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Research Article

A photostable fluorescent marker for the superresolution live imaging of the dynamic structure of the mitochondrial cristae

Chenguang Wang, View ORCID ProfileMasayasu Taki, Yoshikatsu Sato, Yasushi Tamura, Hideyuki Yaginuma, Yasushi Okada, and View ORCID ProfileShigehiro Yamaguchi
  1. aInstitute of Transformative Bio-Molecules, Nagoya University, 464-8601 Nagoya, Japan;
  2. bPrecursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 332-0012 Kawaguchi, Saitama, Japan;
  3. cDepartment of Material and Biological Chemistry, Faculty of Science, Yamagata University, 990‐8560 Yamagata, Japan;
  4. dLaboratory for Cell Polarity Regulation, Center for Biosystems Dynamics Research, RIKEN, 565-0874 Osaka, Japan;
  5. eDepartment of Physics, Graduate School of Science, The University of Tokyo, 113-0033 Tokyo, Japan;
  6. fUniversal Biology Institute, Graduate School of Science, The University of Tokyo, 113-0033 Tokyo, Japan;
  7. gInternational Research Center for Neurointelligence, The University of Tokyo, 113-0033 Tokyo, Japan;
  8. hDepartment of Chemistry, Graduate School of Science, Nagoya University, 464-8602 Nagoya, Japan;
  9. iIntegrated Research Consortium on Chemical Sciences, Nagoya University, 464-8602 Nagoya, Japan

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PNAS August 6, 2019 116 (32) 15817-15822; first published July 23, 2019; https://doi.org/10.1073/pnas.1905924116
Chenguang Wang
aInstitute of Transformative Bio-Molecules, Nagoya University, 464-8601 Nagoya, Japan;
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Masayasu Taki
aInstitute of Transformative Bio-Molecules, Nagoya University, 464-8601 Nagoya, Japan;
bPrecursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 332-0012 Kawaguchi, Saitama, Japan;
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  • ORCID record for Masayasu Taki
  • For correspondence: taki@itbm.nagoya-u.ac.jp y.okada@riken.jp yamaguchi@chem.nagoya-u.ac.jp
Yoshikatsu Sato
aInstitute of Transformative Bio-Molecules, Nagoya University, 464-8601 Nagoya, Japan;
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Yasushi Tamura
cDepartment of Material and Biological Chemistry, Faculty of Science, Yamagata University, 990‐8560 Yamagata, Japan;
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Hideyuki Yaginuma
dLaboratory for Cell Polarity Regulation, Center for Biosystems Dynamics Research, RIKEN, 565-0874 Osaka, Japan;
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Yasushi Okada
dLaboratory for Cell Polarity Regulation, Center for Biosystems Dynamics Research, RIKEN, 565-0874 Osaka, Japan;
eDepartment of Physics, Graduate School of Science, The University of Tokyo, 113-0033 Tokyo, Japan;
fUniversal Biology Institute, Graduate School of Science, The University of Tokyo, 113-0033 Tokyo, Japan;
gInternational Research Center for Neurointelligence, The University of Tokyo, 113-0033 Tokyo, Japan;
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  • For correspondence: taki@itbm.nagoya-u.ac.jp y.okada@riken.jp yamaguchi@chem.nagoya-u.ac.jp
Shigehiro Yamaguchi
aInstitute of Transformative Bio-Molecules, Nagoya University, 464-8601 Nagoya, Japan;
hDepartment of Chemistry, Graduate School of Science, Nagoya University, 464-8602 Nagoya, Japan;
iIntegrated Research Consortium on Chemical Sciences, Nagoya University, 464-8602 Nagoya, Japan
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  • For correspondence: taki@itbm.nagoya-u.ac.jp y.okada@riken.jp yamaguchi@chem.nagoya-u.ac.jp
  1. Edited by Stefan W. Hell, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany, and approved June 24, 2019 (received for review April 6, 2019)

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Significance

Stimulated emission depletion (STED) microscopy is one of the most appealing tools to visualize nanoscale cellular structures and dynamics in living cells. However, its practical utility is significantly limited by the rapid photobleaching of fluorescent dyes under the ultrastrong depletion laser. In this context, superphotostable fluorescent probes, which enable repeated recording of the STED images, are crucial chemical tools to extend the application of STED microscopy in live-cell imaging. Herein, we report a developed fluorescent probe, MitoPB Yellow, which has the characters of outstanding photostability, long fluorescence lifetime, and mitochondrial inner membrane selectivity for staining. In combination with a time-gated-STED microscopy, the mitochondrial inner-membrane structures and dynamics can be impressively visualized in living cells.

