microscopy (STED)2,3 and structured illumination microscopy (SIM),4 which are based on point spread function modulation, as well as photoactivated localization microscopy (PALM)5 and stochastic optical reconstruction microscopy (STORM),6 which are based on single molecule localization. Blinking-based statistical nanoscopy, such as super-resolution optical fluctuation imaging (SOFI),7−9 which is based on the statistical analysis of independent temporal fluorescence fluctuations of photoblinking probes. SOFI has attracted great interest because of its numerous characteristics, such as rational

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balancing between high spatiotemporal resolution, backgroundfree optical sectioning capabilities, and its technical simplicity.9 Notably, fluorescent probes are essential for achieving highquality super-resolution fluorescence images.10 Various photoblinking probes have been recently developed for use in SOFI, and these can be divided into three categories: fluorescent proteins (FPs), organic dyes, and inorganic nanoparticles. Blinking FPs, such as Dronpa (and its variants)9,11 and SkylanS,12 have been extensively used in SOFI owing to their excellent biocompatibility, but their performance in regard to brightness and photostability are lacking. In contrast, different types of organic dyes, such as the Alexa series,13,14 have demonstrated improved brightness and photostability when compared to FPs and have been tested for use in SOFI. However, the brightness and photostability of FPs and organic dyes are still Received: April 26, 2017 Accepted: July 11, 2017 Published: July 11, 2017 8084

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Figure 1. Properties of narrow emissive small photoblinking Pdots. (a) Chemical structures of relative narrow emissive semiconductor polymers for synthesis of small photoblinking Pdots. (b,c) The size distributions of two types of small photoblinking Pdots, blue PFO (blue) and carmine PFTBT5 (magenta) Pdots, respectively, measured by DLS (the mean sizes are (b) 10 nm and (c) 13 nm, respectively). (d,e) Absorption and emission spectra of PFO Pdots and PFTBT5 Pdots, respectively, in water. (f,g) Normalized single particle brightness of PFO (f) and PFTBT5 (g) small Pdot nanoparticles compared to AF 405 (f) and Qdots 655 (g), respectively. (h) A photograph of aqueous small photoblinking Pdot suspensions (top) and under UV light illumination (bottom).

unsatisfactory in regard to their use in SOFI.15 To achieve fluorescent probes featuring ultrahigh brightness and very strong photostability, different types of photoblinking inorganic nanoparticles, such as quantum dots (Qdots)16−18 and carbon dots (CNDs),19 have been developed for use in SOFI. However, the performance of these probes are lacking due to trade-offs in brightness, photostability, biocompatibility, and labeling capability. Semiconducting Pdots have demonstrated great promise for use in fluorescent labeling owing to their outstanding optical performance, and they have displayed broad applications in bioimaging.20−28 Recently, our research group developed two types of small photoblinking Pdots (green PFBT and red CNPPV Pdots) for use in single-color SOFI. Both of these featured higher brightness, stronger photostability, superior biocompatibility, and excellent labeling capabilities compared to conventional Qdots and CNDs.29 Despite this progress, multicolor super-resolution fluorescence imaging is essential for investigating protein−protein interactions and multiple subcellular structures.30 Unfortunately, multicolor SOFI remains a challenge, as conventional FPs, organic dyes, CNDs, and small photoblinking Pdots demonstrate very broad emissions and heavy spectrum crosstalk.29,31 Therefore, it is essential to develop small photoblinking Pdots with different spectral windows and narrow fluorescent emission bands to promote multicolor SOFI. Here we present two types of small photoblinking Pdots, PFO and PFTBT5 Pdots, with relatively narrow blue and carmine emissive windows, respectively. These Pdots feature ultrahigh brightness, very strong photostability, superior biocompatibility, and obvious fluorescence fluctuation. The values of emission at full width at half-maximum are about 40 and 70 nm for the PFO and PFTBT5 Pdots, respectively. Furthermore, we demonstrate that these Pdots function as excellent labels in both single and dual color SOFI of specific subcellular structures. Our results indicate that these small Pdots greatly enrich the photoblinking Pdots family and

promise wide utilization in super-resolution bioimaging and super-resolution multiplexed detection.

