Cryogenic Fluorescence Localization Microscopy of Spectrally

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Cryogenic Fluorescence Localization Microscopy of Spectrally Selected Individual FRET Pairs in a Water Matrix Hiroaki Tabe, Kei Sukenobe, Toru Kondo, Atsunori Sakurai, Minako Maruo, Akari Shimauchi, Mitsuharu Hirano, Shin-nosuke Uno, Mako Kamiya, Yasuteru Urano, Michio Matsushita, and Satoru Fujiyoshi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b03977 • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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The Journal of Physical Chemistry

Cryogenic Fluorescence Localization Microscopy of Spectrally Selected Individual FRET pairs in a Water Matrix Hiroaki Tabe,† Kei Sukenobe,† Toru Kondo,† Atsunori Sakurai,† Minako Maruo,† Akari Shimauchi,† Mitsuharu Hirano,† Shin-nosuke Uno,‡ Mako Kamiya,⊥,§ Yasuteru Urano,‡,⊥,¶ Michio Matsushita,† Satoru Fujiyoshi*,†,§ † §

Department of Physics, Tokyo Institute of Technology, Meguro, Tokyo, 152-8550, Japan. PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan.

Graduate School of Pharmaceutical Sciences and ⊥Graduate School of Medicine, The University of Tokyo, Tokyo, 1130033, Japan. ¶ CREST, Japan Agency for Medical Research and Development (AMED), Chiyoda, Tokyo, 100-0004, Japan. ‡

ABSTRACT: We prepared a pair of visible-absorbing donor dye and near-infrared fluorescing acceptor dye. The donor and the acceptor were covalently linked close enough for Förster resonance energy transfer to occur. Under cryogenic conditions at 1.7 K, we observed the fluorescence excitation spectra of the individual pairs in a water matrix. We tested one Rhodamine, two Bodipy, and one Carbopyronine derivatives as the donor. Among these donors, Bodipy derivatives show the narrowest spectral width of the individuals with respect to the ensemble width. Thus, Bodipy dyes were favorable as the donor for the spectral selection of individual pairs. At 1.7 K, from the several Bodipy-acceptor pairs in the diffraction-limited volume, an individual pair was selected by the fluorescence excitation spectrum of the donor. The spectrally selected pair was localized using the near-infrared fluorescence of the acceptor.

INTRODUCTION Cryogenic (cryo-) fluorescence localization is a highly promising method to visualize molecular-level arrangements of biomolecules in a whole cell. Fluorescence microscopy is noninvasive and suitable for three-dimensional (3D) whole-cell observation. Cryogenic immobilization of a cellular sample allows the long signal accumulation time necessary for highprecision imaging. Structural damage of the sample is minimized by quick freezing below the glass transition temperature so that the cell remains near its native state (vitrification). Cryogenic measurement offers another benefit to fluorescence imaging: drastic reduction in the photobleaching of fluorophores. However, in practice, even in cellular cryo-microscopy of the highest precision, 1 the localization precision remains at approximately 10 nm, which is one order of magnitude lower than the molecular level. In this experiment, organelles in a 0.2-µm-thick cryo-section of a cell were visualized by photoactivatable green fluorescent proteins (PA-GFPs) at a temperature of approximately 100 K.1 The precision was limited to approximately 10 nm by the total photon number detected for PA-GFP before its deactivation (103 photons). In fluorescence microscopy, fluorescent dyes are the alternative to the fluorescent proteins. Owing to recent developments in molecular cell biology, target biomolecules in living cells can be labeled with fluorescent dyes by tag proteins.2 The target molecules in a cell can be localized in the fluorescence image of the labeling dye. The total photon number of com-

mercially available fluorescent dyes is 105 – 106 photons under cryogenic conditions.3,4 The limit of the total photon number is no longer a problem.5,6 Recently, 3D localization precision at the subnanometer level in all three directions has been demonstrated for cryogenic fluorescence localization microscopy of individual dyes in a water matrix.7 The precision has now reached high enough for 3D visualization of arrangements of molecules and their complexes in a vitrified cell. Here, we used a chemically linked pair of dyes, a donor absorbing in the visible wavelength region and an acceptor fluorescing in the near-infrared (NIR), separated by a distance short enough for Förster resonance energy transfer (FRET) to occur efficiently. For simplicity, the paired dye system of the chemically linked donor and acceptor molecules will be called ‘FRET pair’ for the rest of the present paper. In the design of the FRET pairs, we focused on the suppression of background signal from cellular samples and the identification of individual molecules within the focal volume. The first is the suppression of the background signal, i.e., photons emitted from cell contents residing in the excitation volume.8 This so-called autofluorescence is inevitable in whole-cell observation because the photoexcitation volume is filled with the cell contents. B. Liu et al. suppressed the autofluorescence background by using a thin cryo-section (0.2 µm thick).1 However, the thickness of mammalian cells is typically 10 µm, and whole-cell imaging is impossible using thin sections. The measurements of whole cells have to be modified to reduce the

