Article pubs.acs.org/JACS
Super-Photostable Phosphole-Based Dye for Multiple-Acquisition Stimulated Emission Depletion Imaging Chenguang Wang,† Masayasu Taki,*,† Yoshikatsu Sato,*,† Aiko Fukazawa,*,‡ Tetsuya Higashiyama,†,§ and Shigehiro Yamaguchi*,†,‡ †
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo, Chikusa, Nagoya 464-8501, Japan Department of Chemistry, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan § Division of Biological Science, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan ‡
S Supporting Information *
ABSTRACT: As stimulated emission depletion (STED) microscopy can provide structural details of cells with an optical resolution beyond the diffraction limit, it has become an indispensable tool in cell biology. However, the intense STED laser beam usually causes rapid photobleaching of the employed fluorescent dyes, which significantly limits the utility of STED microscopy from a practical perspective. Herein we report a new design of super-photostable dye, PhoxBright 430 (PB430), comprising a fully ring-fused π-conjugated skeleton with an electron-accepting phosphole P-oxide unit. We previously developed a super-photostable dye C-Naphox by combining the phosphole unit with an electron-donating triphenylamine moiety. In PB430, removal of the amino group alters the transition type from intramolecular charge transfer character to π−π* transition character, which gives rise to intense fluorescence insensitive to molecular environment in terms of fluorescence colors and intensity, and bright fluorescence even in aqueous media. PB430 also furnishes high solubility in water, and is capable of labeling proteins with maintaining high fluorescence quantum yields. This dye exhibits outstanding resistance to photoirradiation even under the STED conditions and allows continuous acquisition of STED images. Indeed, using a PB430-conjugated antibody, we succeed in attaining a 3-D reconstruction of super-resolution STED images as well as photostability-based multicolor STED imaging of fluorescently labeled cytoskeletal structures.
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INTRODUCTION Fluorescence microscopy is one of the most powerful and versatile techniques to investigate biological environments. In particular, super-resolution nanoscopy has emerged as a promising technique to gain a detailed understanding of the complex dynamics of biomolecules, as it allows distinguishing different objects beyond the diffraction limit.1−8 Compared to several other types of super-resolution imaging techniques, stimulated emission depletion (STED) microscopy offers a set of advantages, such as fast acquisition speed, wide tolerance of sample thickness and medium, as well as fewer restrictions on dye properties.9−11 Therefore, STED microscopy has been increasingly used for the visualization of biological samples, and also for the examination of various physical and chemical processes.12−17 However, the necessity to irradiate samples with a donutshaped depletion laser beam (STED laser) with extremely high power (>10 MW/cm2)18,19 usually leads to rapid photobleaching of the organic dyes,20−22 which severely limits STED microscopy from a practical perspective. Low bleaching resistance imposes severe limitations especially on time-lapse and three-dimensional (3-D) STED imaging, because these © 2017 American Chemical Society
imaging techniques require prolonged or repeated exposure of the dyes to the STED beam.23−26 The major bleaching pathway has been considered to involve the generation of a long-lived excited triplet-state of the dyes.19,22,27 To avoid accumulation of these long-lived triplet states, several methods, such as RESCue (Reduction of Excitation and Signal suppression Cycles)STED,28 T-Rex (Triplet Relaxation)-STED,18,29 and more recently MOST (Multiple Off State Transitions) microscopy,30 have been developed. The addition of redox reagents31−34 or triplet quenchers35,36 may also represent effective strategies to reduce the photobleaching rate. Along with the development of new apparatus and methodologies to mitigate the limitations of the dyes, the development of inherently more photostable fluorophores is also indispensable in order to establish STED nanoscopy as an essential, practical, and widely applicable bioimaging tool. In this context, several design strategies have been examined in order to create fluorophores with high photostability. For example, self-healing fluorophores, which were originally Received: April 30, 2017 Published: July 25, 2017 10374
DOI: 10.1021/jacs.7b04418 J. Am. Chem. Soc. 2017, 139, 10374−10381
Article
