Letter Cite This: ACS Macro Lett. 2018, 7, 1432−1437
pubs.acs.org/macroletters
A Polymerizable Photoswitchable Fluorophore for Super-Resolution Imaging of Polymer Self-Assembly and Dynamics Zhe Qiang,†,§ Kevin M. Shebek,†,§ Masahiro Irie,‡ and Muzhou Wang*,† †
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States Department of Chemistry and Research Center for Smart Molecules, Rikkyo University, Nishi-Ikebukuro 3-34-1, Toshimaku, Tokyo 171-8501, Japan
‡
ACS Macro Lett. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 11/20/18. For personal use only.
S Supporting Information *
ABSTRACT: Single-molecule super-resolution microscopy has become a standard imaging tool in the life sciences for visualizing nanostructures in situ, but the application of this technique in polymer science is much less explored. A key bottleneck is the lack of fluorophores and simple covalent attachment strategies onto polymer chains. Here, we report a functional diarylethene-based photoswitchable fluorophore that can be directly incorporated into polymer backbones through copolymerization, which significantly streamlines the labeling strategy, with no further postcoupling reactions or purifications needed. The attachment of fluorophores onto selectively labeled polymers enables super-resolution imaging of a series of model polymer blend systems with different nanostructures and chemical compositions. As each individual fluorophore is able to switch several times on average between its bright and dark state, multiple time-lapse images can be acquired to observe the dynamic nanostructural evolution of polymer blends upon solvent vapor annealing. With this demonstration of a universal, simplified labeling strategy and the ability to image polymer assembly under native conditions, this reported fluorophore may promote the widespread use of super-resolution microscopy in the polymer community.
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covalently incorporated into polymers can promote superresolution microscopy in this community, particularly for investigating nanoscale polymer dynamics in real space and real time, which is difficult through other characterization methods. Here, we develop a photoswitchable fluorophore for superresolution microscopy that can be directly copolymerized into polymer chains, which greatly streamlines labeling strategies into a single step. We demonstrate this scheme by polymerizing with radical monomers such as styrene and methyl methacrylate, which are labeled with our fluorophore with no further postfunctionalization reactions or purifications needed. The molecule is based on recently explored disulfone derivatives of diarylethenes that are highly photostable and compatible with organic environments.30 These fluorophores can undergo photoreactions based on cyclization of a central six-membered ring (Figure 1a). This leads to open- and closedring isomers, both of which are indefinitely stable at room temperature, but only the closed-ring form absorbs at visible wavelengths and emits with excellent fluorescence quantum yield (Figure 1b).19,31−35 Unlike many aqueous fluorophore systems that require special buffers to promote transient dark
anostructural characterization is of critical importance for understanding the complex relationship between chemical composition, structure, property, and function for advanced materials design.1,2 Many modern imaging methods such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM), provide subnanometer resolutions,3−5 but each technique has limitations, including high vacuum requirements, poor electron contrast, or difficulty in probing subsurface features.6,7 In the past decade, single-molecule super-resolution microscopy has become a standard tool for visualizing nanostructures in the life sciences under native conditions.8−15 In this technique, a small fraction of spatially separated fluorophores are activated from a nonemissive to an emissive state. Images of these bright molecules are then fit to their point-spread functions, which determines their positions with tens of nanometer precision, after which the molecules are then deactivated to their nonemissive state. By repeating this process, a super-resolution image is reconstructed from the positions of all identified emitters over many thousands of frames. While this method has been routinely used in the biological community,11,16−18 super-resolution imaging in polymer science is much less reported.19−25 A key barrier to widespread adoption is the lack of suitable fluorophore chemistry in organic systems that are common in polymers.26−29 A photoswitchable fluorophore that can be easily © XXXX American Chemical Society
Received: September 7, 2018 Accepted: October 22, 2018
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DOI: 10.1021/acsmacrolett.8b00686 ACS Macro Lett. 2018, 7, 1432−1437
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Figure 1. (a) Photographs of the open- (colorless) and closed-ring forms (yellow-green fluorescent color) of the compound (V-DAE) in THF with their corresponding structures that convert upon irradiation with 365 nm and 473 nm wavelengths. (b) absorption spectra of V-DAE (open-ring form, black dashed line; closed-ring form, block solid line) and emission spectrum of V-DAE (closed-ring form, green solid line) in THF under irradiation with 475 nm light; The open-ring form of V-DAE has two absorption peaks at 322 nm (ε = 2.4×104 M−1cm−1) and 338 nm (ε = 2.2×104 M−1cm−1) and the closed-ring form has a broad absorption peak in the visible wavelength range with a maximum at 475 nm (ε = 6.1×104 M−1cm−1). The peak of emission spectrum of closed ring form V-DAE is at 548 nm. (c) Synthesis of V-DAE.