Abstract

Stimulation emission depletion (STED) microscopy enables ultrastructural imaging of organelle dynamics with a high spatiotemporal resolution in living cells. For the visualization of the mitochondrial membrane dynamics in STED microscopy, rationally designed mitochondrial fluorescent markers with enhanced photostability are required. Herein, we report the development of a superphotostable fluorescent labeling reagent with long fluorescence lifetime, whose design is based on a structurally reinforced naphthophosphole fluorophore that is conjugated with an electron-donating diphenylamino group. The combination of long-lived fluorescence and superphotostable features of the fluorophore allowed us to selectively capture the ultrastructures of the mitochondrial cristae with a resolution of ∼60 nm when depleted at 660 nm. This chemical tool provides morphological information of the cristae, which has so far only been observed in fixed cells using electron microscopy. Moreover, this method gives information about the dynamic ultrastructures such as the intermembrane fusion in different mitochondria as well as the intercristae mergence in a single mitochondrion during the apoptosis-like mitochondrial swelling process.

  • superresolution
  • STED microscopy
  • fluorescence probe
  • mitochondrial cristae
  • live-cell imaging

In eukaryotic cells, mitochondria play pivotal roles in not only the production of energy, but also in various other cellular events including apoptosis and Ca2+ homeostasis as well as the biosynthesis of heme, amino acids, and phospholipids (1, 2). To control these versatile functions, mitochondria have a tightly regulated double-membrane structure, consisting of the inner and outer mitochondrial membranes (IM and OM, respectively), which differ with respect to shape and properties. Whereas the OM appears smooth, the IM is highly folded on the inside to form crista structures that maximize the surface area, which contributes to efficient oxidative phosphorylation. Transmission electron microscopy (TEM) has provided numerous insights into the morphological changes of mitochondrial membrane structures with a spatial resolution of 1 to 2 nm. However, for the preparation of TEM specimen, chemical fixation, dehydration, and embedding processes may potentially introduce artifacts into the intact mitochondrial structure. More importantly, the fixation stops all dynamic biological processes, resulting in a loss of temporal information.

In this context, superresolution nanoscopy techniques have become increasingly important as tools to visualize spatiotemporal dynamics of cellular structures, such as the mitochondrial membrane (3⇓–5), the nuclear pore complex (6, 7), and the cytoskeletons (8, 9), on the nanoscale in living cell. Among various superresolution methods, stimulated emission depletion (STED) microscopy is arguably the most prevalent for live-cell imaging as it offers the highest spatiotemporal resolution (10). However, the poor photostability of current mitochondrial fluorescent markers toward the intense laser irradiation used for excitation and depletion has been the principal technical limitation for observing mitochondrial membrane dynamics using STED microscopy.

To solve this issue, we have recently proposed a structural reinforcement strategy of electron-deficient π-conjugated molecular skeletons, which significantly improves the photostability of fluorophores (11). Based on this strategy, superphotostable phosphole-based fluorescent molecules have been developed, and these allow multiple acquisition of STED images under physiological conditions (12). As the next generation of such superphotostable fluorescence tools, we designed MitoPB Yellow (Fig. 1A), which enabled us to visually distinguish nanoscale cristae structures in mitochondrial IM in living cells, and to perform the long-term time-lapse STED imaging of the cristae dynamics.

Fig. 1.
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Fig. 1.

Properties of phosphole dyes. (A) Chemical structures of MitoPB Yellow and its simplified (1) and water-soluble (2) derivatives. (B) Normalized emission spectra of 1 in organic solvents and 2 in a PBS buffer (pH = 7.4). The dotted line indicates the depletion wavelength used in this study. (C) Comparison of the photostability of live HeLa cells labeled with MitoPB Yellow and cells stained with Rh123 or MTG. Confocal images were recorded under the same acquisition conditions (λex = 488 nm). Signal intensities of each image (I) relative to the initial value (I0) are plotted as a function of the recorded number. (D) Comparison of the fluorescence lifetime of MitoPB Yellow with Rh123 and MTG in live HeLa cells. Signal intensities (I) detected in each delay time (tg) are normalized to the initial value (I0) and plotted as a function of tg. The fluorescence lifetime (τ) of each dye in mitochondria were determined by a single-exponential-decay fitting.

Results

Molecular Design of a Superphotostable Fluorescent Mitochondrial Probe.