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autofluorescence. When cells are excited in the visible region, the intensity of the autofluorescence decreases in the nearinfrared (NIR) region.8 The fluorescence maxima of Alexa Fluor 750 (Ax750), sulfo-cyanine 7 (sCy7), and cyanine 7.5 (Cy7.5) are around 800 nm. Thus, these cyanine dyes were chosen as the acceptor. The detection window of the fluorescence was set to 750 – 900 nm. The second focus of our work is the identification of individual FRET pairs residing in the diffraction-limited excitation volume. In a nonpolar matrix, the spectral width of individual dyes is almost lifetime limited (approximately 20 MHz).5,6,9 The individual width is 3-4 orders of magnitude narrower than the ensemble width. In such a nonpolar matrix, the dyes can be individually selected in a spectrum and localized in an image.5,6 In the present paper, we have initiated the present work with a search for a FRET pair better suited to cryo-fluorescence localization microscopy. EXPERIMENTAL SECTION The light source was supercontinuum white light generated by a combination of a femtosecond pulse laser (λ = 800 nm, pulse width ∼ 100 fs, repetition rate = 80 MHz; Mai Tai, Spectra Physics) and photonic crystal fiber (FemtoWHITE 800, NKT Photonics). To record a fluorescence excitation spectrum, the excitation light has to be monochromatic and tunable. To this end, the white light was dispersed with a 60°-prism (NSF11 glass; PS855, Thorlabs). After being dispersed by the prism, the light was coupled into a polarization-maintaining single-mode fiber (mode field diameter of 4.5 ± 0.5 µm at 630 nm; PM-630HP, Thorlabs) and was delivered to a home-built microscope setup. The core of the fiber functions as a pinhole. The spectral width of the output light was 0.2–0.3 nm (full width at half maximum, FWHM) in the wavelength range from 530 to 700 nm. For fluorescence imaging by broadband excitation, the prism was replaced by a bandpass filter (center

Figure 1. FRET pair of B581–sCy7. (a) Chemical structure. (b) Ensemble absorption and fluorescence spectra in a DMSO solution. The excitation wavelength (λex) was 532 nm.

wavelength = 560 nm, FWHM = 20 nm; FF01-561/14, Semrock). The details of the cryo-microscope have been described previously.7,10-14 Scanning of the laser focus in the sample was performed with two concave mirrors that were arranged in a telecentric configuration. The focal position on the xy-plane at the sample was controlled by the linear movement of one of the concave mirrors.11 The reflecting objective was placed in superfluid helium. The numerical aperture, NA, was 0.93 in superfluid helium (NA = nsinθ, refractive index n = 1.027),15 and the focal length was 2.52 mm. The excitation light was spectrally cleaned using a shortpass filter (FESH0700, Thorlabs). The excitation light and the fluorescence of the donors were blocked by a couple of longpass filters (FELH700, Thorlabs).

Figure 2. Individual fluorescence excitation spectra at 1.7 K (top), ensemble absorption spectra at 297 K (middle), and chemical structures of the donor molecules of the four FRET pairs, from left to right, (a) AT565–Ax750, (b) B558–sCy7, (c) B581–sCy7 and (d) Ax750– AT647N. The fluorescence excitation spectrum at the top and the absorption spectrum in middle were taken in a phosphate buffer matrix and in solution, respectively.


