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A photostable fluorescent marker for thesuperresolution live imaging of the dynamic structureof the mitochondrial cristaeChenguang Wanga, Masayasu Takia,b,1, Yoshikatsu Satoa, Yasushi Tamurac, Hideyuki Yaginumad, Yasushi Okadad,e,f,g,1,and Shigehiro Yamaguchia,h,i,1
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; cDepartment of Material and Biological Chemistry, Faculty of Science, YamagataUniversity, 990‐8560 Yamagata, Japan; dLaboratory for Cell Polarity Regulation, Center for Biosystems Dynamics Research, RIKEN, 565-0874 Osaka, Japan; eDepartmentof Physics, Graduate School of Science, The University of Tokyo, 113-0033 Tokyo, Japan; fUniversal Biology Institute, Graduate School of Science, The University ofTokyo, 113-0033 Tokyo, Japan; gInternational Research Center for Neurointelligence, The University of Tokyo, 113-0033 Tokyo, Japan; hDepartment of Chemistry, GraduateSchool of Science, Nagoya University, 464-8602 Nagoya, Japan; and iIntegrated Research Consortium on Chemical Sciences, Nagoya University, 464-8602 Nagoya, Japan
Edited by Stefan W. Hell, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany, and approved June 24, 2019 (received for review April 6, 2019)
Stimulation emission depletion (STED) microscopy enables ultrastruc-tural imaging of organelle dynamics with a high spatiotemporalresolution in living cells. For the visualization of the mitochondrialmembrane dynamics in STED microscopy, rationally designed mito-chondrial fluorescent markers with enhanced photostability arerequired. Herein, we report the development of a superphotostablefluorescent labeling reagent with long fluorescence lifetime, whosedesign is based on a structurally reinforced naphthophospholefluorophore that is conjugated with an electron-donating dipheny-lamino group. The combination of long-lived fluorescence andsuperphotostable features of the fluorophore allowed us to selec-tively capture the ultrastructures of the mitochondrial cristae with aresolution of ∼60 nm when depleted at 660 nm. This chemical toolprovides morphological information of the cristae, which has so faronly been observed in fixed cells using electron microscopy. More-over, this method gives information about the dynamic ultrastruc-tures such as the intermembrane fusion in different mitochondria aswell as the intercristae mergence in a single mitochondrion duringthe apoptosis-like mitochondrial swelling process.
superresolution | STED microscopy | fluorescence probe | mitochondrialcristae | live-cell imaging
In eukaryotic cells, mitochondria play pivotal roles in not onlythe production of energy, but also in various other cellular
events including apoptosis and Ca2+ homeostasis as well as thebiosynthesis of heme, amino acids, and phospholipids (1, 2). Tocontrol these versatile functions, mitochondria have a tightly reg-ulated double-membrane structure, consisting of the inner andouter mitochondrial membranes (IM and OM, respectively), whichdiffer with respect to shape and properties. Whereas the OM ap-pears smooth, the IM is highly folded on the inside to form cristastructures that maximize the surface area, which contributes toefficient oxidative phosphorylation. Transmission electron micros-copy (TEM) has provided numerous insights into the morpholog-ical changes of mitochondrial membrane structures with a spatialresolution of 1 to 2 nm. However, for the preparation of TEMspecimen, chemical fixation, dehydration, and embedding processesmay potentially introduce artifacts into the intact mitochondrialstructure. More importantly, the fixation stops all dynamic bi-ological processes, resulting in a loss of temporal information.In this context, superresolution nanoscopy techniques have
become increasingly important as tools to visualize spatiotemporaldynamics of cellular structures, such as the mitochondrial mem-brane (3–5), the nuclear pore complex (6, 7), and the cytoskele-tons (8, 9), on the nanoscale in living cell. Among varioussuperresolution methods, stimulated emission depletion (STED)microscopy is arguably the most prevalent for live-cell imaging asit offers the highest spatiotemporal resolution (10). However, the
poor photostability of current mitochondrial fluorescent markerstoward the intense laser irradiation used for excitation and de-pletion has been the principal technical limitation for observingmitochondrial membrane dynamics using STED microscopy.To solve this issue, we have recently proposed a structural re-
inforcement strategy of electron-deficient π-conjugated molecularskeletons, which significantly improves the photostability of fluo-rophores (11). Based on this strategy, superphotostable phosphole-based fluorescent molecules have been developed, and these allowmultiple acquisition of STED images under physiological condi-tions (12). As the next generation of such superphotostable fluo-rescence tools, we designed MitoPB Yellow (Fig. 1A), whichenabled us to visually distinguish nanoscale cristae structures inmitochondrial IM in living cells, and to perform the long-termtime-lapse STED imaging of the cristae dynamics.