Our design strategy for the generation of superphotostable MitoPB Yellow is based on a robust π-conjugated skeleton that contains an electron-withdrawing phosphine oxide (P = O) moiety. We have previously reported that the donor–π–acceptor-type dye C-Naphox, which consists of a naphthophosphole P-oxide skeleton and an electron-donating triphenylamine moiety, exhibits sufficient photoresistance to allow successive STED scans (11). While this dye showed significantly decreased fluorescence quantum yields (ΦF) in aqueous media due to its intramolecular charge transfer (ICT) character in the excited state, intense fluorescence was revived in nonpolar environments. We envisioned that such ICT-based fluorophores would emit bright fluorescence with a comparatively long fluorescence lifetime when incorporated into a lipid bilayer, while its fluorescence intensity would be diminished in aqueous environments, such as the mitochondrial matrix and cytosol, resulting in the possibility to observe the membrane morphology with high contrast. To turn the naphthophosphole P-oxide scaffold into a mitochondrial fluorescent marker, we employed a naphthalene-1,2-diyl–fused core skeleton, since this regioisomer should exhibit longer absorption and emission maxima compared with its naphthalene-2,3-diyl counterpart (12). Moreover, the lipophilic cationic triphenylphosphonium group and a highly reactive epoxide group were introduced to the naphthophosphole scaffold to achieve selective and stable labeling of the mitochondrial membrane. Once the dye is accumulated in the mitochondrial membrane, the proximal membrane proteins would be covalently labeled with the dye (13), enabling the visualization of the mitochondrial morphologies even after the loss of the membrane potential or fixation of the cells.

We first prepared a model compound of MitoPB Yellow 1 and its water-soluble derivative 2 to evaluate the effect of the environmental polarity on the photophysical properties of the core skeleton (Fig. 1A). Irrespective of the solvent, 1 exhibited an absorption maximum at 465 to 480 nm with a molar absorption coefficient of ∼1.5 × 104 M−1⋅cm−1, whereas the maximum emission wavelength (λem) and the fluorescence quantum yield (ΦF) depend strongly on the solvent polarity (Fig. 1B and SI Appendix, Table S1 and Fig. S1). In, e.g., toluene, 1 showed an emission maximum at λem = 570 nm with a high quantum yield (ΦF = 0.93), while λem shifted bathochromically with increasing solvent polarity (λem = 643 nm in acetonitrile). In PBS (pH = 7.4), 2 showed a further bathochromic shift of the emission wavelength (λem = 667 nm) and a decreased quantum yield (ΦF = 0.13). Based on the polarity sensitivity, MitoPB Yellow is supposed to have a fluorogenic character upon localizing the mitochondrial membrane.

The decreased ΦF in water is mainly due to the acceleration of the nonradiative decay processes from the lowest excited singlet state (S1). The fluorescence lifetime (τ = 7.3 ns) of 1 in toluene was 3× longer than that (τ = 2.4 ns) of 2 in water. The radiative (kr) and nonradiative (knr) decay rate constants from S1 were determined based on the τ and ΦF values (SI Appendix, Table S1). The knr value of 2 in water (3.6 × 108 s−1) is substantially larger than that (0.10 × 108 s−1) of 1 in toluene, while their kr values vary to a lesser extent dependent on the solvent polarity.

Mitochondrial Imaging with MitoPB Yellow.

The performance of MitoPB Yellow as a mitochondrial fluorescent marker was evaluated by conducting live-cell imaging experiments with various cell lines (HeLa, HepG2, MCF-7, HaCaT, and NMuMG). Each cell was incubated with 500 nM of MitoPB Yellow for 2 h, followed by staining with the commercially available mitochondria marker MitoTracker Deep Red FM. Irrespective of the cell types, the images thus obtained showed high colocalization of both dyes with Pearson’s correlation coefficient values of 0.86 to 0.93 (SI Appendix, Fig. S2). In a concentration range of 50 nM to 1.5 µM, which did not significantly affect the cell viability based on the MTT assay (SI Appendix, Fig. S3), MitoPB Yellow could efficiently stain the mitochondria. The background fluorescence intensity in the cytoplasm was quite low, even without washing process in the live‐cell imaging (SI Appendix, Figs. S4 and S5). These results demonstrate that MitoPB Yellow exhibits sufficient membrane penetration ability and mitochondrial selectivity.

To demonstrate the superior utility of MitoPB Yellow over other representative mitochondrial fluorescent markers, the performance of MitoPB Yellow was compared with that of MitoTracker Green FM (MTG), MitoTracker Red CMX (MTR), MitoTracker Deep Red FM (MTDR), and Rhodamine 123 (Rh123). First, HeLa cells were stained with each dye under appropriate conditions and then fixed with 4% paraformaldehyde (15 min) followed by washing with a PBS buffer. In the imaging with MitoPB Yellow, more than 70% of the fluorescence signal was retained after fixation; conversely, the remaining fluorescence signal was much weaker when using MTG (24%), MTR (29%), MTDR (37%), and Rh123 (17%) (SI Appendix, Fig. S6). The MitoPB Yellow signal was sufficiently strong to see mitochondrial morphology remained even after permeabilization with Triton X-100. Indeed, multicolor staining, which needs the permeabilization process, was successfully accomplished by fixation and permeabilization of MitoPB Yellow-labeled cells, followed by immunolabeling of α-tubulin with Alexa-Fluor-680–conjugated IgG, and staining the nucleus with Hoechst 33342 (SI Appendix, Fig. S7). Similar results were also observed when the dye-stained cells were treated with CCCP (2 µM), which is a proton ionophore that dissipates the membrane potential across the IM. The fluorescent intensity of the image with MitoPB Yellow showed the highest retention ratio (92% persistence) among these mitochondria markers (SI Appendix, Fig. S8). These results would reflect the covalent linkage of MitoPB Yellow to the mitochondrial proteins via its epoxide moiety.