RESULTS AND DISCUSSION Previously we developed a general synthesis strategy to prepare small photoblinking Pdots.29,32 To develop small photoblinking Pdots of different colors with relatively narrow emission spectra for multicolor super-resolution imaging, we utilized a highthroughput screening process.21,33 We then selected blue fluorescent polymer PFO and carmine fluorescent polymers PFTBT5 to produce relatively narrow-emissive small photoblinking Pdots. The chemical structures and acronyms related to the conjugated polymers presented in this paper are shown in Figure 1 a. Figure 1b,c shows that the average size of the PFO and PFTBT5 Pdots was approximately 10 and 13 nm, respectively, as measured by dynamic light scattering (DLS) and consistent with the results of transmission electron microscope (TEM) analysis (Figure S1, Supporting Information). The approximately spherical morphologies of the PFO and PFTBT5 Pdots were further verified using atomic force microscope (AFM) imaging (Figure S2, Supporting Information). Both PFO and PFTBT5 Pdots exhibited a broad absorption range from 300 to 400 nm, demonstrated peak emission wavelengths at 430 and 650 nm, and featured 40 and 70 nm emission values at full width at half-maximum, respectively (Figure 1d,e). We prepared the small photoblinking Pdots by optimizing the mass ratio of the fluorescent polymer (PFO or PFTBT5) and the PSMA polymer in the nanoprecipitation procedure. Furthermore, the concentration of the polymer solutions (in tetrahydrofuran) injected into the deionized water under vigorous ultrasonic conditions was modified relative to that previously reported,29,32 which resulted in only one or two PFO or PFTBT5 polymer chains curled into the Pdots dispersed in water. With optimized synthesis methods, PFO and PFTBT5 Pdots with robust fluorescence fluctuation/blinking were developed. To achieve quantitative brightness comparisons, bright Alexa Fluor 405 8085

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Figure 2. SOFI nanoscopy of PFO and PFTBT5 small photoblinking Pdots. (a) Typical conventional averaged wide-field image of PFO Pdots. (b) Second-order SOFI analysis of 300 raw data from panel a). (c, d) Magnified view of the white box in panels a and b, respectively. (e) Intensity profiles of the particle indicated by white arrows in panels c and d. (f) Typical conventional averaged wide-field image of PFTBT5 Pdots. (g) Second-order SOFI analysis of 300 raw data points from panel f. (h, i) Magnified view of the white box in panels f and g, respectively. (j) Intensity profiles of the particle indicated by white arrows in panels h and i. Scale bar = 1 μm in panels a, b, f, and g and 500 nm in panels c, d, h, and i.

Figure 3. Fluorescence images of different subcellular structures in BS-C-1 cells labeled with PFO and PFTBT5 small photoblinking Pdots. (a,b) Single color imaging of microtubule filaments and mitochondria, respectively, labeled with PFO Pdots. (c,d) Single color imaging of microtubule filaments and clathrin coated vesicles, respectively, labeled with PFTBT5 Pdots. (e) Dual-color imaging of microtubule filaments and clathrin coated vesicles labeled using PFTBT5 (magenta) and PFO (green) Pdots, respectively. (f) Dual-color imaging of alpha and acetylated microtubule labeled using PFO (green) and PFTBT5 (magenta) Pdots, respectively. (g) Dual-color imaging of microtubule and mitochondria using PFO (green) and PFTBT5 (magenta) Pdots, respectively. (h) Dual-color imaging of microtubule filaments and clathrin coated vesicles labeled using PFO (green) and PFTBT5 (magenta) Pdots, respectively. Scale bar = 5 μm in panels a−h.

(AF 405) and Qdots 655 were chosen as references for the PFO and PFTBT5 Pdots, respectively, due to their similar spectra. PFO and PFTBT5 Pdots show a 4.3-fold and 2.4-fold brightness improvement compared to AF 405 and Qdots 655, respectively, under the same imaging conditions (405 nm for excitation and detection; Figure 1f,g). Typical blinking trajectories of PFO and PFTBT5 Pdots are shown in Figure S3 (Supporting Information). We then measured the fluorescence fluctuations of the PFO and PFTBT5 Pdots, which demonstrated power-law blinking properties similar to other small photoblinking Pdots (Figure 1h−k). Importantly,

our observations indicated the potential for PFO and PFTBT5 Pdots to be the first blue and carmine blinking fluorescent probes for use in super-resolution imaging based on blinking statistics. In addition, no photodecay was observed during the acquisition (>25 s), which demonstrated their excellent photostability (Figure S4, Supporting Information). Noncytotoxic Pdots show potential for fluorescence imaging and tracking in living cells (Figure S5 and Table S1, Supporting Information). As shown in Figure 1 h, along with the previously reported PFBT and CN-PPV Pdots, blue FPO and carmine PFTBT5 Pdots greatly enrich the small photoblinking Pdots 8086

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Figure 4. Single-color SOFI imaging of subcellular structures labeled with single-color narrow emissive small photoblinking Pdots. (a) Wide field imaging of microtubule structure labeled with PFO Pdots. (b) Second-order SOFI analysis of 600 frames of raw data from panel a. (c,d) The intensity profiles of the white arrows shown in panels a and b. (e) Wide field imaging of clathrin-coated pit (CCP) structure labeling with PFTBT5 Pdots. (f) SOFI analysis of 500 frames of raw data from panel e. (g) Magnified area in the square in panel f. (h) Second-order SOFI image generated by analyzing 500 frames of raw data from panel g. (i,j) The intensity profiles of the white arrows i and ii shown in panels g and h. Scale bar = 5 μm in panels a, b, e, and f and 1 μm in panels g and h.