ResultsMolecular Design of a Superphotostable Fluorescent MitochondrialProbe. Our design strategy for the generation of superphotostableMitoPB Yellow is based on a robust π-conjugated skeleton that
Significance
Stimulated emission depletion (STED) microscopy is one of themost appealing tools to visualize nanoscale cellular structuresand dynamics in living cells. However, its practical utility is sig-nificantly limited by the rapid photobleaching of fluorescent dyesunder the ultrastrong depletion laser. In this context, super-photostable fluorescent probes, which enable repeated recordingof the STED images, are crucial chemical tools to extend the ap-plication of STED microscopy in live-cell imaging. Herein, we re-port a developed fluorescent probe, MitoPB Yellow, which has thecharacters of outstanding photostability, long fluorescence life-time, and mitochondrial inner membrane selectivity for staining.In combination with a time-gated-STED microscopy, the mito-chondrial inner-membrane structures and dynamics can be im-pressively visualized in living cells.
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/analytictools; 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.
Published under the PNAS license.1To whom correspondence may be addressed. Email: [email protected], [email protected], or [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1905924116/-/DCSupplemental.
Published online July 23, 2019.
www.pnas.org/cgi/doi/10.1073/pnas.1905924116 PNAS | August 6, 2019 | vol. 116 | no. 32 | 15817–15822
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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 skeletonand an electron-donating triphenylamine moiety, exhibits sufficientphotoresistance to allow successive STED scans (11). While this dyeshowed significantly decreased fluorescence quantum yields (ΦF) inaqueous media due to its intramolecular charge transfer (ICT)character in the excited state, intense fluorescence was revivedin nonpolar environments. We envisioned that such ICT-basedfluorophores would emit bright fluorescence with a comparativelylong fluorescence lifetime when incorporated into a lipid bilayer,while its fluorescence intensity would be diminished in aqueousenvironments, such as the mitochondrial matrix and cytosol,resulting in the possibility to observe the membrane morphologywith high contrast. To turn the naphthophosphole P-oxide scaffoldinto a mitochondrial fluorescent marker, we employed a naphtha-lene-1,2-diyl–fused core skeleton, since this regioisomer should ex-hibit longer absorption and emission maxima compared with itsnaphthalene-2,3-diyl counterpart (12). Moreover, the lipophilic cat-ionic triphenylphosphonium group and a highly reactive epoxidegroup were introduced to the naphthophosphole scaffold to achieveselective and stable labeling of the mitochondrial membrane. Oncethe dye is accumulated in the mitochondrial membrane, the prox-imal membrane proteins would be covalently labeled with the dye(13), enabling the visualization of the mitochondrial morphologieseven 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 envi-ronmental polarity on the photophysical properties of the coreskeleton (Fig. 1A). Irrespective of the solvent, 1 exhibited anabsorption maximum at 465 to 480 nm with a molar absorptioncoefficient of ∼1.5 × 104 M−1·cm−1, whereas the maximumemission wavelength (λem) and the fluorescence quantum yield(ΦF) depend strongly on the solvent polarity (Fig. 1B and SIAppendix, 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 increasingsolvent polarity (λem = 643 nm in acetonitrile). In PBS (pH =7.4), 2 showed a further bathochromic shift of the emissionwavelength (λem = 667 nm) and a decreased quantum yield(ΦF = 0.13). Based on the polarity sensitivity, MitoPB Yellow issupposed to have a fluorogenic character upon localizing themitochondrial membrane.The decreased ΦF in water is mainly due to the acceleration of
the nonradiative decay processes from the lowest excited singletstate (S1). The fluorescence lifetime (τ = 7.3 ns) of 1 in toluenewas 3× longer than that (τ = 2.4 ns) of 2 in water. The radiative(kr) and nonradiative (knr) decay rate constants from S1 weredetermined based on the τ and ΦF values (SI Appendix, TableS1). The knr value of 2 in water (3.6 × 108 s−1) is substantiallylarger than that (0.10 × 108 s−1) of 1 in toluene, while their krvalues vary to a lesser extent dependent on the solvent polarity.