Moreover, the photostability of MitoPB Yellow in mitochondria was evaluated. Under the same excitation conditions (λex = 488 nm), Rh123 and MTG suffered from rapid photobleaching during the repeated recording of confocal images, while MitoPB Yellow demonstrated unsurpassed photostability. Even after the acquisition of 50 images, no significant decrease in fluorescence intensity was observed for MitoPB Yellow (Fig. 1C and SI Appendix, Fig. S10).

Photophysical Properties of MitoPB Yellow in Mitochondria.

Spectral imaging provided valuable information on the local environmental polarity of MitoPB Yellow in mitochondria. The emission spectrum observed for the MitoPB Yellow-labeled-HeLa cells was comparable to that measured in toluene (SI Appendix, Fig. S1C). This clearly indicates that in mitochondria, the dye is surrounded by a quite hydrophobic microenvironment, such as the lipid bilayer and/or hydrophobic pockets of proteins. The fluorescence lifetime (τ) of MitoPB Yellow in living HeLa cells was determined using picosecond-pulsed laser excitation and gated detection (14). After HeLa cells were stained with MitoPB Yellow, fluorescence images of the same area were recorded at various gate delay times (tg), which are the delays between the excitation pulse and the start of the detection window for fluorescence collection, while taking a constant gate width (Δt = 3.5 ns) (SI Appendix, Fig. S11). The sums of the fluorescence signal intensity for the collected area were plotted as a function of the tg value and fitted by a single exponential decay function (Fig. 1D). The thus-calculated fluorescence lifetime of MitoPB Yellow in mitochondria (τ = 7.5 ns) is comparable to that obtained in toluene (τ = 7.3 ns) and substantially longer than those of Rh123 (τ = 3.3 ns) and MTG (τ = 0.75 ns) in mitochondria (Fig. 1D). Notably, the longer τ of MitoPB Yellow should be an important advantage to gain images with higher contrast in the time-gated mode or fluorescence lifetime imaging microscopy, as most of the short-lived background signals, including cell autofluorescence and scattered excitation laser light, can be effectively eliminated.

Live-Cell STED Microscopy Using MitoPB Yellow.

The long fluorescence lifetime of MitoPB Yellow and its remarkable photostability suggested its promising utility for STED microscopy (15, 16). Therefore, we examined MitoPB Yellow-stained cells with a gated continuous wave (CW) STED microscope that was equipped with a pulsed white-light laser (WLL) for excitation and a CW laser (660 nm) for depletion. Initially, we recorded confocal images of the mitochondria under excitation at 488 nm, before we applied the maximum power of the STED beam (270 mW at the back aperture of the objective lens) for detection using a delay time (tg) of 3 ns (Fig. 2A). The live-STED image clearly revealed a ladder-like pattern of the mitochondrial cristae that could not be seen in the confocal image, exhibiting a full width at half maximum (FWHM) of 60 nm. The contrast of image was further improved by removing the haze and the noise in the raw image data by deconvolution with a theoretical point-spread function (PSF) using the commercial program package Huygens (Fig. 2B). The final image was sufficiently clear to accurately count the number of individual cristae from the line profiles of the intensity (Fig. 2C).

Fig. 2.
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Fig. 2.

STED imaging of mitochondria of living HeLa cells stained with MitoPB Yellow (λex = 488 nm; λSTED = 660 nm). (A) Comparison of scanning images of mitochondria for the confocal (Left) and STED (Right; tg = 3 ns; PSTED = 270 mW) recording. (Scale bar, 2 µm.) (B) STED images of mitochondria before and after deconvolution. (Scale bar, 1 µm.) (C) Intensity profile plot along the white lines in the raw (black) and deconvoluted (blue) STED images. (D) The FWHM resolution as a function of STED power PSTED. The solid line indicates a theoretical fit of the data to the equation for STED microscopy. (E) FWHM as a function of delay time tg. Solid lines show fits of the data to the equation for gated CW STED with τ = 7.5 ns for PSTED = 108 mW (open square) and PSTED = 270 mW (filled square).