Journal of the American Chemical Society pioneered by the Lüttke and co-workers37 and recently rediscovered by Blanchard and co-workers for live-cell and single-molecule imaging, exhibited the enhancement of the brightness and photostability as a result of intramolecular quenching of the triplet and/or radical species by a covalently linked triplet-quenching molecule.38 Lavis and co-workers reported that the replacement of dialkylamino groups in rhodamine and coumarin dyes with four-membered azetidinyl groups effectively increases the quantum yield and photostability by preventing the formation of reactive diradical intermediates.39 The most commonly encountered strategy is the introduction of electron-withdrawing substituents, such as fluorine atoms40−42 or sulfonate groups,43,44 on the fluorophore skeleton. In contrast, our strategy is based on a fully ring-fused π-conjugated skeleton that contains an electron-withdrawing phosphine oxide (PO). We previously developed the naphthophosphole P-oxide-based fluorescent dye C-Naphox (Figure 1),45 which has a donor-π-acceptor (D-π-A) type
moreover contains a functional group that allows bioconjugation to antibodies for immunolabeling. The lack of ICT character in the excited state resulted in a high fluorescence quantum yield (ΦF = 0.67) for a dye-conjugated antibody, even in aqueous media. Most importantly, the outstanding photostability of PB430 enabled multiple consecutive acquisitions of STED images of microtubules in fixed cells, whereby the presence of antifade agents was not required.46,47 Using PB430, we have achieved z-stack imaging for 3-D reconstruction in STED microscopy and, moreover, multicolor STED imaging. The distinct difference in the photostability between PB430 and other conventional dyes allowed us to distinguish the fluorescence signals of PB430 from those of conventional fluorescent probes, such as Alexa Fluor 430, with similar excitation and emission properties using a single set of excitation and depletion lasers.
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RESULTS Molecular Design of the Super-photostable Fluorescent Probe PhoxBright 430. The key molecular design feature for the exceptional photostability for PB430 (Figure 1) is the reinforcement of the molecular skeleton with the PO and methylene bridges. In order to enable bioconjugation and mitigate the dependence of the photophysical properties on the molecular environment, the electron-donating diphenylamino group in C-Naphox is replaced with a 3-carboxyphenyl group in PB430. This structural modification, however, would induce a significant blue-shift of the longest absorption band. Therefore, the naphthalene-2,3-diyl-fused structure employed in CNaphox is replaced with a naphthalene-1,2-diyl-fused structure in PB430 to compensate for the unfavorable hypsochromic shift (Figure S1). In order to enhance the hydrophilicity and to suppress aggregation in aqueous media, sulfonate groups (SO3H) are introduced. The weakly nucleophilic sulfonates do not impede the selective conversion of the carboxylic acid into an N-hydroxysuccinimidyl (NHS) ester, which is crucial for conjugation to biomolecules.40 To assess the effect of the removal of the electron-donating diphenylamino group and the positional effect of the fused naphthalene moiety on the photophysical properties, we initially synthesized two naphthophosphole derivatives, the naphthalene-2,3-diyl and the naphthalene-1,2-diyl-fused phosphole derivatives 1 and 2 (Figure 1), respectively. Unlike the Dπ-A type C-Naphox, the absorption and fluorescence spectra of both compounds 1 and 2 were insensitive to solvent polarity indicative of its π−π* transition character of the lowest-energy transition (Figure 2, Figures S2 and S3, and Table S1). Moreover, the high fluorescence quantum yields (ΦF = ∼ 0.85) of 1 and 2 were maintained in various organic solvents and even in aqueous medium such as CH3OH/H2O (v/v = 7/3) (Figures S4 and S5), whereas that of C-Naphox substantially decreased (ΦF = 0.43) in CH3OH/H2O (v/v = 7/3) (Figure S6). These results clearly demonstrate that the switching of the excited state character from ICT to π−π* is crucial for attaining high ΦF in an aqueous medium. The replacement of the electron-donating diphenylamino group in C-Naphox with the phenyl group in 1 resulted in a substantial blue-shift of the absorption band to the ultraviolet region (λabs = 371 nm in CH3CN) as expected, which indicates a relatively low excitation cross section in the visible region. In contrast, change in the fused mode of naphthalene from 1 to 2 gave rise to a substantial red shift of the absorption band. The absorption maximum (λabs) of 2 was shifted to 418 nm with the
Figure 1. Molecular design of PB430 based on the rational structural modification of the first-generation super-photostable dye C-Naphox.
