Whole-cell, 3D and multi-color STED imaging with exchangeable

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Whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores Christoph Spahn, Jonathan B. Grimm, Luke D. Lavis, Marko Lampe, and Mike Heilemann Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b04385 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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Whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores Christoph Spahn1, Jonathan B. Grimm2, Luke D. Lavis2, Marko Lampe3,*, Mike Heilemann1,* 1

Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7,

60438 Frankfurt, Germany 2

Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia

20147, USA 3

Advanced Light Microscopy Facility, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117

Heidelberg, Germany

Correspondence: [email protected], [email protected] Lead contact: [email protected]

Abstract We demonstrate STED microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multi-color and live cell STED microscopy. Key words: exchange-based STED microscopy, PAINT, fluorogenic labels, multicolor imaging, volumetric imaging, live-cell STED microscopy Main Fluorophore bleaching is a fundamental challenge in super-resolution STED microscopy. Minimizing photobleaching comes with more fluorophores that contribute to signal-to-noise and thus resolution. This is particularly beneficial for super-resolution imaging, especially in live-cell and 3D imaging. In the context of STED microscopy 1, 2, this challenge was addressed, for example, by developing optical schemes that dynamically tune the excitation light during image acquisition 3, by developing photostable fluorophores (reviewed in Zheng & Lavis 4) or by using fluorescent proteins with multiple off-states as in protected STED 5. Finding solutions to bypass fluorophore bleaching can also advance STED microscopy towards whole-cell imaging, since bleaching of out-of-focus fluorophores is reduced and a large-volume imaging can be achieved. Another important application of reduced photobleaching is long-term live cell STED imaging, as shown recently for STED imaging of extracellular space of living brain slices 6. 1 / 11

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In single-molecule localization microscopy 7, photobleaching can be bypassed using fluorophores that reversibly bind to their target structure, a concept that originally was realized in Point Accumulation for Imaging in Nanoscale Topography (PAINT) 8. We repurposed this idea for STED microscopy by using fluorophores that reversibly bind to and dynamically exchange from their target structure (Figure 1a). This procedure efficiently replaces fluorophores that eventually photobleached during imaging by new and intact fluorophores. This concept requires fast exchange kinetics of the fluorophores and a sufficiently large reservoir of unbleached (intact) fluorophores. It must also ensure high-density labeling of the target structure. We first investigated whether fluorogenic PAINT fluorophores can in principle be used for STED imaging, using the membrane stain Nile Red 8 and the DNA stain JF646-Hoechst 9. We demonstrate subdiffraction spatial resolution STED imaging with these exchangeable fluorophores in E. coli cells (Figure 1b). In contrast to single-molecule PAINT imaging, where a low labeling density ensures the detection of single emitters, our concept requires higher concentrations of fluorophores in the buffer (~100 nM – 1 µM) in order to ensure a constantly high labeling density despite the exchange dynamics. Next, we investigated whether a constant and dynamic exchange of fluorophores occurs, which should enable multiple rounds of STED imaging of the same cell without any loss of fluorescence signal. For this purpose, we again chose E. coli cells as cellular test system for imaging, because of their small size and regular shape that facilitates quantitative intensity analysis. We recorded a sequence of STED images of the bacterial cell envelope using either the exchangeable label Nile Red, or the stationary label Alexa-Fluor 594-conjugated wheat germ agglutinin (WGA-AF594). The quantification of the fluorescence intensity over multiple imaging frames showed photobleaching for the stationary label, WGA-AF594, and a constant fluorescence signal for the exchangeable label, Nile Red (Figure 1b). We next recorded a sequence of STED images of the bacterial nucleoid, using either the exchangeable label JF646-Hoechst, or the same fluorophore, JF646, as stationary label targeting DNA through click labeling of EdU-treated cells

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Again, we observed photobleaching for the stationary label and a constant

fluorescence signal for the exchangeable label at moderate illumination intensities (Figure 1b). Under similar imaging conditions, photobleaching was also observed for ATTO647N (Figure S1), a fluorophore that is sufficiently photostable to enable single-molecule STED imaging 11. The labeling density observed for the stationary and the exchangeable labels appeared very similar, which demonstrates efficient high-density labeling. Compared to single-molecule PAINT imaging12, higher concentrations of these exchangeable labels in the imaging buffer were used. However, we did not observe a significant increase the background signal in STED experiments, which we attribute to the fluorogenic properties of Nile Red 13 and JF646-Hoechst 9.