states,27 switching between the two diarylethene states is controlled by relative intensities of ultraviolet and visible illumination, enabling super-resolution imaging through a photoactivated localization microscopy (PALM) scheme. We apply this technique to image the nanostructures of fluorophore-labeled polymer blends and validate these images through correlation with AFM of the same region. As each individual fluorophore can be switched several times, multiple time-lapse images can be acquired for observing the dynamic nanostructural evolution, which we demonstrate using polymer blends swollen with solvent vapor. A 6,6′-diiodo derivative of 1,2-bis(2-ethyl-1,1,-dioxidobenzothiophene-3-yl)perfluorocyclopentene was first synthesized following as previously reported31 and then reacted with a mixture of 4-(2-methoxycarbonylethyl)benzeneboronic acid and 4-vinylphenylboronic acid by a Suzuki-Miyaura coupling reaction. Reaction with two different boronic acids leads to three different derivatives containing 0, 1, and 2 vinyl groups through a single batch reaction, which are easily separated by chromatography. Only the derivative with one vinyl functional group (vinyl-terminated diarylethene, V-DAE, Figure 1c) will be used and discussed for the rest of the study. Upon irradiation with 375 nm light, the initially colorless fluorophore isomerizes into a bright yellow state, which can revert back to its colorless state when exposed to 473 nm light (Figure 1a). The bright state is highly fluorescent with a quantum yield of 0.82 (in THF) and an emission band from 500 to 680 nm (Figure 1b). The vinyl group in V-DAE enables copolymerization of this fluorophore with standard monomers through conventional controlled free-radical polymerization, significantly simplifying fluorophore-labeling strategies. We demonstrate this by synthesizing functionalized polystyrene (PS) via reversible
addition−fragmentation polymerization (RAFT) with a target molecular weight of 200 kg/mol.36,37 Figure 2a shows the GPC traces of as-synthesized V-DAE labeled PS with peaks shifting toward higher molecular weights with increasing reaction time (see Supporting Information). The symmetric, monomodal peaks with narrow distributions (PDI < 1.2) indicate a wellcontrolled polymerization in the presence of V-DAE. While the
Figure 2. (a) GPC traces of as-synthesized V-DAE labeled polystyrene sampled at different times from a polymerization reaction and (b) the reaction conversion (determined by Mn obtained from GPC measurements) and the relative fluorophore concentration (wt %) in the PS as a function of reaction time. Reaction conditions: [M]0/[chain transfer agent (CTA)]0 = 2000/1 (molar) at 120 °C. 1433
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resolution imaging technique can completely describe the polymer nanostructures. In PALM, the ultimate resolution is determined by both the uncertainty of each single-molecule localization and the number of localization events acquired for each image.13,38,39 In order to acquire multiple time-lapse images, it is important to understand the total number of localization events that can be collected from a given concentration of V-DAE molecules before permanent photobleaching. These localization events can then be optimally distributed over many separate image acquisitions throughout a dynamic process. The total number of localization events N in a given area is given by,
conversion of styrene monomer to PS gradually increases to 38% after polymerization at 120 °C for 8 h, the relative fluorophore concentration in the polymers remains constant at approximately 0.018 wt % regardless of polymerization time. This suggests that the incorporation of V-DAE into functionalized polymers is simultaneous with PS chain growth during polymerization, leading to a random distribution of fluorophores along the polymer backbones. Furthermore, the concentration of fluorophores in the as-synthesized functional polymers can be precisely tuned by adjusting the molar ratio of monomer to fluorophore, which is usually more difficult in postfunctionalization labeling strategies. We also demonstrated that V-DAE can be copolymerized with other monomers such as methyl methacrylate (MMA) through the same strategy (Supporting Information). These results indicate the generalizability of this fluorophore (V-DAE) to