The properties of MitoPB Yellow in STED microscopy were further evaluated by changing the acquisition conditions. For a single, well-separated crista in the relatively large mitochondria, the FWHM could be measured even when using the confocal microscopy, affording dc = 243 ± 31 nm (n = 10). Upon increasing the STED beam power (PSTED) at 660 nm, the resolution increased and the FWHM of the crista image decreased to 91 ± 12 nm (n = 10) when a depletion power of PSTED = 270 mW was applied (SI Appendix, Fig. S12). The average FWHMs, calculated from the image data, were plotted as a function of PSTED, and the data were fitted using the theoretical resolution formula for STED microscopy (Fig. 2D) (17), from which the effective saturation power Psat required for the half-depletion of MitoPB Yellow fluorescence at 660 nm was determined to be 37 mW. Notably, the time-gated detection further improved the spatial resolution in the CW STED microscopy (15). In good agreement with the theory for the gated CW STED (16), the resolution for mitochondrial membrane increased with decreasing fluorescence intensity upon increasing the gated time tg (SI Appendix, Fig. S13). The FWHM value was saturated at ∼58 ± 6 nm at tg = 3 ns and PSTED = 270 mW (Fig. 2E). It should be noted here that the FWHM values reported above would be larger than the optical resolution (FWHM of PSF), since the size of crista is not negligible compared with the optical resolution. To give a more accurate estimate of resolution, we used the nested-loop ensemble PSF fitting method (18). The estimated resolution was 45 ± 5 nm under the best imaging conditions (Fig. 2E). Importantly, cristae could not be visualized in the STED microscopy when using MTG and Rh123, due to their poor photostability and/or low membrane selectivity, clearly demonstrating the superiority of MitoPB Yellow over these conventional dyes (SI Appendix, Fig. S14).

The superresolution image suggests that MitoPB Yellow would localize specifically to the mitochondrial IM in living cells. To confirm its selectivity toward IM rather than the OM, OM and IM were counterstained using mitochondrial proteins TOMM20 and OPA1, respectively. Their localization in mitochondria is well established, and the full-length TOMM20 protein and the N-terminal mitochondrial targeting signal (1-123 aa) of OPA1 (mtsOPA1) were used (19). HaloTag was used to label these mitochondrial marker proteins with tetramethylrhodamine (TMR), because the large difference in Stokes shift between MitoPB Yellow and TMR [Δ(1/λ) = 3,416 cm−1, SI Appendix, Fig. S15] is beneficial to gaining good separation in multicolor STED imaging (20). To minimize cross-talks between the dyes, laser wavelengths of 470 and 540 nm were chosen for the excitation of MitoPB Yellow and TMR, respectively. Two-color STED images were acquired via a line-by-line sequential scanning of 2 channels with one detector (λem = 550 to 650 nm; detector gate time = 3.0 to 7.5 ns) to suppress the positional deviation of 2 images caused by rapid mitochondrial movement in living cells. In the comparison of fluorescent signals of TMR labeled to the OM and IM (TOMM20-TMR and mtsOPA1-TMR, respectively) with MitoPB Yellow, TOMM20-TMR signal (Fig. 3B) always surrounded the MitoPB Yellow signal (Fig. 3A). The line profiles across the mitochondrial tubule section in the overlay image (Fig. 3C) indicated that the fluorescence of MitoPB Yellow appears inside the TMR-labeled OM (Fig. 3D). The FWHMs of the structures in the 2-color STED image were determined to be 68 ± 3 nm (n = 10) and 77 ± 4 nm (n = 10) for MitoPB Yellow and TMR, respectively (SI Appendix, Fig. S16). Contrastingly, mtsOPA1-TMR showed good colocalization with MitoPB Yellow (SI Appendix, Fig. S17C). These results not only confirm that MitoPB Yellow signals come from the IM rather than OM, intermembrane space, or matrix, but also demonstrate the utility of MitoPB Yellow in 2-color STED live imaging as a mitochondrial IM-selective fluorescent marker.

Fig. 3.
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Fig. 3.

Two-color STED image: (A) mitochondrial inner membrane labeled with MitoPB Yellow (λex = 470 nm; green); (B) outer membrane labeled with TOMM20-TMR (λex = 540 nm; magenta); (C) merged image; (D) signal intensity profile across the mitochondrial membranes for the two channels. The images were deconvoluted using Huygens deconvolution software. Zoomed views of boxed regions in white are shown in Insets [Scale bar, 1 µm (white) and 0.5 µm (yellow for Inset).]

Visualization of Mitochondrial Cristae Remodeling.

To demonstrate the practical utility of MitoPB Yellow for live-cell imaging, we monitored the various changes of the mitochondrial morphology, which have been so far studied using TEM. Stress derived from nutrient deprivation is known to induce an increase in fusion activity, resulting in mitochondrial elongation. A TEM analysis revealed an increased number of cristae in the hyperfused mitochondria (21). It has been proposed that the elongation of mitochondria might increase the efficiency of the adenosine 5′-triphosphate (APT) synthesis and protect mitochondria from autophagosomal degradation to enhance cellular survival during starvation (22). However, a method to visualize the ultrastructure of hyperfused mitochondria in living samples remains elusive. Therefore, we employed MitoPB Yellow to this end.