We next demonstrated super-resolution imaging of the bacterial nucleoid and envelope in two-color, 2D-STED (Figure 1c), as well as in 2-color, 3D-STED mode (Figure 1d; Supplementary Video 1). Cells 2 / 11


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were labeled with both exchangeable fluorophores, Nile Red and JF646-Hoechst, simultaneously. The 3D STED images clearly reveal the radial positioning of DNA (red hot) in proximity of the bacterial plasma membrane (green) (Figure 1d), potentially mediated by coupling of transcription, translation and membrane insertion (transertion) of proteins

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We note that Nile Red and JF646-Hoechst allowed

recording STED images using the same depletion laser (emitting at 775 nm), which simplifies the optical setup and image acquisition. Another advantage of these two exchangeable fluorophores is that no specific cellular treatment is needed (compared to e.g. EdU incorporation for high-density labeling of DNA), further simplifying sample preparation and avoiding undesired stress for cells. We next applied the exchangeable labels Nile Red and JF646-Hoechst to STED imaging of eukaryotic cells (Figure 2). Hereby, the constant label-exchange allows a higher degree of optimization of imaging parameters for single-color high-resolution STED imaging in fixed specimen. This is limited if static labels are used, due to irreversible photobleaching affecting any STED image recorded with high irradiation intensities. By optimizing imaging parameters for the individual exchangeable labels, we were able to visualize nano-scale structural details of mitochondria (Nile Red, Figure 2a, Figure S2) and mitotic chromosomes (JF646-Hoechst, Figure 2b, Figure S3). Similar results were obtained with the large Stokes-shift membrane dye FM4-64 (Fig. S4) and the DNA stain SiR-Hoechst (Fig. S5)

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While

mitochondrial cristae membranes were previously investigated using electron microscopy (EM) and provided a high level of structural information 16, they have to our knowledge not been resolved to a comparable level as shown in this work using fluorescence microscopy. We observed a spacing between cristae membrane structures of about 100 – 140 nm (Figures 2a and S4) and a width of ~ 80 nm for cristae membranes (Figure S4) in good agreement with the mitochondrial morphology described in EM studies 17. We note that with the obtained spatial resolution, we can not reveal whether the observed structures are single cristae or cristae stacks. As another example for optimized single-color STED in fixed cells, we visualized the mitotic chromosome in fixed HeLa cells using the exchangeable label JF646-Hoechst, followed by deconvolution of the resulting STED image (Figure 2e, Figure S6). The images show a grained chromosome structure (Figure 2e, i) and small DNA loops (Figure 2e, ii and iii). These key features of mitotic chromosomes are also known from EM studies 18, but were just recently visualized by fluorescence microscopy using lattice light sheet-PAINT microscopy 9. Next to transiently binding membrane- and DNA-targeted PAINT probes, short peptides were used for super-resolution imaging in image reconstruction by integrating exchangeable single-molecule localization (IRIS) Here, the Lifeact peptide

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exhibits the fastest binding kinetics, with a binding time of 23 ms 19. We

demonstrated that an Alexa-Fluor 594 (AF594) conjugated Lifeact peptide also is applicable to exchange-based STED imaging. Indeed, the actin cytoskeleton could be super-resolved by applying Lifeact-AF594 at comparably high concentration (1 µM, Figure 2c). Similar to membrane- and DNAtargeted PAINT probes, the constant exchange of molecules provided a stable signal over 50 imaging frames when using imaging conditions providing high-resolution STED images (Figure S6). Consequently, the application of Lifeact at high concentrations also allowed for whole-cell STED 3 / 11


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imaging (Figure S7). In single-molecule based PAINT imaging, whole cell imaging is only achievable when using techniques that provide optical sectioning, such as lattice-light-sheet microscopy or by focusing on the basolateral membrane using TIRF-microscopy 9, 19. To test the limits of exchange-based STED imaging, we performed time-lapse STED imaging at a high imaging rate of conventional STED microscopes (~1 frame/s) with varying times of light exposure (1/3, 2/3 and full frame duration) (Figure S8). Longer light exposure should lead to increased bleaching rates, which manifests in lower equilibrium intensities. This effect is observed for all tested labels and can be used to evaluate the label performance in exchange-based STED imaging. Here, Nile Red and JF646Hoechst showed higher equilibrium intensities compared to FM4-64 and SiR-Hoechst. The high equilibrium intensity provided by Lifeact indicates the highest exchange rate of all labels tested. Based on our experiments, we estimate that fluorophore labels with affinities in the lower µM range (lifeact, Kd = 2.2 µM