Mitochondrial Imaging with MitoPB Yellow. The performance ofMitoPB Yellow as a mitochondrial fluorescent marker was eval-uated by conducting live-cell imaging experiments with various celllines (HeLa, HepG2, MCF-7, HaCaT, and NMuMG). Each cellwas incubated with 500 nM of MitoPB Yellow for 2 h, followed bystaining with the commercially available mitochondria markerMitoTracker Deep Red FM. Irrespective of the cell types, theimages thus obtained showed high colocalization of both dyes withPearson’s correlation coefficient values of 0.86 to 0.93 (SI Ap-pendix, Fig. S2). In a concentration range of 50 nM to 1.5 μM,which did not significantly affect the cell viability based on theMTT assay (SI Appendix, Fig. S3), MitoPB Yellow could effi-ciently stain the mitochondria. The background fluorescence in-tensity in the cytoplasm was quite low, even without washingprocess in the live‐cell imaging (SI Appendix, Figs. S4 and S5).These results demonstrate that MitoPB Yellow exhibits sufficientmembrane penetration ability and mitochondrial selectivity.To demonstrate the superior utility of MitoPB Yellow over
other representative mitochondrial fluorescent markers, theperformance of MitoPB Yellow was compared with that ofMitoTracker Green FM (MTG), MitoTracker Red CMX(MTR), MitoTracker Deep Red FM (MTDR), and Rhodamine123 (Rh123). First, HeLa cells were stained with each dye un-der appropriate conditions and then fixed with 4% para-formaldehyde (15 min) followed by washing with a PBS buffer.In the imaging with MitoPB Yellow, more than 70% of thefluorescence signal was retained after fixation; conversely, theremaining fluorescence signal was much weaker when usingMTG (24%), MTR (29%), MTDR (37%), and Rh123 (17%) (SIAppendix, Fig. S6). The MitoPB Yellow signal was sufficientlystrong to see mitochondrial morphology remained even afterpermeabilization with Triton X-100. Indeed, multicolor staining,which needs the permeabilization process, was successfully ac-complished by fixation and permeabilization of MitoPB Yellow-labeled cells, followed by immunolabeling of α-tubulin withAlexa-Fluor-680–conjugated IgG, and staining the nucleus withHoechst 33342 (SI Appendix, Fig. S7). Similar results were alsoobserved when the dye-stained cells were treated with CCCP(2 μM), which is a proton ionophore that dissipates the mem-brane potential across the IM. The fluorescent intensity of theimage with MitoPB Yellow showed the highest retention ratio(92% persistence) among these mitochondria markers (SI Ap-pendix, Fig. S8). These results would reflect the covalent link-age of MitoPB Yellow to the mitochondrial proteins via itsepoxide moiety.Moreover, the photostability of MitoPB Yellow in mitochon-
dria was evaluated. Under the same excitation conditions (λex =488 nm), Rh123 and MTG suffered from rapid photobleachingduring the repeated recording of confocal images, while MitoPBYellow demonstrated unsurpassed photostability. Even after the
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Fig. 1. Properties of phosphole dyes. (A) Chemical structures of MitoPBYellow and its simplified (1) and water-soluble (2) derivatives. (B) Normalizedemission spectra of 1 in organic solvents and 2 in a PBS buffer (pH = 7.4). Thedotted line indicates the depletion wavelength used in this study. (C)Comparison of the photostability of live HeLa cells labeled with MitoPBYellow and cells stained with Rh123 or MTG. Confocal images were recordedunder the same acquisition conditions (λex = 488 nm). Signal intensities ofeach image (I) relative to the initial value (I0) are plotted as a function of therecorded number. (D) Comparison of the fluorescence lifetime of MitoPBYellow with Rh123 and MTG in live HeLa cells. Signal intensities (I) detectedin each delay time (tg) are normalized to the initial value (I0) and plotted as afunction of tg. The fluorescence lifetime (τ) of each dye in mitochondriawere determined by a single-exponential-decay fitting.