For that purpose, HeLa cells were stained with MitoPB Yellow, starved in Hanks’ balanced salt solution (HBSS), and STED images were acquired. After the deconvolution process, the densities of cristae in the hyperfused mitochondria were assessed by counting the number of cristae per unit of length. Compared with the cells cultured in Dulbecco’s modified Eagle’s medium (DMEM), the cells that were starved for 3 h showed elongated mitochondria, in which the cristae were sparsely arranged (Fig. 4 A, Top and Middle, and SI Appendix, Fig. S18). Conversely, after 12 h of starvation, the formation of significantly dense cristae was observed in much thinner tubular mitochondria (Fig. 4 A, Bottom). The average density of cristae almost remained unchanged for the 3-h-starved cells, whereas that of the 12-h-starved cells was 1.5× higher than that for the untreated cells (Fig. 4B), which is consistent with a previous study that reports an ∼1.8× higher density for mouse embryonic fibroblasts observed by TEM (21).

Fig. 4.
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Fig. 4.

Morphological changes of the mitochondrial inner membrane captured by STED microscopy (λex = 488 nm; λSTED = 660 nm). (A) Deconvoluted STED images showing changes in the mitochondrial morphology under concomitant change of the cristae density upon nutrition starvation for 3 and 12 h (HBSS containing Ca2+ and Mg2+). (Scale bar, 2 µm.) (B) Comparison of the number of cristae per micrometer of mitochondrial length before and after incubation for 3 and 12 h under starvation conditions (n = 20). (C) STED image of cristae in HeLa cells, pretreated with 10 µM mitochondrial DNA replication inhibitor (ddC) for 5 d followed by staining with MitoPB Yellow. (Scale bar, 2 µm.)

Mitochondrial dysfunction gives rise to an unusual cristae morphology (23), which can be visualized using MitoPB Yellow. HeLa cells were treated for 5 d with the mitochondrial DNA (mtDNA) replication inhibitor 2′,3′-dideoxycytidine (ddC), and subsequently stained with MitoPB Yellow before images were recorded (Fig. 4C and SI Appendix, Fig. S19). As previously observed by TEM (24), concentrically remodeled cristae, which have never been observed in untreated cells, were clearly visible in some living cells using STED microscopy. We also succeeded in gaining images of live cells treated with etoposide, which induces the mitochondrial apoptotic event by disrupting mitochondrial membranes (25). In this experiment, the mitochondria were labeled with MitoPB Yellow before etoposide treatment. As MitoPB Yellow remains in the membrane even after the loss of the mitochondrial membrane potential thanks to the immobilization at its epoxide functional group, ultrastructural morphological changes of the inner membrane can be recorded in the live cells undergoing apoptosis. In fact, we observed fewer cristae and partial vesicular morphologies in swollen mitochondria as well as circumference of fragmented mitochondria, which were previously identified using TEM (26) (SI Appendix, Fig. S20). Such etoposide-induced morphological changes in mitochondria were prevented when the cells were treated with the general caspase inhibitor Z-VAD-fmk (27), indicating that the imaging results are unlikely to be artifacts of MitoPB Yellow (SI Appendix, Fig. S20). These are observations of cristae remodeling in living cells.

Time-Lapse STED Imaging of Mitochondria.

One of the crucial advantages of superresolution microscopy over TEM is that it enables one to visualize the structural details as they happen in real time. In particular, the time-lapse STED imaging should provide morphological and temporal information of organelle dynamics; however, currently available fluorescent organelle markers usually suffer from photobleaching. The superphotostable MitoPB Yellow circumvented this obstacle and enabled recording >1,000 frames of the IM dynamics in the time-lapse sequence when images were recorded at a frame rate of 1.54 fps (frames per second) (Movie S1). From these image sequences, we have successfully observed a rapid intercristae mergence (<2 s) in a single mitochondrion (Fig. 5A and Movie S2) and the intermitochondrial fusion (Fig. 5B and Movie S3). These images strongly demonstrate the potential of live-cell STED imaging with MitoPB Yellow. However, during a long-term observation over 90 s, unexpected morphological changes of the mitochondria started to occur, even though the fluorescence intensity remained unchanged (Fig. 5C). The mitochondria began to swell with the loss of cristae, and in some cases, the membrane eventually ruptured (Movie S4) (28). It should be noted that photobleaching might not be the source of this damage to mitochondria, because the fluorescence signals from MitoPB Yellow remained unchanged. Instead, the extremely strong STED laser might have damaged the mitochondria. Indeed, similar morphological changes were also observed under the irradiation only with the STED laser at 660 nm, i.e., in the absence of the excitation at 488 nm, although MitoPB Yellow does not absorb the STED beam (SI Appendix, Fig. S21). It is well known that mitochondria are easily damaged during fluorescence live imaging. When mitochondria are stained with conventional dyes, the photodamage is usually accompanied by photobleaching of the dyes. Therefore, radical formation during photobleaching of the mitochondria-staining dyes has been surmised as the source of the photodamage. Our results with photoresistant MitoPB Yellow has separated the photodamage from photobleaching, and it enabled us to visualize the photoinduced damaging process of mitochondria at high spatial resolution.