label different polymers through free-radical polymerization synthesis. To demonstrate this fluorophore in super-resolution imaging of self-assembled features, we use a model system of PS (Mn = 40500 g/mol, PDI: 1.3) /PMMA (Mn = 120000 g/mol) homopolymer blends where PS is selectively labeled at approximately 0.48 wt % fluorophore concentration. To provide the best quality images, this fluorophore content was selected to be higher than the functionalized PS reported in Figure 2b, by increasing the ratio of V-DAE to styrene monomer during synthesis. Figure 3a shows the superresolution image of PS/PMMA (1:3 by mass) blends, in which white spherical domains are fluorophore-labeled PS and the dark matrix is nonfluorescent PMMA. Inverting the weight ratio of PS/PMMA to 3:1 leads to a structure of dark voids (PMMA) embedded in a bright continuous PS phase (Figure 3b). A more direct confirmation of the nanostructure is achieved by comparing with AFM of PS/PMMA blends at a mass ratio of 1:2 (Figure 3c), where the AFM phase image is superimposable with the super-resolution image with near perfect matching. This result indicates that our super-
N = fmx + b where x is the number of fluorophore molecules added in the area, f is the fraction of fluorophores whose emissions can be detected, m is the average number of cycles of each dye has between the open- and closed-ring forms before photobleaching, and b is the number of localization events from fluorescent contaminates in the cast film. We have counted the number of total localization events at four different concentrations of V-DAE labeled PS embedded in nonlabeled PS films over a large area of 16.5 μm × 16.5 μm (Figure 4). A linear relationship between N and x is observed with slope of f m = 7.35. This f m describes the relationship between the total number of localization events observed for a given amount of added molecules. However, each factor within the product (f m) can independently impact the performance of the fluorophore in time-lapse imaging experiments. Through careful counting experiments (Supporting Information), we observed that f = 0.34 in a thin PS film, indicating that some fluorophores added will be “wasted” due to either being stuck in the dark state, having undergone photobleaching prior to the experiment, or having insufficient brightness to overcome background in the experimental configuration. The dark state corresponds to the open-ring form of the diarylethene core, whose pendant thiophenes can adopt multiple conformations of which only one is readily isomerized into the bright state.40 Interconversion between these conformations happens readily in solution, but is kinetically hindered in a glassy polymer matrix. We observed an increase in the f value to 0.6 though a preactivation strategy, where a majority of the open-ring form dyes are reacted to the closed-ring form in solution prior
Figure 3. Super-resolution images of thin films of PS/PMMA blends with mass ratios of (a) 1:3 and (b) 3:1. PS was selectively labeled with V-DAE at 0.48 wt % and, thus, appears bright. (c) Images of a 1:2 PS/ PMMA blend. Left is an AFM phase image, right is a super-resolution image and they are partially overlaid in the middle. Film thickness for imaging is 28 nm and scale bars are 1 μm.
Figure 4. Number of localization events detected as a function of number of added dye molecules in 20 nm thick PS film, upon simultaneous UV (375 nm) and visible light (473 nm) excitation. A linear relationship (red dashed line) was used to fit all the data points and the slope (f m, see text) equals 7.35. 1434
DOI: 10.1021/acsmacrolett.8b00686 ACS Macro Lett. 2018, 7, 1432−1437
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Figure 5. Time lapse super-resolution images of PS/PMMA blends thin film (1/2; w/w) under (a) anisole vapor annealing (p/p0 = 0.95) for 0, 60, 120, and 360 min, and (b) toluene vapor annealing (p/p0 = 1) for 0, 90, 180, and 450 min. The images were acquired at a lower relative vapor pressure of p/p0 = 0.5 over 2 min, to minimize rearrangement during imaging. All scale bars are 4 μm. The corresponding zoomed-in regions below in (a) are magnified 2-fold from the red areas in the above images, with highlighted features in blue visualizing specific features that evolve or remain fixed during annealing. The film thickness is approximately 28 nm.