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acquisition of 50 images, no significant decrease in fluorescenceintensity was observed for MitoPB Yellow (Fig. 1C and SI Ap-pendix, Fig. S10).
Photophysical Properties of MitoPB Yellow in Mitochondria. Spectralimaging provided valuable information on the local environ-mental polarity of MitoPB Yellow in mitochondria. The emis-sion spectrum observed for the MitoPB Yellow-labeled-HeLacells was comparable to that measured in toluene (SI Appendix,Fig. S1C). This clearly indicates that in mitochondria, the dye issurrounded by a quite hydrophobic microenvironment, such asthe lipid bilayer and/or hydrophobic pockets of proteins. Thefluorescence lifetime (τ) of MitoPB Yellow in living HeLa cellswas determined using picosecond-pulsed laser excitation andgated detection (14). After HeLa cells were stained with MitoPBYellow, fluorescence images of the same area were recorded atvarious gate delay times (tg), which are the delays between theexcitation pulse and the start of the detection window for fluo-rescence collection, while taking a constant gate width (Δt = 3.5ns) (SI Appendix, Fig. S11). The sums of the fluorescence signalintensity for the collected area were plotted as a function of thetg value and fitted by a single exponential decay function (Fig.1D). The thus-calculated fluorescence lifetime of MitoPB Yel-low in mitochondria (τ = 7.5 ns) is comparable to that obtainedin toluene (τ = 7.3 ns) and substantially longer than those ofRh123 (τ = 3.3 ns) and MTG (τ = 0.75 ns) in mitochondria (Fig.1D). Notably, the longer τ of MitoPB Yellow should be an im-portant advantage to gain images with higher contrast in thetime-gated mode or fluorescence lifetime imaging microscopy,as most of the short-lived background signals, including cellautofluorescence and scattered excitation laser light, can beeffectively eliminated.
Live-Cell STED Microscopy Using MitoPB Yellow. The long fluores-cence lifetime of MitoPB Yellow and its remarkable photo-stability suggested its promising utility for STED microscopy (15,16). Therefore, we examined MitoPB Yellow-stained cells with agated continuous wave (CW) STED microscope that wasequipped with a pulsed white-light laser (WLL) for excitationand a CW laser (660 nm) for depletion. Initially, we recordedconfocal images of the mitochondria under excitation at 488 nm,before we applied the maximum power of the STED beam (270 mWat the back aperture of the objective lens) for detection using adelay time (tg) of 3 ns (Fig. 2A). The live-STED image clearlyrevealed a ladder-like pattern of the mitochondrial cristae thatcould not be seen in the confocal image, exhibiting a full widthat half maximum (FWHM) of 60 nm. The contrast of image wasfurther improved by removing the haze and the noise in the rawimage data by deconvolution with a theoretical point-spreadfunction (PSF) using the commercial program package Huygens(Fig. 2B). The final image was sufficiently clear to accurately countthe number of individual cristae from the line profiles of the in-tensity (Fig. 2C).The properties of MitoPB Yellow in STED microscopy were
further evaluated by changing the acquisition conditions. For asingle, well-separated crista in the relatively large mitochondria,the FWHM could be measured even when using the confocalmicroscopy, affording dc = 243 ± 31 nm (n = 10). Upon in-creasing the STED beam power (PSTED) at 660 nm, the resolu-tion increased and the FWHM of the crista image decreased to91 ± 12 nm (n = 10) when a depletion power of PSTED = 270 mWwas applied (SI Appendix, Fig. S12). The average FWHMs, cal-culated from the image data, were plotted as a function of PSTED,and the data were fitted using the theoretical resolution formulafor STED microscopy (Fig. 