Fig. 5.
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Fig. 5.

(A–C) Time-lapse STED imaging of mitochondrial dynamics in MitoPB Yellow-labeled cells; acquisition conditions: λex = 488 nm; λSTED = 660 nm; PSTED = 108 mW; tg = 3 ns; line average = 1; scan speed = 100 Hz; frame rate = 0.77 fps. (Scale bar, 2 µm; Huygens deconvolution was applied.)

Discussion

In this study, we developed a molecular tool that enables imaging of the mitochondrial ultrastructure in living cells. The fluorescent marker MitoPB Yellow was designed to overcome the 2 principal drawbacks of conventional dyes, i.e., photostability and mitochondrial membrane selectivity, which have limited the use of STED microscopy for the investigation of the mitochondrial morphology. Firstly, the insufficient photostability of classical fluorescent dyes has been the most crucial problem for their application in superresolution microscopy, which requires intense laser irradiation. The rapid photobleaching of conventional fluorescent dyes usually hampers the repeated acquisition of images at a superresolution level. To address this issue, we employed a robust π-conjugated skeleton containing an electron-withdrawing P = O moiety, which attained significantly higher photostability. Secondly, to gain a fluorogenic response to the mitochondrial membrane, an electron-donating group was directly conjugated to the fluorophore skeleton. The donor–π–acceptor skeleton gives rise to an environment polarity-sensitive character of fluorescence. The thus-obtained large difference in ΦF of MitoPB Yellow between polar and nonpolar environments results in enhanced fluorescence contrast of the membrane structure in mitochondrial imaging. The exceptionally high photostability of MitoPB Yellow allows using the STED laser power at the maximum intensity possible in commercially available microscopes. In combination with its membrane-selective staining characteristics, MitoPB Yellow can thus provide hitherto unprecedented resolution in the mitochondrial IM imaging in living cells.

The resolution in the STED microscopy (d) is approximately proportional to the inverse square root of the saturation factor ISTED/Isat. Here, ISTED is the STED laser power, while Isat is the effective saturation intensity, at which the population of the excited electron is depleted by half. Therefore, dyes with a lower Isat are beneficial to increasing the resolution with a given maximum STED laser power. Isat for MitoPB Yellow is estimated to be 1.3 MW/cm2 at 660 nm on the assumption of the typical doughnut area (A ∼ 3 × 10−9 cm2) and the losses of the STED laser power in the objective lens (decreased by ∼12%) (29). In other words, MitoPB Yellow is efficiently depleted at the maximum CW STED laser power of the commercial STED microscope (∼10 MW/cm2). In general, the lower Isat values are attributable to the larger stimulated emission cross-section (σSTED) and/or the longer fluorescent lifetime (τ), because the Isat value is inversely proportional to these parameters by the following formula: Isat = hc/(λSTED σSTED τ), where h and c refer to the Plank constant and the speed of light, respectively. The σSTED is calculated as 3.23 × 10−17 cm2 by using the values for the lifetime τ = 7.5 ns and the depletion wavelength λSTED = 660 nm. This σSTED value is in good agreement with the value theoretically derived from the emission spectrum of MitoPB Yellow in PMMA (σSTED = 3.39 × 10−17 cm2; SI Appendix, Fig. S1D). As this value falls in a common range for many fluorophores (σSTED = 10−16 to 10−18 cm2) (30), the major contribution to the lower Isat for MitoPB Yellow should be the longer fluorescent lifetime (τ = 7.5 ns in mitochondria).

The development of MitoPB Yellow allowed us to examine a number of aspects regarding the IM structure and dynamics in mitochondria. Most importantly, our microscopic analyses revealed the dynamic changes of mitochondrial ultrastructure in living cells. In particular, we observed a drastic crista development following mitochondrial hyperfusion upon starvation. We also confirmed a thinning of mitochondrial tubules with increase of cristae density under starvation conditions, which was previously observed by TEM analyses (Fig. 4A) (31), demonstrating the great utility of our live-cell STED imaging. Thus, MitoPB Yellow would be an invaluable molecular tool for further biological studies such as the mechanism how the morphological changes are accomplished in the IM.