including cytoskeleton microtubule filaments (Figure 3a,c), mitochondrial outer membranes (Figure 3b), and clathrincoated vesicles (Figure 3d). These fluorescence images clearly showed staining patterns consistent with other widely used fluorescent proteins and organic dyes. Benefiting from the ultrahigh brightness of these Pdots, all fluorescence images exhibited high signal-to-noise ratios. We also evaluated the possibility of performing dual color labeling using PFO and PFTBT5 Pdots. We labeled clathrin-coated vesicles and microtubule filaments with PFO and PFTBT5 Pdots, respectively (Figure 3e). Subsequent imaging indicated that clathrin-coated vesicles were randomly distributed around the microtubule filaments, which is consistent with previous reports.4,34 These observations are equivalent to the results obtained using PFO-labeled microtubules and PFTBT5-labeled clathrin (Figure 3h). Prior studies have indicated that the acetylation of microtubules is predominant at their minus end.35 When we specifically labeled α-tubulin and acetylated tubulin with PFO and PFTBT5 Pdots, respectively, our results indicated that the growing end of the microtubule filament lacked acetylated tubulin, which is consistent with prior studies implying the plus end of microtubules is more dynamic than other regions (Figure 3f). 35 In addition, we labeled mitochondria and microtubule filaments with PFO and PFTBT5 Pdots, respectively (Figure 3g). Subsequently, the distribution of the mitochondria appeared heterogeneous, demonstrating a much denser distribution near the nucleus. In addition, we were also able to differentiate different mitochondria structures, including elongated, tubular, and globular morphologies. When simultaneously imaging microtubules, we found that a large proportion of the mitochondria were diffused along the microtubules, consistent with prior

family and together completely cover the visible spectrum for blinking-based statistical super-resolution imaging methods. To investigate the possibility of performing super-resolution imaging using these small photoblinking Pdots, 300 frames of raw data were acquired and analyzed (Figure 2). Considering the power-law blinking properties of the PFO and PFTBT5 Pdots, second-order cross-cumulant SOFI analysis was utilized.19 For the PFO Pdots, the spatial resolution of conventional wide-field microscopy was approximately 270 ± 42 nm (n = 20), while the spatial resolution was improved to approximately 152 ± 12 nm (n = 20) with second-order SOFI analysis, which corresponded to an approximate 1.8-fold spatial resolution enhancement (Figure 2a−e and Figure S6a, Supporting Information). For the PFTBT5 Pdots, the conventional resolution was approximately 323 ± 30 nm (n = 20), while the spatial resolution was improved to approximately 198 ± 37 nm (n = 20) using second-order SOFI analysis, which corresponded to an approximate 1.6-fold spatial resolution enhancement (Figure 2f−j and Figure S6b, Supporting Information). Furthermore, the signal-to-background ratios were also improved for both the PFO and PFTBT5 Pdots using SOFI analysis compared to wide-field imaging. Single particle super-resolution results indicated that these two types of small photoblinking Pdots have the capacity to be excellent fluorescent markers for subcellular super-resolution imaging. To satisfy the Nyquist sampling rate, higher labeling density is usually required for super-resolution fluorescence imaging. We thus investigated the performance of these small Pdots in subcellular labeling. After optimization of the labeling methods, both streptavidin-conjugated PFO and PFTBT5 Pdots specially targeted specific subcellular structures with high labeling density in green monkey kidney epithelia (BS-C-1) cells, 8087

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Figure 5. Dual-color SOFI nanoscopy of subcellular structures labeled with narrow emissive small photoblinking Pdots. (a) Wide field imaging of clathrin coated pits labeled with PFO Pdots and microtubule labeled with PFTBT5 Pdots. (b) Magnified region show in white box in panel a. (c) Second-order SOFI image generated by analyzing 500 frames of raw data from panel panel b. (d,e) The intensity profiles of the white arrows i and ii shown in panels b and c. Scale bar = 1 μm in panel a and 500 nm in panels b and c.

observations indicating that mitochondria are predominantly transported along microtubules in mammalian cells.36 We then tested SOFI performance via single-color subcellular labeling using these two types of small photoblinking Pdots. About 600 frames of raw data were acquired from microtubule core component α-tubulin labeled using PFO Pdots (Figure 4a,b). With second-order cross-cumulant SOFI analysis, SOFI images exhibited a higher degree of detail and enhanced signal-to-background ratio compared to average conventional wide-field images. The intensity profiles shown in Figure 4c,d indicated that an approximate 1.93-fold enhancement of spatial resolution was achieved. Next, we labeled clathrin-coated pits (CCPs) using PFTBT5 Pdots (Figure 4e−h) and collected 500 frames of raw data. Previous studies have demonstrated that the maximum diameter of mature CCPs is between 90 and 240 nm.4 Therefore, Pdotlinked CCPs were always found using wide-field imaging due to the poor spatial resolution (>300 nm). With second-order cross-cumulant SOFI analysis, some ring structure characterization (white arrow i in Figure 4h) of mature CCPs was resolved with enhanced spatial resolution and signal-tobackground ratio. The intensity profiles shown in Figure 4i,j indicated an approximate 1.89-fold enhancement of spatial resolution was achieved. We next performed indirect immunofluorescence imaging of the microtubule network and CCPs in BS-C-1 cells via the use of small Pdots to achieve dual-color labeling, as shown in Figure 5. Cells were immune-stained with primary antibodies and streptavidin-conjugated small Pdots. PFO and PFTBT5 Pdots were used to identify clathrin and microtubules, respectively (Figure 4a). Our observations indicated that PFO and PFTBT5 perform equally well as labels in this immunofluorescence imaging. In contrast to conventional wide-field images, detailed colocalization of individual CCPs and microtubule filaments were clearly resolved with enhanced spatial resolution and

signal-to-background ratio following second-order SOFI analysis (Figure 5b,c). Based on intensity profile statistics, an approximate 1.67-fold enhancement of spatial resolution was obtained and finer structures were resolved (Figure 5d,e).