2D) (17), from which the effectivesaturation power Psat required for the half-depletion of MitoPBYellow 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 agreementwith the theory for the gated CW STED (16), the resolution formitochondrial membrane increased with decreasing fluorescenceintensity upon increasing the gated time tg (SI Appendix, Fig.S13). The FWHM value was saturated at ∼58 ± 6 nm at tg = 3 nsand PSTED = 270 mW (Fig. 2E). It should be noted here that theFWHM values reported above would be larger than the opticalresolution (FWHM of PSF), since the size of crista is not neg-ligible compared with the optical resolution. To give a moreaccurate estimate of resolution, we used the nested-loop en-semble PSF fitting method (18). The estimated resolution was45 ± 5 nm under the best imaging conditions (Fig. 2E). Impor-tantly, cristae could not be visualized in the STED microscopywhen using MTG and Rh123, due to their poor photostabilityand/or low membrane selectivity, clearly demonstrating the su-periority of MitoPB Yellow over these conventional dyes (SIAppendix, 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, OMand IM were counterstained using mitochondrial proteinsTOMM20 and OPA1, respectively. Their localization in mito-chondria is well established, and the full-length TOMM20 pro-tein and the N-terminal mitochondrial targeting signal (1-123 aa)of OPA1 (mtsOPA1) were used (19). HaloTag was used to label
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Fig. 2. STED imaging of mitochondria of living HeLa cells stained withMitoPB Yellow (λex = 488 nm; λSTED = 660 nm). (A) Comparison of scanningimages of mitochondria for the confocal (Left) and STED (Right; tg = 3 ns;PSTED = 270 mW) recording. (Scale bar, 2 μm.) (B) STED images of mito-chondria before and after deconvolution. (Scale bar, 1 μm.) (C) Intensityprofile 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 STEDmicroscopy. (E) FWHM as a function of delay time tg. Solid lines show fits ofthe data to the equation for gated CW STED with τ = 7.5 ns for PSTED = 108mW (open square) and PSTED = 270 mW (filled square).
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these mitochondrial marker proteins with tetramethylrhodamine(TMR), because the large difference in Stokes shift betweenMitoPB Yellow and TMR [Δ(1/λ) = 3,416 cm−1, SI Appendix,Fig. S15] is beneficial to gaining good separation in multicolorSTED imaging (20). To minimize cross-talks between the dyes,laser wavelengths of 470 and 540 nm were chosen for the exci-tation of MitoPB Yellow and TMR, respectively. Two-colorSTED images were acquired via a line-by-line sequential scan-ning of 2 channels with one detector (λem = 550 to 650 nm;detector gate time = 3.0 to 7.5 ns) to suppress the positionaldeviation of 2 images caused by rapid mitochondrial movementin living cells. In the comparison of fluorescent signals of TMRlabeled to the OM and IM (TOMM20-TMR and mtsOPA1-TMR, respectively) with MitoPB Yellow, TOMM20-TMR sig-nal (Fig. 3B) always surrounded the MitoPB Yellow signal (Fig.3A). The line profiles across the mitochondrial tubule section inthe overlay image (Fig. 3C) indicated that the fluorescence ofMitoPB Yellow appears inside the TMR-labeled OM (Fig. 3D).The FWHMs of the structures in the 2-color STED image weredetermined to be 68 ± 3 nm (n = 10) and 77 ± 4 nm (n = 10) forMitoPB Yellow and TMR, respectively (SI Appendix, Fig. S16).Contrastingly, mtsOPA1-TMR showed good colocalization withMitoPB Yellow (SI Appendix, Fig. S17C). These results not onlyconfirm that MitoPB Yellow signals come from the IM ratherthan OM, intermembrane space, or matrix, but also demonstratethe utility of MitoPB Yellow in 2-color STED live imaging as amitochondrial IM-selective fluorescent marker.