Time-lapse imaging may be able to capture a fusion event between distinct cristae protruding from opposing sides of the mitochondrion, although we cannot rule out the possibility that two opposing cristae happen to come close each other without fusion (Fig. 5A). However, previous electron microscopy (EM) studies strongly suggest such fusion events to occur between distinct cristae or between a crista and the inner boundary membrane (IBM). Specifically, multiple connections between a single crista and the IBM as well as cristae connecting to both opposing sides of the IBMs are frequently observed by EM analyses (32).

In summary, MitoPB Yellow surpasses currently used fluorescent mitochondrial markers in terms of photostability, fluorescence lifetime, and signal-to-noise ratio for membrane-staining, which enables STED imaging of the mitochondrial inner-membrane structures in living cells at unprecedented resolution (<60 nm). Moreover, the large Stokes shift of this probe, combined with another small-Stokes-shift dye, allows multicolor STED imaging with a single STED laser. The molecular design strategy based on this superphotostable fluorophore should thus be applicable to the STED imaging of membrane dynamics in different cellular organelle, and fluorescent markers thus developed should also be expected to find a wide range of applications in other superresolution techniques.

Materials and Methods

For chemical synthesis of probe MitoPB Yellow, spectral measurements, cell culture, mitochondrial staining, and confocal imaging, see SI Appendix.

STED Imaging.

The cells were stained in DMEM+ containing 500 nM MitoPB Yellow and 0.5% dimethyl sulfoxide for 2 h in a CO2 incubator. Then, the cells were washed 3 time with fresh medium to remove the free dye, and kept in DMEM+ for imaging. The Leica TCS SP8 STED 3× system with 2 CW lasers at 592 and 660 nm and a pulsed laser at 775 nm for the depletion was used for STED imaging. An HyD detector and an STED WHITE objective (100×/1.40 OIL or 93×/1.30 GLYC) were employed. Unless otherwise noted, the STED images were acquired with excitation at 488 nm (WLL), emission in the range of 500 to 640 nm, depletion at 660 nm (CW-STED, 270 mW), and gated detection at tg = 3 ns. In general, the images were recorded with a pixel resolution of 22.7 nm × 22.7 nm or higher, a scan speed of 100 Hz, a bidirectional mode, a line average of 3, and a pinhole size of 151.63 µm. The images were processed using ImageJ. In some cases, the STED images were deconvoluted with a theoretical PSF using a commercial program package Huygens.

Acknowledgments

We thank the following people for their technical assistance or sharing materials: A. Fukazawa (Kyoto University) for discussing the synthesis, K. Kuwata (Nagoya University) for measuring the HPLC-MS, T. Imamura (Ehime University) for providing HaCaT and NMuMG cell lines, Miho Ohsugi (Tokyo University) for the selected clone of HeLa cell, Michael Davidson and David Chan for the plasmids (Addgene 54282 and 26047), and Junko Asada for her technical assistance including plasmid construction and cell culture. This work was supported by JST PRESTO JPMJPR16F5 (M.T.) and JST CREST JPMJCR15G2 (Y.O.), as well as JSPS KAKENHI Grants 16H05119 (Y.O.), 17H19511 (Y.O.), 18K14721 (H.Y.), and JP16H06280 (Advanced Bioimaging Support).

Footnotes

  • ↵1To whom correspondence may be addressed. Email: taki{at}itbm.nagoya-u.ac.jp, y.okada{at}riken.jp, or yamaguchi{at}chem.nagoya-u.ac.jp.
  • Author contributions: C.W., M.T., and S.Y. designed research; C.W., Y.S., H.Y., and Y.O. performed research; M.T., Y.T., H.Y., Y.O., and S.Y. contributed new reagents/analytic tools; C.W., M.T., Y.S., Y.T., H.Y., Y.O., and S.Y. analyzed data; and C.W., M.T., Y.T., Y.O., and S.Y. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1905924116/-/DCSupplemental.

Published under the PNAS license.

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A photostable fluorescent marker for the superresolution live imaging of the dynamic structure of the mitochondrial cristae
Chenguang Wang, Masayasu Taki, Yoshikatsu Sato, Yasushi Tamura, Hideyuki Yaginuma, Yasushi Okada, Shigehiro Yamaguchi
Proceedings of the National Academy of Sciences Aug 2019, 116 (32) 15817-15822; DOI: 10.1073/pnas.1905924116

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A photostable fluorescent marker for the superresolution live imaging of the dynamic structure of the mitochondrial cristae
Chenguang Wang, Masayasu Taki, Yoshikatsu Sato, Yasushi Tamura, Hideyuki Yaginuma, Yasushi Okada, Shigehiro Yamaguchi
Proceedings of the National Academy of Sciences Aug 2019, 116 (32) 15817-15822; DOI: 10.1073/pnas.1905924116
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