CONCLUSIONS In conclusion, we have reported on two types of small photoblinking Pdots with different colors and relatively narrow emissive spectra: blue PFO and carmine PFTBT5 Pdots. These small photoblinking Pdots fill prior color gaps in the labels used in multicolor statistical super-resolution imaging. Bioconjugation and specific subcellular targeting using these Pdots exhibited excellent capability for multicolor blinking-based statistical nanoscopy, such as multicolor SOFI. Several challenges, however, remain unaddressed. First, it is important to reduce the size of these Pdots down to 5 nm so as to approach the size of FPs. Second, synthesis of Pdots with emissive values at full width at half-maximum of less than 30 nm should be explored. Third, to facilitating fast superresolution imaging for assessment of live-cell dynamics the blinking rate of small Pdots must be further enhanced. In addition, the development of an effective multiplexing labeling method utilizing small photoblinking Pdots would greatly improve our ability to identify different subcellular structures. The blue PFO and carmine PFTBT5 small photoblinking Pdots presented in this paper greatly enrich the small photoblinking Pdots family, which now covers the entire visible spectrum, and show substantial promise for use in blinking-based statistical super-resolution imaging37 and many multiplexed biological detections.38−42 MATERIALS AND METHODS Preparation of Functioning Semiconducting Polymer Dots. The blue emission polymer poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO, average molecular weight, Mw 147 000; polydispersity, 3.0) was 8088

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Technology), the two different color fluorescence signals were passed through two different cleanup filters (ET450/50m for PFO and ET667/30m for PFTBT5 small Pdots, Chroma Technology) and then imaged on a scientific complementary metal oxide semiconductor (sCMOS, Flash 4.0, Hamamatsu). Cell Immunostaining. BS-C-1 cells (African Green monkey kidney epithelial cells) were seeded in a glass bottomed 35 mm dish and cultured with Dulbecco’s modified Eagle medium (Gibco), supplemented with 10% fetal bovine serum (Gibco), nonessential amino acids (Gibco) and antibiotics (penicillin and streptomycin; Life Technologies). BS-C-1 cells were harvest when the confluency reached about 80−90%. For microtubule imaging, cells were washed with PBS twice and extracted with 0.2% Triton X-100 dissolved in prewarmed extracting buffer containing 0.1 M piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 1 mM ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), and 1 mM MgCl2 at room temperature for about 30 s. Next, cells were fixed with a mixture of 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde (GA) for 15 min and then reduced with 0.1% sodium borohydride (NaH4B) for 7 min. Cells were permeabilized with 0.5% Trion-X100 for 5 min and blocked with blocking buffer containing 5% bovine serum albumin (BSA) and 0.5% Triton-X100 for 30 min. BS-C-1 cells were stained with biotin conjugated anti-α-tubulin antibody (ab74696; Abcam) dissolved in blocking buffer for about 45 min. Cells were washed with PBS and stained with streptavidin conjugated small Pdots in blocking buffer for 1 h at room temperature or 4 °C overnight. After that, the labeled cells were extremely washed and stored at 4 °C until use. For nuclear membrane, mitochondrial, and clathrin-coated pit staining, BS-C-1 cells were washed with PBS and fixed with 4% PFA for 15 min at room temperature. After washing with PBS, cells were permeabilized with 0.5% Triton X-100 for 5 min and then blocked with 5% BSA and 0.1% Triton X-100 for 30 min. Next, cells were stained with the primary antibody, such as mouse anti-Tom20 antibody (612278; BD), mouse anti-Lamin A/C antibody (sc-7292, Santa Cruz Biotechnology), or mouse anticlathrin antibody (ab21679, Abcam) for 1 h. After washing with PBS, BS-C-1 cells were stained with the customized biotinylated secondary antibody for 1 h. After washing with PBS, cells were incubated with about 10 nM small Pdot−streptavidin conjugates in blocking buffer for 2 h. The stained samples were extremely washed and stored at 4 °C before until use. For dual color imaging, the antibody incubation step was repeated as described above. Because the concentration of streptavidin-conjugated Pdots used excessive compared with the biotinylated antibody, crosstalk between different structures was not observed. Samples were stored at 4 °C before imaging. Data Processing. For brightness comparison, the brightness was defined as brightness = brightness of images − background of images to reducing the influence of background. For blinking statics, the drift of the raw data was first corrected by a subpixel drift correction algorithm written in home-written Matlab 2013a code (Mathworks Inc., USA),44 then normalized, and the off state interval calculated using STaSI methods.45 For SOFI images, the raw data was also first corrected using the same method, and then second-order crosscumulant SOFI analysis with shortest lag time and Richardson−Lucy algorithm (3 iterations) was implemented using Localizer software package in Igor Pro (WaveMetrics).46 Characterization of Functionalized Small Pdots. UV−vis absorption spectra of small PFO and PFTBT5 Pdots were measured with an ultraviolet−visible spectrophotometer (Schimadzu UV-2550) using a standard 1 cm quartz cuvettes. Fluorescence spectra were measured using a Hitachi F-4600 fluorescence spectrophotometer. The size distribution of small Pdots in bulk solution was characterized by a malvern NANO ZS dynamic light scattering (DLS) instrument. The morphology of small Pdots was investigated by a Hitachi H-600 transmission electron microscope (TEM) with 100 kV acceleration voltage. For atomic force microscopy (AFM) experiments, a freshly cleaved mica substrate was immersed for 2 min with 0.05 mg/mL poly(L-lysine) (molecular weight ≥ 300 000, Sigma, P1524). A drop of Pdots dispersion was deposited onto mica for 2 min. The coverslip was gently washed with deionized water and dried under a stream of

