Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.
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We thank the following people for plasmids and reagents: F. Bottanelli, L. Schroeder, S. Baguley, Z. Xi and I. Kukic (Yale University School of Medicine), E.T. Spiliotis (Drexel University), M. Davidson (Florida State University), and C.G. Giraudo (Children's Hospital of Philadelphia). We thank J. Coleman and S.C. Alexander for advice and help with liposomes preparations. This work was supported by a Wellcome Trust Foundation grant (095927/A/11/Z, D.T., J.B., J.E.R.), the NIH (R01GM83257 to A.S., RO1GM118486 to D.T. and J.B., and P30 DK45735) and partly by grants AMED Brain/MINDS and JSPS Core-to-Core Program, A. Advanced Research Networks to Y.U., and by a MEXT KAKENHI grant number JP15H05951 to M.K. H.T. was supported by a JSPS postdoctoral fellowship for research abroad. R.S.E. was supported by an Advanced Postdoc Mobility fellowship from the Swiss National Science Foundation. A.D.T. was supported by the National Institutes of Health Ruth L. Kirschstein NRSA (F31GM119259). S.U. was supported by a JSPS postdoctoral fellowship.
J.B. discloses financial interest in Bruker Corp. and Hamamatsu Photonics. R.S.E., A.D.T., D.T. and A.S. disclose a pending patent for lipid probes.
Supplementary Figures 1–20, Supplementary Table 1 and Supplementary Data. (PDF 14429 kb)
Monte Carlo simulations support the hypothesis that combining a high density labeling with an ultra-low ON fraction improves apparent photostability.
Monte Carlo simulations were performed as described in Methods (section 2.3). The movies shown illustrate the time-lapse image expected using various labeling densities and ON fractions. (i) Images expected when labeling density = 10 (low) and ON fraction = 1% (low); (ii) Images expected when labeling density = 500 (high) and ON fraction = 1% (low); (iii) Images expected when labeling density is 10 (low) and ON fraction is 0.01% (ultra-low); (iv) Images expected when labeling density is 500 (high) and ON fraction is 0.01% (ultra-low). Images (i) and (iv) represent the parameters expected for HMSiR-conjugated proteins and lipids, respectively. Selected frames from these movies are shown in Supplementary Fig. 6. (MOV 4482 kb)
Live HeLa cells were labeled with Cer-TCO followed by HMSiR-Tz and imaged live on a custom 2D wide-field SMS microscope (see Fig. 2 c-f). The movie was recorded at 400 frames per second, reconstructed with a 2 s temporal resolution (800 frames per movie frame) and Kalman-filtered using default parameters. Laser intensity: 9.9 kW/cm2. Scale bar: 1 μm. (MOV 24600 kb)
The movie illustrates the area of interest labeled d in Fig. 2c; individual frames are shown as Fig. 2d. The yellow arrow identifies a loop growing out of a single ER tubule. The blue and pink arrows identify the fission and fusion of two separate ER tubules. The green arrow identifies the elongation of an ER tubule and fusion with a neighboring tubule. Scale bar: 500 nm. (MOV 16909 kb)
Five movies generated from the same raw data but with different temporal resolution. The number above each panel represents the temporal resolution (time per frame) of that movie. The movie with a 2.0 s temporal resolution corresponds to the single image shown as Fig. 2c; stills are shown in Supplementary Fig. 9. Scale bar: 1 μm. (MOV 46692 kb)
Direct comparison between long time-lapse SMS and diffraction-limited images of the ER in a live HeLa cell labeled with Cer-HMSiR.
Side-by-side comparison of ER dynamics observed under SMS and diffraction limited conditions. Scale bar: 1 μm. (MOV 10738 kb)
Direct comparison between SMS images obtained using HMSiR conjugated to a lipid (Cer-HMSiR) or a protein (Sec61β-HMSiR) within the ER in live HeLa cells.
Live HeLa cells were labeled with Cer-TCO and HMSiR-Tz or transfected Halo-Sec61β and labeled with HMSiR-CA, and subsequently imaged live on a custom 2D wide-field SMS microscope. The data from this movie was used to prepare the plot shown as Fig. 2g. Scale bar: 1 μm. (MOV 1689 kb)
Live HeLa cells were labeled with RhoB-TCO followed by HMSiR-Tz and imaged live on a custom 2D wide-field SMS microscope. The movie was recorded at 400 frames per second, reconstructed with a 2 s temporal resolution (800 frames per movie frame) and Kalman-filtered with default parameters. This movie corresponds to the time-colored image shown in Fig. 3c. Laser intensity: 4.4 kW/cm2. Scale bar: 1 μm. (MOV 12047 kb)
Live HeLa cells were labeled with DiI-TCO followed by HMSiR-Tz and imaged live on a custom 2D wide-field SMS microscope. A still image of an early movie frame is shown in Fig. 4b. Time-stamped snapshots from the movie are shown in Fig. 4d. The movie was recorded at 400 frames per second, reconstructed with a 2 s temporal resolution (800 frames per movie frame) and Kalman-filtered with default parameters. Snapshots from this movie are shown in Supplementary Fig. 16. Laser intensity: 9.9 kW/cm2. Scale bar: 1 μm. (MOV 8730 kb)
Live HeLa cells were labeled with DiI-C6-TCO followed by HMSiR-Tz and imaged live on a custom 2D wide-field SMS microscope. The movie was recorded at 400 frames per second, reconstructed with a 2 s temporal resolution (800 frames per movie frame) and Kalman-filtered with default parameters. Data from this movie was used to generate Fig. 4e. Snapshots from this movie are shown in Supplementary Fig. 16. Laser intensity: 9.9 kW/cm2. Scale bar: 1 μm. (MOV 23786 kb)
Live HeLa cells were labeled with Cer-TCO followed by HMSiR-Tz and imaged live on a custom 3D wide-field SMS microscope. Movie shows multiple 3D perspectives of the area of interest shown in the pink box in Fig. 6a and b; stills from this movie are shown as Fig. 6c and d. A cartoon schematic of this region is shown as Fig. 6e. Movie is depth-colored. Laser intensity 9.7 kW/cm2. Scale bar: 1 μm. (MOV 1830 kb)
Live HeLa cells were labeled with Cer-TCO followed by HMSiR-Tz and imaged live on a custom 3D wide-field SMS microscope. A depth-colored image of an early frame of this movie is shown as Fig. 6a. Laser intensity 9.7 kW/cm2. Scale bar: 1 μm. (MOV 20423 kb)
Supplementary Movie 11 rendered for observation with 3D glasses. (MOV 20504 kb)
Live HeLa cells were labeled with Cer-TCO followed by HMSiR-Tz and imaged live on a custom 3D wide-field SMS microscope. Pink arrow shows tubule elongation spanning different z-layers and fusion with a neighboring tubule. Images from this movie are shown as Fig. 6f. Movie is depth-colored. Laser intensity 9.7 kW/cm2. Scale bar: 4 μm. (MOV 8345 kb)
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Takakura, H., Zhang, Y., Erdmann, R. et al. Long time-lapse nanoscopy with spontaneously blinking membrane probes. Nat Biotechnol 35, 773–780 (2017). https://doi.org/10.1038/nbt.3876
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