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SiR-Hoechst, Kd = 8.4 µM 9) are suitable candidates for exchange-based STED

microscopy. Next, we demonstrate two-color STED microscopy with sub-diffraction spatial resolution in eukaryotic cells (Figure 2d). The STED images of the Nile Red channel show structural features of various membrane-surrounded cellular organelles, e.g. lipid droplets, mitochondria and the nuclear lamina, which were elusive in the respective CLSM image (Figure 2d). JF646-Hoechst can also be combined with Lifeact-AF594 to perform dual-color STED using the same depletion laser (λdep = 775 nm) as shown for an interphase (Figure 2e) and mitotic cell (Figure S10). A key advantage of exchange-based STED imaging is that a large number of images at different axial positions can be recorded, without compromising the fluorescence signal in out-of-focus imaging planes by the excitation or depletion laser. To demonstrate this, we recorded whole-cell two-color 3D STED images using a 3D optical depletion scheme in both XZ (Figure 2f, Supplementary Videos 2 and 3) and XY scanning mode (Figure S9), with a constant fluorescence signal over time. Without dynamic fluorophore exchange, the imaging conditions need to consider bleaching as one critical parameter, which requires a compromise in resolution or signal-to-noise ratio. Bypassing fluorophore bleaching would also enable long-term observation of living cells and following the dynamics of cellular features in an (almost) quantitative manner, avoiding misinterpretations arising from photobleached labels. Here, we demonstrate time-lapse STED imaging of a living cell labeled with Nile Red (Figure 2g i; Supplementary Video 4) and followed organelle dynamics while displaying no detectable change in average fluorescent intensity (Figure S11). Using a commercial STED microscope and restricting imaging to smaller field of views allowed STED imaging at a frame rate of 0.4 - 0.8 Hz (Supplementary Videos 5 and 6). Since Nile Red can be effectively depleted using far-red depletion lasers (here λdep = 775 nm), it can be combined with other membrane permeable labels, e.g. live-cell compatible SiR-conjugates 21. We recorded dual-color STED videos of lysosomes (SiR-lysosome) and membrane-labeled organelles and followed their dynamics in living cells (Figure 2g ii, Supplementary 4 / 11


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Video 7). In addition, we visualized the tight association of mitochondria and microtubules using a tubulin-binding SiR-conjugate together with Nile Red in live cells using STED microscopy (Figure 2g iii, Supplementary Video 8). While the fluorescence signal from SiR-tubulin decreased with time, the fluorescence signal from Nile Red remained constant over the whole acquisition (up to 100 min) (Figure S12, Supplementary Video 9). Membrane- and DNA-targeting exchangeable labels can also be combined for 2-color live-cell STED imaging (Supplementary Video 10). An important consideration in live cell microscopy are toxicity of the fluorophore label itself and phototoxic effects induced by using the fluorophore label with a particular microscopy technique. To assess phototoxicity during livecell STED imaging, we used the exchangeable label Nile Red and recorded 25 whole-cell STED images using imaging conditions as in previous live-cell experiments. We followed the cells over at least 24h (Supplementary Video 11), and observed a small increase in cell division defects (multinuclear cells) in STED-imaged cells in comparison to adjacent control cells (Figure S13). The high survival and cell division rate indicates that Nile Red, at the concentrations used in this work, has no obvious long-term cytotoxic effects. In conclusion, we present a simple and powerful experimental protocol for STED microscopy with reduced photobleaching using exchangeable fluorophore labels that transiently bind to their target. We demonstrate that exchangeable labels can achieve high labeling density, circumvent fluorophore bleaching and allow for whole-cell, multi-color and live cell STED imaging. This concept differs from approaches which reduce photobleaching of permanently bound labels by populating additional molecular states 5, yet we envision synergies from combining both strategies. The concept of exchangeable labels for STED microscopy can also be extended to other labels that reversibly bind to their target structure. This might also foster the development of novel suitable labels or lead to the rediscovery of existing labels, and it may profit from fluorophore delivery methods for living cells 22, 23. Finally, this concept opens new opportunities for correlated super-resolution light and electron microscopy 24, 25.