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The Journal of Physical Chemistry Aqueous solutions of the FRET pairs for single-molecule spectroscopy were prepared at pH = 7 in the presence of 0.02M phosphate buffer, 0.03-M n-octyl-β-D-glucopyranoside and 1% wt/wt polyvinyl alcohol. The solution was spin-coated on a CaF2 substrate with a spinning speed of 3000 revolutions per minute (rpm) for a coating time of 60 s. For the FRET pairs, ATTO565 (AT565), ATTO647N (AT647N), BODIPY581/591 (B581), or Bodipy 558/568 (B558) was used as the donor. The AT565 and AT647N molecules are rhodamine and carbopyronine derivatives, respectively. Ax750, sCy7, or Cy7.5 was utilized as the acceptor. Ax750, sCy7, and Cy7.5 are cyanine dye derivatives. The materials and their general information are provided in the Supporting Information. The FRET efficiency (ΦFRET), Förster distance (R0), and fluorescence quantum yield (ΦF_A) of the FRET pairs at 296 K are summarized in Table S1. RESULTS AND DISCUSSION Fluorescence excitation spectrum of individual FRET pairs in a water matrix at 1.7 K. Figure 1a shows the chemical structures of a representative FRET pair, B581 and sCy7. The absorption and fluorescence spectra of this FRET pair are shown in Fig. 1b. The excitation wavelength of the fluorescence spectrum (λex) was 532 nm. The distance between the donor and the acceptor, which corresponds to eighteen C-C bonds, is shorter than the Förster distance (R0) of 6.0 nm, so the photoexcitation energy of the donor effectively transfers to the acceptor (see Table S1). For the absorption spectra of individual FRET pairs, fluorescence excitation spectra were taken as an equivalent of the absorption. Since spectral separation between the absorption and the fluorescence is so large, longpass filters are enough to block the excitation light in the detection path. The top panels of Figs. 2a–d are three examples of the fluorescence excitation spectrum of individual FRET pairs in a water matrix at 1.7 K. We measured (a) AT565–Ax750, (b) B558–sCy7, (c) B581– sCy7, and (d) Ax750–AT647N as the FRET pairs. For reference, the steady-state absorption spectrum at 296 K (middle) and the chemical structure (bottom) of the donor are shown in the figure. The molecular structure of AT647N has been reported.16,17As a general tendency, the spectrum of individuals appears narrower than the ensemble spectrum (compare Figs. 2a–d top and middle). For each of the FRET pairs, 20–30 individuals were measured. In Figs. 3a–d, the results are summarized as histograms of the fluorescence maximum and FWHM. For reference, the steady-state absorption spectrum of the corresponding donor is shown by the solid curve in the left panels. As seen in the left panels of Figs. 3a–d, the histogram of the maximum follows the S1 ← S0 transition of the ensemble, showing that the donor absorption was recorded via the acceptor fluorescence. The right panel of Figs. 3a–d shows a histogram of the FWHM of individual FRET pairs plotted along the axis of the ratio of individual width (Γindividual) to the ensemble width (Γensemble). Γensemble was determined from the absorption spectrum of the solution. The Γensemble values were 31 nm (AT565), 25 nm (B558), 20 nm (B581) and 31 nm (AT647N). The histograms were fitted with a Gaussian function. The fitting results are, in the format of center ± standard deviation, 0.43 ± 0.12 (AT565), 0.26 ± 0.07 (B558), 0.25 ± 0.08 (B581), and 0.40 ± 0.24 (AT647N). In a simple case where all individuals have identical Γindividual values, Γensemble / Γindividual can serve as a measure

Figure 3. The maximum (left) and FWHM (right) of the fluorescence excitation spectra of individual FRET pairs in a water matrix at 1.7 K: (a) AT565–Ax750, (b) B558–sCy7, (c) B558– sCy7, and (d) Ax750–AT647N. For reference, the absorption spectra of the donors in a buffer solution at 296 K are shown in the left graphs. The Gaussian fitting result of FWHM is shown by the solid curves.

Figure 4. (a) Confocal fluorescence image of the FRET pair of B581–Cy7.5 in a water matrix at 1.7 K with excitation by broadband laser light (530–610 nm). The polarization of the excitation light was circular. (b) The excitation spectrum of the FRET pair (spectral width of laser light ∼ 0.2 nm). Spectrum b was observed at the position denoted by the cross in a. The spectrum is an average over the spectra recorded with differently-oriented linearlypolarized lights.