purchased from American Dye Source, Inc.. The carmine emission polymer poly(9,9-dioctylfluorene)-co-(4,7-di-2-thienyl-2,1,3-benzothiadiazole) (PFTBT5) was synthesized as described in a previous report.43 Amphiphilic functional polymer poly(styrene-co-maleic anhydride) (PSMA, terminated by cumene, content of 68% styrene, average molecular weight about 1700) and solvent tetrahydrofuran (THF, anhydrous, 99.9%) were purchased from Sigma-Aldrich. Functionalized small Pdots were prepared using a modified nanoprecipitation method. In a typical preparation, a THF solution (1 mL) containing fluorescent polymer (10 μg) and PSMA (10 μg) was quickly injected into 10 mL of deionized water under vigorous sonication. The THF was then removed by nitrogen bubbling on a 100 °C hot plate. To remove the fraction of aggregates generated in the nanoprecipitation method, the Pdots were then filtered through a 0.22 μm membrane filter. During the formation of Pdots, the maleic anhydride units from the functional polymer PSMA were hydrolyzed generating carboxyl groups on small Pdots in the aqueous environment. Cytotoxicity Assay of Pdots. The cytotoxicity of PFO and PFTBT5 Pdots was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) assays. Hela cells (cervical cancer cell line) were seeded into U-bottom 96-well cell culture plates (Costar, IL, USA) at a density of about 5 × 104 cells/well until adherent. And then the culture medium was replaced with PFO and PFTBT5 Pdots at various mass concentrations (0−100 μg/mL). The Hela cells were incubated at 37 °C under 5% CO2 for 24 h. Subsequently, MTT (20 μL, 5 mg/mL) was added and incubated for an additional 4 h. After the addition of dimethyl sulfoxide (DMSO, 150 μL/well), a microplate reader (BioTek Cytation 3) was used to measure the absorbance value (OD570nm) of each well with background subtraction. The cell viability of Pdot-treated cells is defined as the mean absorbance value of Pdot treatment group divided by the mean absorbance value of untreated group. Biomolecular Conjugation to Functionalized Small Pdots. In this work, we performed bioconjugation utilizing the 1-ethyl-3-(3(dimethylamino)propyl) carbodiimide hydrochloride (EDC, Sigma)catalyzed reaction between carboxyl groups on the small Pdots and amine groups on streptavidin (Sigma). In a typical bioconjugation reaction, 4 mL of a 50 μg/mL small Pdot solution was added into a clean and sterilized 20 mL glass vial, then the following solutions were added sequentially: 80 μL of PEG (5 wt %), mixed well by vortexing; 80 μL of concentrated 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (1 M), mixed well by vortexing; 240 μL of streptavidin (1 mg/mL), mixed well by vortexing. A fresh solution of EDC in Milli-Q water (5 mg/mL) was prepared, and then 80 μL of the EDC solution was added to the small Pdot mixture and the mixture was mixed well by vortexing. The mixture was then magnetically stirred for 4 h at room temperature. The resulting small Pdot−streptavidin bioconjugates were separated from free streptavidin by gel filtration using Sephacryl HR-300 gel media and stored at 4 °C until use. Preparation of Biotinylated Donkey Anti-mouse IgG. We dissolved 100 μL of donkey anti-mouse IgG (H+L) (715-005-151; Jackson ImmunoResearch Laboratories) with 12 μL of a 1 M sodium bicarbonate solution to change the buffer pH to about 8.0. We then added 10 μL of a 1 mM biotin-amidohexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester (biotin-NHS) (B3295, Sigma) to the mixture and vortexed it at room temperature for 30 min. The resulting biotinylated antibodies were separated from free biotin-NHS with illustra NAP-5 columns (GE Healthcare) and stored at 4 °C before use. Optical Setup. Fluorescence imaging was carried out on an inverted Nikon Eclipse Ti microscope (Nikon Instruments) with a TIRF objective (60×, 1.49 NA, Nikon) and additional 1.5× magnification, corresponding to a pixel size of 72 nm. Perfect Focus System (PFS) was used for reducing axial drift. Two lasers were used for excitation: 405 nm (200 mW nominal, Coherent Sapphire); 488 nm (200 mWnominal, MPB). The laser beam was coupled into the microscope objective using a multiband beam splitter (TRF89901-EM, 405, 488, 561, and 640 nm Laser Quad Band Set, Chroma 8089