Acknowledgment We thank the Advanced Light Microscopy Facility (ALMF) at the European Molecular Biology Laboratory (EMBL), Leica Microsystems and Abberior Instruments for support. We thank Petra Freund, George Galea and Juan Jung for help with cell culture and sample preparation, and Hans-Dieter Barth for help in optics questions. MH and CS acknowledge funding by the German Science Foundation (SFB 902 and SFB 1177). Data availability Data is available upon request from the authors. Competing interest statement The authors declare no competing financial and non-financial interests. 5 / 11


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Supporting Information (1)

Experimental Methods and Supplementary Figures 1-13 (PDF)

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Information on Supplementary Videos 1-11 (SI_Guide_Supplementary_Videos) (PDF)

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List of applied experimental and image processing parameters for measurements contributing to Maintext Figures 1 and 2, Supplementary Figures 1-13 and Supplementary Videos 1-11. (Supplementary_Table_1) (Excel spreadsheet)

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Supplementary Video 1 (QuickTime)

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(10) Supplementary Video 7 (QuickTime) (11) Supplementary Video 8 (QuickTime) (12) Supplementary Video 9 (QuickTime) (13) Supplementary Video 10 (QuickTime) (14) Supplementary Video 11 (QuickTime)

References 1. Blom, H.; Widengren, J. Chem Rev 2017, 117, (11), 7377-7427. 2. Vicidomini, G.; Bianchini, P.; Diaspro, A. Nat Methods 2018, 15, (3), 173-182. 3. Heine, J.; Reuss, M.; Harke, B.; D'Este, E.; Sahl, S. J.; Hell, S. W. Proc Natl Acad Sci U S A 2017, 114, (37), 9797-9802. 4. Zheng, Q.; Lavis, L. D. Curr Opin Chem Biol 2017, 39, 32-38. 5. Danzl, J. G.; Sidenstein, S. C.; Gregor, C.; Urban, N. T.; Ilgen, P.; Jakobs, S.; Hell, S. W. Nature Photonics 2016, 10, 122. 6. Tonnesen, J.; Inavalli, V.; Nagerl, U. V. Cell 2018, 172, (5), 1108-1121 e15. 7. Sauer, M.; Heilemann, M. Chem Rev 2017, 117, (11), 7478-7509. 8. Sharonov, A.; Hochstrasser, R. M. Proc Natl Acad Sci U S A 2006, 103, (50), 18911-6. 9. Legant, W. R.; Shao, L.; Grimm, J. B.; Brown, T. A.; Milkie, D. E.; Avants, B. B.; Lavis, L. D.; Betzig, E. Nat Methods 2016, 13, (4), 359-65. 10. Spahn, C.; Endesfelder, U.; Heilemann, M. J Struct Biol 2014, 185, (3), 243-9. 11. Kasper, R.; Harke, B.; Forthmann, C.; Tinnefeld, P.; Hell, S. W.; Sauer, M. Small 2010, 6, (13), 1379-84. 12. Spahn, C. K.; Glaesmann, M.; Grimm, J. B.; Ayala, A. X.; Lavis, L. D.; Heilemann, M. Sci Rep 2018, 8, (1), 14768. 13. Greenspan, P.; Fowler, S. D. J Lipid Res 1985, 26, (7), 781-9. 14. Bakshi, S.; Choi, H.; Weisshaar, J. C. Front Microbiol 2015, 6, 636. 15. Lukinavicius, G.; Blaukopf, C.; Pershagen, E.; Schena, A.; Reymond, L.; Derivery, E.; GonzalezGaitan, M.; D'Este, E.; Hell, S. W.; Gerlich, D. W.; Johnsson, K. Nat Commun 2015, 6, 8497. 16. Zick, M.; Rabl, R.; Reichert, A. S. Biochim Biophys Acta 2009, 1793, (1), 5-19. 17. Ikon, N.; Ryan, R. O. Biochim Biophys Acta 2017, 1859, (6), 1156-1163. 18. Marsden, M. P.; Laemmli, U. K. Cell 1979, 17, (4), 849-58. 6 / 11


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19. Kiuchi, T.; Higuchi, M.; Takamura, A.; Maruoka, M.; Watanabe, N. Nat Methods 2015, 12, (8), 743-6. 20. Riedl, J.; Crevenna, A. H.; Kessenbrock, K.; Yu, J. H.; Neukirchen, D.; Bista, M.; Bradke, F.; Jenne, D.; Holak, T. A.; Werb, Z.; Sixt, M.; Wedlich-Soldner, R. Nat Methods 2008, 5, (7), 605-7. 21. Lukinavicius, G.; Reymond, L.; D'Este, E.; Masharina, A.; Gottfert, F.; Ta, H.; Guther, A.; Fournier, M.; Rizzo, S.; Waldmann, H.; Blaukopf, C.; Sommer, C.; Gerlich, D. W.; Arndt, H. D.; Hell, S. W.; Johnsson, K. Nat Methods 2014, 11, (7), 731-3. 22. Kollmannsperger, A.; Sharei, A.; Raulf, A.; Heilemann, M.; Langer, R.; Jensen, K. F.; Wieneke, R.; Tampe, R. Nat Commun 2016, 7, 10372. 23. Wieneke, R.; Raulf, A.; Kollmannsperger, A.; Heilemann, M.; Tampe, R. Angew Chem Int Ed Engl 2015, 54, (35), 10216-9. 24. Perkovic, M.; Kunz, M.; Endesfelder, U.; Bunse, S.; Wigge, C.; Yu, Z.; Hodirnau, V. V.; Scheffer, M. P.; Seybert, A.; Malkusch, S.; Schuman, E. M.; Heilemann, M.; Frangakis, A. S. J Struct Biol 2014, 186, (2), 205-13. 25. de Souza, N. Nature Methods 2014, 12, 37.