to casting a film (see Supporting Information for a detailed discussion).41 However, we found it optimal to proceed with 5 ms acquisition times without preactivation for reasons of overall imaging speed and the ability to rapidly attain conditions where overlap of single-molecule images was minimized for the super-resolution reconstruction. To visualize dynamic evolution of polymer nanostructures at different length scales using this technique, films of PS/PMMA (1:2 by mass) blends were annealed under two different conditions. Annealing a film with unsaturated anisole vapor (p/p0 = 0.95, where p0 is the anisole vapor pressure at room temperature) limits the large-size coarsening behavior of polymer blends with the average feature sizes consistently under optical diffraction limit (∼200 nm) even after hours of annealing time. Figure 5a shows the morphological evolution from an as-cast film composed of locally ordered cylindrical and spherical patterns, where both local coalescence and fission of the bright PS domains are easily visible. We also performed a more aggressive annealing condition under saturated toluene vapor (p/p0 = 1), showing domain sizes increasing significantly from ∼100 nm to ∼4 μm (Figure 5b). After annealing for 450 min, there are still many bright spots (representing PS) that are locally trapped in the PMMA matrix, possibly visualizing the transit of PS chains as they migrate between the larger domains during assembly. The current acquisition time of 50 s is sufficiently fast to observe the dynamic phenomena relevant in this process, though further improvements are easily possible by increasing the frame rate and illumination intensity, or improving the fluorophore kinetics.42 These results confirm that this photoswitchable fluorophore is particularly promising for observing polymer self-assembly in situ by super-resolution imaging.
The value of f m = 7.35 (Figure 4) predicts a total of 28 images that can be obtained before permanent photobleaching of all fluorophores. This calculation is based on the required areal density per image of approximately 7.4 × 10−3 nm−2 to sufficiently resolve the nanoscale features (see Supporting Information for details),13 compared to the overall localizations available from the 0.48 wt % of added dyes, which in this case corresponds to an areal density of 2.8 × 10−2 nm−2. Higher fluorophore concentrations will result in more images. In our experiments, only eight images were acquired before image quality deteriorated (see Supporting Information for discussion). This could be due to heterogeneity in blinking kinetics, where the number of on−off cycles for a given molecule has a broad distribution, with some blinking many times and others blinking significantly fewer over the course of a dynamic experiment. Although a quantitative effect of this issue on image quality is beyond the scope of this paper and will be explored in future work, we expect that more images can generally be obtained using higher fluorophore concentrations or under conditions with a higher f m. Nevertheless, this investigation of in situ nanostructural evolution provides a great opportunity for understanding complex polymer assembly dynamics in the native environment. In conclusion, we reported a functional photoswitchable diarylethene-based fluorophore for single-molecule superresolution imaging of polymeric materials. This fluorophore can be directly incorporated into polymer chains through a copolymerization strategy with no postfunctionalization or purification needed, providing a simple way to label and image polymer systems. We have imaged various nanostructures in a model self-assembled polymer blends system. Furthermore, as each individual fluorophore can be switched between the dark 1435
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and bright states several times, we successfully visualized dynamic morphological evolution upon external stimuli via multiple time-lapse super-resolution images. With the advantages of a simple, universal labeling methodology and the ability to visualize polymer dynamic assembly in situ, we hope this fluorophore will provide a platform for promoting the widespread of this super-resolution microscopy technique to the polymer community.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.8b00686. Experimental details (dye synthesis, optical apparatus, single-molecule data acquisition, and analysis), photos of custom sample chamber, NMR and mass spectra of VDAE, GPC traces of fluorophore-labeled PMMA and fluorophore-labeled PS (RAFT polymerization for 4 h) before and after Soxhlet extraction for removing unreacted V-DAE, discussion about how to determine f and m individually, and the effect of preactivating fluorophore solution for increasing f (PDF).
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Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Masahiro Irie: 0000-0002-5644-3818 Muzhou Wang: 0000-0002-7054-3022 Author Contributions §
Z.Q. and K.M.S. contributed equally to the manuscript and this manuscript was written through contributions of all authors. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Profs. Julia Kalow and John Torkelson for access to their equipment and useful discussions. We thank Prof. Masakazu Morimoto for his assistance in dye synthesis. We acknowledge the Donors of the American Chemical Society Petroleum Research Fund for partial support of this research. Software for localization analysis was developed with the help of Charles Laughlin and support from Northwestern’s Summer Internship Grant Program. This work made use of the SPID facility of Northwestern University’s NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); the MRSEC program (NSF DMR1720139) at the Materials Research Center; the International Institute for Nanotechnology (IIN); the Keck Foundation; and the State of Illinois, through the IIN. This work made use of NMR and MS instrumentation at the Integrated Molecular Structure Education and Research Center (IMSERC) at Northwestern University, which has received support from the NSF (NSF-CHE 9871268). This work was performed in collaboration with award 70NANB14H012 from U.S. Department of Commerce, National Institute of Standards and Technology as part of the Center for Hierarchical Materials Design (CHiMaD). 1436
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