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ACS Nano nitrogen gas. AFM experiments were conducted with Bioscope Resolve (Bruker, USA). Scanasyst-Air probes (Bruker, USA) with a spring constant of 0.4 N/m and a resonance frequency of 70 kHz were used to image the Pdots. Images were taken in PeakForce QNM mode at a maximal force less than 200 pN and a resolution of 256 × 256 pixels. AFM data was processed with NanoScope Analysis 1.8 software.

(6) Rust, M. J.; Bates, M.; Zhuang, X. Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM). Nat. Methods 2006, 3, 793−796. (7) Chen, X.; Zeng, Z.; Zhang, X.; Xu, P.; Xi, P. Optical Nanoscopy with SOFI. In Super-Resolution Imaging in Biomedicine; Diaspro, A., Zandvoort, M., Eds.; Taylor and Francis: New York, 2016; pp 241− 257. (8) Dertinger, T.; Colyer, R.; Iyer, G.; Weiss, S.; Enderlein, J. Fast, Background-Free, 3D Super-Resolution Optical Fluctuation Imaging (SOFI). Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 22287−22292. (9) Dedecker, P.; Mo, G. C.; Dertinger, T.; Zhang, J. Widely Accessible Method for Superresolution Fluorescence Imaging of Living Systems. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 10909−10914. (10) Yang, Z.; Sharma, A.; Qi, J.; Peng, X.; Lee, D. Y.; Hu, R.; Lin, D.; Qu, J.; Kim, J. S. Super-Resolution Fluorescent Materials: an Insight into Design and Bioimaging Applications. Chem. Soc. Rev. 2016, 45, 4651−4667. (11) Duwe, S.; De Zitter, E.; Gielen, V.; Moeyaert, B.; Vandenberg, W.; Grotjohann, T.; Clays, K.; Jakobs, S.; Van Meervelt, L.; Dedecker, P. Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-Resolution Microscopy. ACS Nano 2015, 9, 9528−9541. (12) Zhang, X.; Chen, X. Z.; Zeng, Z. P.; Zhang, M. S.; Sun, Y. J.; Xi, P.; Peng, J. X.; Xu, P. Y. Development of a Reversibly Switchable Fluorescent Protein for Super-Resolution Optical Fluctuation Imaging (SOFI). ACS Nano 2015, 9, 2659−2667. (13) Dertinger, T.; Heilemann, M.; Vogel, R.; Sauer, M.; Weiss, S. Superresolution Optical Fluctuation Imaging with Organic Dyes. Angew. Chem., Int. Ed. 2010, 49, 9441−9443. (14) Geissbuehler, S.; Sharipov, A.; Godinat, A.; Bocchio, N. L.; Sandoz, P. A.; Huss, A.; Jensen, N. A.; Jakobs, S.; Enderlein, J.; Gisou van der Goot, F.; Dubikovskaya, E. A.; Lasser, T.; Leutenegger, M. Live-Cell Multiplane Three-Dimensional Super-Resolution Optical Fluctuation Imaging. Nat. Commun. 2014, 5, 5830. (15) Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Quantum Dots versus Organic Dyes as Fluorescent Labels. Nat. Methods 2008, 5, 763−775. (16) Zeng, Z. P.; Chen, X. Z.; Wang, H. N.; Huang, N.; Shan, C. Y.; Zhang, H.; Teng, J. L.; Xi, P. Fast Super-Resolution Imaging with Ultra-High Labeling Density Achieved by Joint Tagging SuperResolution Optical Fluctuation Imaging. Sci. Rep. 2015, 5, 8459. (17) Chen, X. Z.; Zeng, Z. P.; Wang, H. N.; Xi, P. ThreeDimensional Multimodal Sub-Diffraction Imaging with Spinning-Disk Confocal Microscopy Using Blinking/Fluctuating Probes. Nano Res. 2015, 8, 2251−2260. (18) Chen, X.; Zong, W.; Li, R.; Zeng, Z.; Zhao, J.; Xi, P.; Chen, L.; Sun, Y. Two-Photon Light-Sheet Nanoscopy by Fluorescence Fluctuation Correlation Analysis. Nanoscale 2016, 8, 9982−9987. (19) Chizhik, A. M.; Stein, S.; Dekaliuk, M. O.; Battle, C.; Li, W.; Huss, A.; Platen, M.; Schaap, I. A.; Gregor, I.; Demchenko, A. P.; Schmidt, C. F.; Enderlein, J.; Chizhik, A. I. Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots. Nano Lett. 2016, 16, 237−242. (20) Pu, K.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J.; Gambhir, S. S.; Bao, Z.; Rao, J. Semiconducting Polymer Nanoparticles as Photoacoustic Molecular Imaging Probes in Living Mice. Nat. Nanotechnol. 2014, 9, 233−239. (21) Wu, C.; Chiu, D. T. Highly Fluorescent Semiconducting Polymer Dots for Biology and Medicine. Angew. Chem., Int. Ed. 2013, 52, 3086−3109. (22) Sun, K.; Tang, Y.; Li, Q.; Yin, S.; Qin, W.; Yu, J.; Chiu, D. T.; Liu, Y.; Yuan, Z.; Zhang, X.; Wu, C. in vivo Dynamic Monitoring of Small Molecules with Implantable Polymer-Dot Transducer. ACS Nano 2016, 10, 6769−6781. (23) Sun, Z.; Liu, S.; Liu, Z.; Qin, W.; Chen, D.; Xu, G.; Wu, C. FRET Acceptor Suppressed Single-Particle Photobleaching in Semiconductor Polymer Dots. Opt. Lett. 2016, 41, 2370−2373.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b02893. The characterizations of PFO and PFTBT5 Pdots (transmission electronic microscope, atomic force microscope), typical fluorescence intensity fluctuation and offtime interval statistics, photostability and cytotoxicity of PFO and PFTBT5 small Pdots, and full width at halfmaximum values comparison of wide field and secondorder SOFI imaging of PFO and PFTBT5 Pdots (PDF)