Visualization of Mitochondrial Cristae Remodeling. To demonstratethe practical utility of MitoPB Yellow for live-cell imaging, wemonitored the various changes of the mitochondrial morphology,which have been so far studied using TEM. Stress derived fromnutrient deprivation is known to induce an increase in fusion
activity, resulting in mitochondrial elongation. A TEM analysisrevealed an increased number of cristae in the hyperfused mi-tochondria (21). It has been proposed that the elongationof mitochondria might increase the efficiency of the adenosine5′-triphosphate (APT) synthesis and protect mitochondria fromautophagosomal degradation to enhance cellular survival dur-ing starvation (22). However, a method to visualize the ultra-structure of hyperfused mitochondria in living samples remainselusive. Therefore, we employed MitoPB Yellow to this end.For that purpose, HeLa cells were stained with MitoPB Yel-
low, starved in Hanks’ balanced salt solution (HBSS), and STEDimages were acquired. After the deconvolution process, thedensities of cristae in the hyperfused mitochondria were assessedby counting the number of cristae per unit of length. Comparedwith the cells cultured in Dulbecco’s modified Eagle’s medium(DMEM), the cells that were starved for 3 h showed elongatedmitochondria, in which the cristae were sparsely arranged (Fig. 4A, Top and Middle, and SI Appendix, Fig. S18). Conversely, after12 h of starvation, the formation of significantly dense cristae wasobserved in much thinner tubular mitochondria (Fig. 4 A, Bot-tom). The average density of cristae almost remained unchangedfor the 3-h-starved cells, whereas that of the 12-h-starved cellswas 1.5× higher than that for the untreated cells (Fig. 4B), whichis consistent with a previous study that reports an ∼1.8× higherdensity for mouse embryonic fibroblasts observed by TEM (21).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), andsubsequently stained with MitoPB Yellow before images wererecorded (Fig. 4C and SI Appendix, Fig. S19). As previouslyobserved by TEM (24), concentrically remodeled cristae, whichhave never been observed in untreated cells, were clearly visiblein some living cells using STED microscopy. We also succeededin gaining images of live cells treated with etoposide, which in-duces the mitochondrial apoptotic event by disrupting mito-chondrial membranes (25). In this experiment, the mitochondriawere labeled with MitoPB Yellow before etoposide treatment.As MitoPB Yellow remains in the membrane even after the lossof the mitochondrial membrane potential thanks to the immo-bilization at its epoxide functional group, ultrastructural mor-phological changes of the inner membrane can be recorded inthe live cells undergoing apoptosis. In fact, we observed fewercristae and partial vesicular morphologies in swollen mitochon-dria as well as circumference of fragmented mitochondria, whichwere previously identified using TEM (26) (SI Appendix, Fig.S20). Such etoposide-induced morphological changes in mito-chondria were prevented when the cells were treated with thegeneral caspase inhibitor Z-VAD-fmk (27), indicating that theimaging results are unlikely to be artifacts of MitoPB Yellow (SIAppendix, Fig. S20). These are observations of cristae remodel-ing in living cells.
Time-Lapse STED Imaging of Mitochondria. One of the crucial ad-vantages of superresolution microscopy over TEM is that it en-ables one to visualize the structural details as they happen in realtime. In particular, the time-lapse STED imaging should providemorphological and temporal information of organelle dynamics;however, currently available fluorescent organelle markers usu-ally suffer from photobleaching. The superphotostable MitoPBYellow circumvented this obstacle and enabled recording >1,000frames of the IM dynamics in the time-lapse sequence whenimages were recorded at a frame rate of 1.54 fps (frames persecond) (Movie S1). From these image sequences, we havesuccessfully observed a rapid intercristae mergence (<2 s) in asingle mitochondrion (Fig. 5A and Movie S2) and the inter-mitochondrial fusion (Fig. 5B and Movie S3). These imagesstrongly demonstrate the potential of live-cell STED imaging
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Fig. 3. Two-color STED image: (A) mitochondrial inner membrane labeledwith MitoPB Yellow (λex = 470 nm; green); (B) outer membrane labeled withTOMM20-TMR (λex = 540 nm; magenta); (C) merged image; (D) signal in-tensity 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).]