of the maximum number of individual molecules whose spectra can be recorded without mutual overlap. Thus, BODIPY molecules were found to be favorable for the spectral selection of individual FRET pairs. For the rest of the present work, we concentrated the Bodipy-acceptor pairs. The spectral widths of the individual FRET pairs were 0.2 – 0.4 with respect to the ensemble widths. This width is five orders of magnitude broader than that of individual aromatic molecules in a nonpolar matrix.9,18 The spectral broadening is due to (1) frequent structural changes of the water matrix near the fluorescent molecule and (2) the lifetime broadening due to the FRET process. First, the spectral peak of a fluorescent molecule in the water matrix is frequently shifted by fluctuation of the electrostatic interaction between the fluorescent molecule and water. In numerous single-molecule spectroscopic studies in polar matrixes, the spectral width of individual fluorescent molecules is as broad as that of their ensemble width.10,11,19,20 Figures S1 and S2 show the fluorescence spectra of individual free dyes in a water matrix at 1.7 K. Figure S1 shows that of AT565 and Ax750 constituting the FRET pair of Figs. 2a and 3b. The spectral widths ranged from several nanometers to a few tens of nanometers, which are similar to those of the donors in the FRET pairs shown in Figs. 2a–d, top. The broadening in the water matrix might be due to the rearrangements of hydrogen bonds near the fluorescent dye. The phenomena have been extensively studied in spectral hole-burning experiments.21-23 Second, the lifetime of the donors becomes shorter when FRET occurs. In the absence of FRET, the τD of donors is typically 5 ns. The FRET efficiency ΦFRET of the FRET pairs measured in the present work is 0.8 – 0.9 (see Table S1). The lifetime of the donor in the FRET pair is approximately 0.5 ns. The spectral broadening of the donor expected from its lifetime is approximately 0.4 pm at the center wavelength of 600 nm (0.3 GHz in frequency or approximately 10 cm–1 in wavenumber), which is 4 orders of magnitude narrower than the observed width shown in Figs. 3a–d, right. Therefore, the fluctuation of the interaction with the water matrix determined the width of the donors in the FRET pairs. Spectral-selective cryo-localization microscopy of individual FRET pairs in a water matrix at 1.7 K. Using Bodipyacceptor pairs, we performed spectral-selective localization microscopy at 1.7 K. One example for B581–Cy7.5 is shown in Figs. 4 and 5, and another for B581 and two data for B558 are shown in Figs. S3 – S5. First, a fluorescence image of the FRET pairs was observed with circularly polarized broadband light. The image is shown in Fig. 4a. The concentration of the sample solution for spin coating was set at approximately 10–8 M, which was one hundred times higher than the condition under which individual molecules appear as well-separated fluorescence spots in an image.14 Consequently, the fluorescence spots of the individual FRET pairs overlapped with each other. Second, at the position indicated by a sky-blue cross in Fig. 4a, the fluorescence excitation spectrum was recorded with the narrowband light (the spectral width of the laser light ∼ 0.2 nm). The spectrum of Fig. 4b is an average over the spectra with differently-oriented linearly polarized lights. A 580-nm-centered peak was observed in the spectrum. Smaller peaks also seem to be present. Third, linearly polarized narrowband light with a polarization angle of θex was irradiated at the position as that indicated by the sky-blue cross in Fig. 4a.

Figure 5. Spectral-selective localization of individual FRET pairs of B581–Cy7.5 in a water matrix at 1.7 K. (a) The polarization dependence of the fluorescence excitation spectrum. (b,c) Confocal fluorescence image of the FRET pair at (b) θex = 0° and λex = 603 nm and (c) θex = 90° and λex = 580 nm. Circles having a 1/e2 radius of the fitted 2D Gaussian function are shown by (b) red and (c) green. A circle having the 1/e2 radius of the fitted Gaussian function. (d) The localized positions (cross) and the circles having the 1/e2 diameter (∼640 nm) of the individual FRET pairs in b and c.


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The Journal of Physical Chemistry Figure 5a shows the θex-dependence of the fluorescence excitation spectra of individual FRET pairs. As seen in these spectra, three FRET pairs are identified as spectral peaks at (1) θex = 60° and λex = 603 nm, (2) θex = 150° and λex = 580 nm, and (3) θex = 90° and λex = 589 nm. Fourth, confocal fluorescence images of these FRET pairs recorded under the excitation and polarization conditions of (1) and (2) are shown in Figs. 5b–c and 5c. The position of the two molecules within the excitation spots was determined by measuring the centroids. The 2D Gaussian fitting of these spots was performed, and the fitting results show circles having a 1/e2 diameter of the fitted functions. The 1/e2 diameter was approximately 640 nm. The two circles are presented in the same figure (Fig. 5d), and the centroids obtained from the fitting are overlaid by crosses. The xdistance from the FRET pair under the condition (1) to that of (2) was 127 ± 8 nm, and the y-distance was 2 ± 8 nm. The two FRET pairs residing in the excitation spot were individually selected by the excitation wavelength as well as by the polarization, and the NIR fluorescence localization determined their positions with the standard error of 8 nm. In the present work, we observed the individual FRET pairs by a confocal laserscanning fluorescence microscope. T. Furubayashi et al. reported that the localization precision of individual molecules by confocal imaging is worse than the precision by CCD imaging due to the blinking noise (see Fig. S2 in reference 7).7 To realize nanometer-precision localization, we are developing spectral-selective cryo-localization microscopy by imaging with a CCD camera.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Photophysical properties of the FRET pair (Table S1); Fluorescence spectrum of individual dyes at 1.7 K (Figs. S1 and S2); three examples for spectral-selective localization at 1.7 K (Figs. S3 – S5); supporting materials and general information (PDF).

AUTHOR INFORMATION Corresponding Author * [email protected]

ACKNOWLEDGMENT This work was financially supported by Grants-in-Aid for Scientific Research (16H04094 and 15H03765). We thank Hiromu Kashida for the helpful comments of the design of single-stranded DNA of AT647N-Ax750.

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The Journal of Physical Chemistry

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Cryogenic Fluorescence Localization Microscopy of Spectrally

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