AUTHOR INFORMATION Corresponding Authors

*[email protected] *[email protected] *[email protected] ORCID

Xuanze Chen: 0000-0002-0304-2391 Peng Xi: 0000-0001-6626-4840 Changfeng Wu: 0000-0001-6797-9784 Author Contributions ξ

X.C., Z.L. and R.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Mr. Jiaxi Zhao (Tsinghua University) for his assistance with blinking statistics. This work is supported by grants from National Natural Science Foundation of China (61475010 for P.X.), the Key Program of National Natural Science Foundation of China (61335001) and the Thousand Young Talents Program for C.W., and the Key Program of National Natural Science Foundation of China (21573013, 21390412, 31271423, 31327901) and “863” Program (SS2015AA020406 for Y.S.). REFERENCES (1) Hell, S. W. Far-Field Optical Nanoscopy. Science 2007, 316, 1153−1158. (2) Hell, S. W.; Wichmann, J. Breaking the Diffraction Resolution Limit by Stimulated-Emission-Stimulated-Emission-Depletion Fluorescence Microscopy. Opt. Lett. 1994, 19, 780−782. (3) Yang, X.; Xie, H.; Alonas, E.; Liu, Y.; Chen, X.; Santangelo, P. J.; Ren, Q.; Xi, P.; Jin, D. Mirror-Enhanced Super-Resolution Microscopy. Light: Sci. Appl. 2016, 5, e16134. (4) Li, D.; Shao, L.; Chen, B. C.; Zhang, X.; Zhang, M.; Moses, B.; Milkie, D. E.; Beach, J. R.; Hammer, J. A., 3rd; Pasham, M.; Kirchhausen, T.; Baird, M. A.; Davidson, M. W.; Xu, P.; Betzig, E. Extended-Resolution Structured Illumination Imaging of Endocytic and Cytoskeletal Dynamics. Science 2015, 349, aab3500. (5) Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 2006, 313, 1642−1645. 8090