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with MitoPB Yellow. However, during a long-term observationover 90 s, unexpected morphological changes of the mitochon-dria started to occur, even though the fluorescence intensityremained unchanged (Fig. 5C). The mitochondria began to swellwith the loss of cristae, and in some cases, the membraneeventually ruptured (Movie S4) (28). It should be noted thatphotobleaching might not be the source of this damage to mi-tochondria, because the fluorescence signals from MitoPB Yel-low remained unchanged. Instead, the extremely strong STEDlaser might have damaged the mitochondria. Indeed, similarmorphological changes were also observed under the irradiationonly with the STED laser at 660 nm, i.e., in the absence of theexcitation at 488 nm, although MitoPB Yellow does not absorbthe STED beam (SI Appendix, Fig. S21). It is well known thatmitochondria are easily damaged during fluorescence live im-aging. When mitochondria are stained with conventional dyes,the photodamage is usually accompanied by photobleaching ofthe dyes. Therefore, radical formation during photobleachingof the mitochondria-staining dyes has been surmised as thesource of the photodamage. Our results with photoresistantMitoPB Yellow has separated the photodamage from photo-bleaching, and it enabled us to visualize the photoinduceddamaging process of mitochondria at high spatial resolution.
DiscussionIn this study, we developed a molecular tool that enables imagingof the mitochondrial ultrastructure in living cells. The fluorescentmarker MitoPB Yellow was designed to overcome the 2 principaldrawbacks of conventional dyes, i.e., photostability and mito-chondrial membrane selectivity, which have limited the use ofSTED microscopy for the investigation of the mitochondrialmorphology. Firstly, the insufficient photostability of classicalfluorescent dyes has been the most crucial problem for their ap-plication in superresolution microscopy, which requires intenselaser irradiation. The rapid photobleaching of conventional fluo-rescent dyes usually hampers the repeated acquisition of images ata superresolution level. To address this issue, we employed a ro-bust π-conjugated skeleton containing an electron-withdrawingP = O moiety, which attained significantly higher photostability.Secondly, to gain a fluorogenic response to the mitochondrialmembrane, an electron-donating group was directly conjugated to
the fluorophore skeleton. The donor–π–acceptor skeleton givesrise to an environment polarity-sensitive character of fluorescence.The thus-obtained large difference in ΦF of MitoPB Yellow be-tween polar and nonpolar environments results in enhancedfluorescence contrast of the membrane structure in mitochondrialimaging. The exceptionally high photostability of MitoPB Yellowallows using the STED laser power at the maximum intensitypossible in commercially available microscopes. In combinationwith its membrane-selective staining characteristics, MitoPB Yel-low can thus provide hitherto unprecedented resolution in themitochondrial IM imaging in living cells.The resolution in the STED microscopy (d) is approximately
proportional to the inverse square root of the saturation factorISTED/Isat. Here, ISTED is the STED laser power, while Isat is theeffective saturation intensity, at which the population of theexcited electron is depleted by half. Therefore, dyes with a lowerIsat are beneficial to increasing the resolution with a givenmaximum STED laser power. Isat for MitoPB Yellow is esti-mated to be 1.3 MW/cm2 at 660 nm on the assumption of thetypical doughnut area (A ∼ 3 × 10−9 cm2) and the losses of theSTED laser power in the objective lens (decreased by ∼12%)(29). In other words, MitoPB Yellow is efficiently depleted at themaximum CW STED laser power of the commercial STEDmicroscope (∼10 MW/cm2). In general, the lower Isat values areattributable to the larger stimulated emission cross-section(σSTED) and/or the longer fluorescent lifetime (τ), because theIsat value is inversely proportional to these parameters by thefollowing formula: Isat = hc/(λSTED σSTED τ), where h and c referto the Plank constant and the speed of light, respectively. TheσSTED is calculated as 3.23 × 10−17 cm2 by using the values for thelifetime τ = 7.5 ns and the depletion wavelength λSTED = 660 nm.This σSTED value is in good agreement with the value theoretically
A C
B
Fig. 5. (A–C) Time-lapse STED imaging of mitochondrial dynamics inMitoPB 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.)
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Fig. 4. Morphological changes of the mitochondrial inner membrane cap-tured by STED microscopy (λex = 488 nm; λSTED = 660 nm). (A) DeconvolutedSTED images showing changes in the mitochondrial morphology underconcomitant change of the cristae density upon nutrition starvation for 3and 12 h (HBSS containing Ca2+ and Mg2+). (Scale bar, 2 μm.) (B) Comparisonof the number of cristae per micrometer of mitochondrial length before andafter incubation for 3 and 12 h under starvation conditions (n = 20). (C) STEDimage of cristae in HeLa cells, pretreated with 10 μM mitochondrial DNAreplication inhibitor (ddC) for 5 d followed by staining with MitoPB Yellow.(Scale bar, 2 μm.)