skeleton consisting of an electron-accepting phosphole P-oxide moiety and an electron-donating triphenylamine moiety. This dye showed exceptionally high photoresistance that should most likely be ascribed to the structural reinforcement, i.e., the planar alignment resulting from the PO and methylene bridges. Although the high photostability of C-Naphox enabled us to acquire repeated STED images, it still exhibits several drawbacks that hamper its practical use in cell imaging. For instance, its relatively high hydrophobicity causes nonspecific binding to hydrophobic organelles, such as the endoplasmic reticulum. In addition, the fluorescence quantum yield (ΦF) of C-Naphox is significantly decreased in aqueous media on account of its intramolecular charge transfer (ICT) character in the excited state due to the D-π-A skeleton. The absence of a bioconjugation site on C-Naphox represents another obstacle that needs to be circumvented in order to generate a dye that may be used in a multitude of biological applications. Herein, we report the design of the second-generation superphotostable fluorescent dye PhoxBright 430 (hereafter denoted as PB430), which does not rely on the D-π-A skeleton. PB430 thus produced overcomes all the aforementioned drawbacks for C-Naphox, and thereby significantly expands the practical utility of such fluorescent dyes in STED nanoscopy. The solubility of PB430 in water is sufficiently increased and PB430 10375
DOI: 10.1021/jacs.7b04418 J. Am. Chem. Soc. 2017, 139, 10374−10381
Article
Journal of the American Chemical Society
exhibited a large Stokes shift (5020 cm−1), which is comparable to those of STAR 440SX and STAR 470SXP.48 Subsequently, we evaluated the photostability of PB430 relative to C-Naphox and the representative photostable dye Alexa Fluor 488 in a mixed solvent (DMSO/HEPES buffer, v/v = 7/3, pH = 7.3) (Figures 3b and S8). Photoirradiation of PB430 and C-Naphox in a cuvette using a Xe lamp equipped with a 460 nm band-pass filter (half peak width, 11 nm; illumination intensity, 9 mW cm−2) for 5 h did not induce any detectable photobleaching (99.9% of the dyes remained intact). In contrast, only 46.2% of Alexa Fluor 488 persisted under the same irradiation conditions, demonstrating that the exceptional photostability of C-Naphox is retained in the PB430 skeleton. Application of PhoxBright 430-Conjugated Secondary Antibody for STED Nanoscopy. In order to conduct STED imaging, PB430 was transformed into the corresponding NHS ester, followed by conjugation with goat antimouse IgG antibodies. A degree of labeling (DOL) of 2.8 was determined for the sample prepared from 0.50 mg of the IgG-antibodies and 20 μg of the NHS ester in 0.25 mL of a labeling buffer (pH = 8.3). The photophysical properties of the PB430-conjugated antibody were almost identical to those of antibody-free PB430 in PBS (pH 7.4), indicative of the absence of interaction between the dyes or with the amino acid residues of the antibody (Figure S9 and Table S2). STED imaging experiments were performed using a Leica TCS SP8 STED microscope, equipped with various Ar lasers and a pulsed white-light laser (WLL, 78 MHz, 470−670 nm) for excitation, as well as a continuous wave depletion (CWSTED) laser (592 nm). In order to obtain high-resolution images in gated CW-STED mode, an excitation wavelength of 470 nm was chosen for PB430 from the pulsed WLL.49,50 To confirm the practical utility of PB430 as a fluorescent labeling reagent for proteins, we stained α-tubulin in fixed HeLa cells by indirect immunofluorescence with a PB430-labeled secondary antibody. A confocal image demonstrated that microtubules were successfully stained with negligible nonspecific binding, as evident from the low background signals (Figure S10). Under STED conditions (λSTED = 592 nm, STED laser power = ∼30 mW, gated detection = 0.5 ns), individual microtubules were well separated from each other (Figure 4a). The full width at half-maximum (fwhm) resolution of STED image was 76 ± 7 nm (Figure S11c). Repeated STED Imaging for 3-D Reconstruction. It was furthermore possible to repeat STED imaging of the tubulin labeled with the PB430-conjugated secondary antibody under retention of the high fluorescence brightness. The total fluorescence signal intensity of the stained cells was monitored during repetitive scanning. After recording five images, PB430 retained more than 80% of the initial fluorescence intensity and clearly visualized the microtubules (Figure 4b). In contrast, when the tubulin was labeled with the Alexa Fluor 488conjugated secondary antibody, significant photobleaching (