DOI: 10.1021/acsnano.7b02893 ACS Nano 2017, 11, 8084−8091

Article

ACS Nano (24) Wu, C.; Szymanski, C.; Cain, Z.; McNeill, J. Conjugated Polymer Dots for Multiphoton Fluorescence Imaging. J. Am. Chem. Soc. 2007, 129, 12904−12905. (25) Massey, M.; Wu, M.; Conroy, E. M.; Algar, W. R. Mind Your P’s and Q’s: The Coming of Age of Semiconducting Polymer Dots and Semiconductor Quantum Dots in Biological Applications. Curr. Opin. Biotechnol. 2015, 34, 30−40. (26) Xie, C.; Zhen, X.; Lei, Q.; Ni, R.; Pu, K. Self-Assembly of Semiconducting Polymer Amphiphiles for in vivo Photoacoustic Imaging. Adv. Funct. Mater. 2017, 27, 1605397. (27) Lyu, Y.; Pu, K. Recent Advances of Activatable Molecular Probes Based on Semiconducting Polymer Nanoparticles in Sensing and Imaging. Adv. Sci. 2017, 4, 1600481. (28) Chan, Y. H.; Wu, P. J. Semiconducting Polymer Nanoparticles as Fluorescent Probes for Biological Imaging and Sensing. Part. Part. Syst. Charact. 2015, 32, 11−28. (29) Chen, X.; Li, R.; Liu, Z.; Sun, K.; Sun, Z.; Chen, D.; Xu, G.; Xi, P.; Wu, C.; Sun, Y. Small Photoblinking Semiconductor Polymer Dots for Fluorescence Nanoscopy. Adv. Mater. 2017, 29, 1604850. (30) Bates, M.; Huang, B.; Dempsey, G. T.; Zhuang, X. Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes. Science 2007, 317, 1749−1753. (31) Ke, C.-S.; Fang, C.-C.; Yan, J.-Y.; Tseng, P.-J.; Pyle, J. R.; Chen, C.-P.; Lin, S.-Y.; Chen, J.; Zhang, X.; Chan, Y.-H. Molecular Engineering and Design of Semiconducting Polymer Dots with Narrow-Band, Near-Infrared Emission for in vivo Biological Imaging. ACS Nano 2017, 11, 3166−3177. (32) Liu, Z.; Sun, Z.; Di, W.; Qin, W.; Yuan, Z.; Wu, C. Brightness Calibrates Particle Size in Single Particle Fluorescence Imaging. Opt. Lett. 2015, 40, 1242−1245. (33) Wu, C.; Bull, B.; Szymanski, C.; Christensen, K.; McNeill, J. Multicolor Conjugated Polymer Dots for Biological Fluorescence Imaging. ACS Nano 2008, 2, 2415−2423. (34) Montagnac, G.; Meas-Yedid, V.; Irondelle, M.; Castro-Castro, A.; Franco, M.; Shida, T.; Nachury, M. V.; Benmerah, A.; Olivo-Marin, J. C.; Chavrier, P. αTAT1 Catalyses Microtubule Acetylation at Clathrin-Coated Pits. Nature 2013, 502, 567−570. (35) Palazzo, A.; Ackerman, B.; Gundersen, G. G. Cell Biology: Tubulin Acetylation and Cell Motility. Nature 2003, 421, 230. (36) Boldogh, I. R.; Pon, L. A. Mitochondria on the Move. Trends Cell Biol. 2007, 17, 502−510. (37) Chen, X.; Wei, M.; Zheng, M. M.; Zhao, J.; Hao, H.; Chang, L.; Xi, P.; Sun, Y. Study of RNA Polymerase II Clustering inside Live-Cell Nuclei Using Bayesian Nanoscopy. ACS Nano 2016, 10, 2447−2454. (38) Lin, C.; Jungmann, R.; Leifer, A. M.; Li, C.; Levner, D.; Church, G. M.; Shih, W. M.; Yin, P. Submicrometre Geometrically Encoded Fluorescent Barcodes Self-Assembled from DNA. Nat. Chem. 2012, 4, 832−839. (39) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Quantum-Dot-Tagged Microbeads for Multiplexed Optical Coding of Biomolecules. Nat. Biotechnol. 2001, 19, 631−635. (40) Lyu, Y.; Fang, Y.; Miao, Q.; Zhen, X.; Ding, D.; Pu, K. Intraparticle Molecular Orbital Engineering of Semiconducting Polymer Nanoparticles as Amplified Theranostics for in vivo Photoacoustic Imaging and Photothermal Therapy. ACS Nano 2016, 10, 4472−4481. (41) Miao, Q.; Lyu, Y.; Ding, D.; Pu, K. Semiconducting Oligomer Nanoparticles as an Activatable Photoacoustic Probe with Amplified Brightness for in vivo Imaging of pH. Adv. Mater. 2016, 28, 3662− 3668. (42) Wu, I.-C.; Yu, J.; Ye, F.; Rong, Y.; Gallina, M. E.; Fujimoto, B. S.; Zhang, Y.; Chan, Y.-H.; Sun, W.; Zhou, X.-H.; et al. Squaraine-Based Polymer Dots with Narrow, Bright Near-Infrared Fluorescence for Biological Applications. J. Am. Chem. Soc. 2015, 137, 173−178. (43) Hou, Q.; Xu, Y.; Yang, W.; Yuan, M.; Peng, J.; Cao, Y. Novel Red-Emitting Fluorene-Based Copolymers. J. Mater. Chem. 2002, 12, 2887−2892. (44) Guizar-Sicairos, M.; Thurman, S. T.; Fienup, J. R. Efficient Subpixel Image Registration Algorithms. Opt. Lett. 2008, 33, 156−158.

(45) Shuang, B.; Cooper, D.; Taylor, J. N.; Kisley, L.; Chen, J.; Wang, W.; Li, C. B.; Komatsuzaki, T.; Landes, C. F. Fast Step Transition and State Identification (STaSI) for Discrete Single-Molecule Data Analysis. J. Phys. Chem. Lett. 2014, 5, 3157−3161. (46) Dedecker, P.; Duwe, S.; Neely, R. K.; Zhang, J. Localizer: Fast, Accurate, Open-Source, and Modular Software Package for Superresolution Microscopy. J. Biomed. Opt. 2012, 17, 126008.

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DOI: 10.1021/acsnano.7b02893 ACS Nano 2017, 11, 8084−8091