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derived from the emission spectrum of MitoPB Yellow in PMMA(σSTED = 3.39 × 10−17 cm2; SI Appendix, Fig. S1D). As this valuefalls in a common range for many fluorophores (σSTED = 10−16 to10−18 cm2) (30), the major contribution to the lower Isat forMitoPB Yellow should be the longer fluorescent lifetime (τ = 7.5ns in mitochondria).The development of MitoPB Yellow allowed us to examine a
number of aspects regarding the IM structure and dynamics inmitochondria. Most importantly, our microscopic analysesrevealed the dynamic changes of mitochondrial ultrastructure inliving cells. In particular, we observed a drastic crista develop-ment following mitochondrial hyperfusion upon starvation. Wealso confirmed a thinning of mitochondrial tubules with increaseof cristae density under starvation conditions, which was pre-viously observed by TEM analyses (Fig. 4A) (31), demonstratingthe great utility of our live-cell STED imaging. Thus, MitoPBYellow would be an invaluable molecular tool for further bi-ological studies such as the mechanism how the morphologicalchanges are accomplished in the IM.Time-lapse imaging may be able to capture a fusion event
between distinct cristae protruding from opposing sides of themitochondrion, although we cannot rule out the possibility thattwo opposing cristae happen to come close each other withoutfusion (Fig. 5A). However, previous electron microscopy (EM)studies strongly suggest such fusion events to occur betweendistinct cristae or between a crista and the inner boundarymembrane (IBM). Specifically, multiple connections between asingle crista and the IBM as well as cristae connecting to bothopposing sides of the IBMs are frequently observed by EManalyses (32).In summary, MitoPB Yellow surpasses currently used fluo-
rescent mitochondrial markers in terms of photostability, fluo-rescence lifetime, and signal-to-noise ratio for membrane-staining,which enables STED imaging of the mitochondrial inner-membranestructures in living cells at unprecedented resolution (<60 nm).
Moreover, the large Stokes shift of this probe, combined with an-other small-Stokes-shift dye, allows multicolor STED imaging witha single STED laser. The molecular design strategy based on thissuperphotostable fluorophore should thus be applicable to theSTED imaging of membrane dynamics in different cellular organelle,and fluorescent markers thus developed should also be expected tofind a wide range of applications in other superresolution techniques.
Materials and MethodsFor chemical synthesis of probe MitoPB Yellow, spectral measurements, cellculture, mitochondrial staining, and confocal imaging, see SI Appendix.
STED Imaging. The cells were stained in DMEM+ containing 500 nM MitoPBYellow and 0.5% dimethyl sulfoxide for 2 h in a CO2 incubator. Then, the cellswere washed 3 time with fresh medium to remove the free dye, and kept inDMEM+ for imaging. The Leica TCS SP8 STED 3× system with 2 CW lasers at592 and 660 nm and a pulsed laser at 775 nm for the depletion was used forSTED imaging. An HyD detector and an STED WHITE objective (100×/1.40 OILor 93×/1.30 GLYC) were employed. Unless otherwise noted, the STED imageswere acquired with excitation at 488 nm (WLL), emission in the range of 500 to640 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 averageof 3, and a pinhole size of 151.63 μm. The images were processed usingImageJ. In some cases, the STED images were deconvoluted with a theoreticalPSF using a commercial program package Huygens.
ACKNOWLEDGMENTS. We thank the following people for their technicalassistance or sharing materials: A. Fukazawa (Kyoto University) for discussingthe 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, MichaelDavidson and David Chan for the plasmids (Addgene 54282 and 26047), andJunko Asada for her technical assistance including plasmid construction andcell culture. This work was supported by JST PRESTO JPMJPR16F5 (M.T.) andJST CREST JPMJCR15G2 (Y.O.), as well as JSPS KAKENHI Grants 16H05119(Y.O.), 17H19511 (Y.O.), 18K14721 (H.Y.), and JP16H06280 (AdvancedBioimaging Support).
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