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Figure 1: STED microscopy with exchanging probes. (a) Fluorophores that dynamically bind and unbind to their target structure ensure a constant exchange with a quasi-infinite pool, compensating for irreversible photobleaching and allowing for long-term STED imaging. (b) Multi-frame STED microscopy comparing stationary and reversibly binding labels targeting the E. coli membrane or nucleoid. Cells were labeled using either a static (left panel) or dynamic labeling approach (right panel). Static labeling was achieved using click-chemistry (DNA, red hot) or sugar-binding lectins (cell wall, green). A high concentration of the exchangeable labels JF646-Hoechst and Nile Red were applied for dynamic labeling of DNA (red hot) and E. coli membranes (green), respectively. Two different sets of settings were applied (red circles: low intensity: 19 nm pixel size, 50% depletion laser power, black circles: high intensity: 11 nm pixel size, maximum depletion laser power; varying excitation laser powers for the different labels, see Supplementary Table 1). Fluorescence intensities were extracted for each imaging frame (data points represent mean values of at least 3 cells and error bars the respective standard deviation) and fitted with a mono-exponential decay or linear fit function. (c) Parallel twocolor STED of E. coli DNA (red hot) and membrane (green) using one depletion wavelength at 775 nm. Both cross-sections of DNA filaments (1) and membrane (2) illustrate the resolution enhancement provided by 2D STED. (d) Two-color 3D STED imaging with near isotropic resolution. The cell shown in the XY-overview image was reconstructed from the XZ-scan. Dashed lines indicate the position of

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XZ cross-sections shown in the right image panel (the full stack is provided as Supplementary Video 1). Scale bars are 1 µm.

Figure 2: Single-, Dual-color 2D and 3D STED imaging in fixed whole cells, and live cell STED imaging using reversibly binding dyes. (a) STED image of mitochondria in a fixed HeLa cell, visualized using Nile Red. (i) Average image (raw images) of 4 imaging planes, corresponding to a 240 nm thick section. (ii) Magnified ROIs from (i), showing mitochondrial cristae. (iii) Normalized intensity profile of the line indicated in (ii). (b) High-resolution, deconvolved STED image of mitotic chromosomes in a fixed HeLa cell, visualized with JF646-Hoechst (i). (ii) Magnified region from (i) showing a small chromatin loop. (iii) Normalized intensity profile of the line indicated in (ii). (c) STED image of the actin cytoskeleton in a fixed HeLa cell, visualized using Lifeact-AF594. (ii) Magnified 9 / 11


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region from (i) showing a region with a dense actin network. (iii) Normalized intensity profile of the line indicated in (ii). (d) 2-color 2D STED image of a fixed HeLa cell (i). DNA (red hot) is labeled with JF646-Hoechst and cellular membranes with Nile Red (green). Various membranous structures can be resolved with Nile Red, such as lipid droplets (ii), vesicular structures (iii), the nuclear lamina (iv) and mitochondria (v). Super-resolution imaging of nano-scale DNA features is achieved with the exchangeable fluorophore JF646-Hoechst (vi). (e) 3D-STED imaging of fixed HeLa cells labeled for membranes (green) and DNA (red), achieving near isotropic resolution (the comparison to the CLSM image stack is shown in Supplementary Video 2). The image stack was recorded in XZ-scanning mode. (f) 2-color 2D STED image of a fixed HeLa cell labeled for actin (lifeact-AF594, cyan hot) and DNA (JF646-Hoechst, red) (i). (ii) Magnified CLSM (left) and STED image (right) of the region indicated in (i). (g) Live-cell STED imaging with long acquisition times of HeLa cells stained with Nile Red (i), Nile Red and SiR-lysosome (ii) or Nile Red and SiR-tubulin (iii). Arrows in (iii) indicate association (yellow) or dissociation (cyan) of mitochondria and microtubules. Scale bars are 5 µm (d i, e i, f), 3 µm (a i, c i), 1 µm (a ii, b i, c ii, dii-vi, e ii, g i-iii) and 250 nm (b ii).

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