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. 2017 Aug;35(8):773-780.
doi: 10.1038/nbt.3876. Epub 2017 Jul 3.

Long Time-Lapse Nanoscopy With Spontaneously Blinking Membrane Probes

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Long Time-Lapse Nanoscopy With Spontaneously Blinking Membrane Probes

Hideo Takakura et al. Nat Biotechnol. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1
Development of a high-density, environment-sensitive (HIDE) probe based on spontaneously blinking HMSiR for long time-lapse and 3D super-resolution imaging in living cells. (a) HMSiR dyes equilibrate between two forms, a closed, intramolecularly cyclized form, which is not emissive, and an open, uncyclized form that is fluorescent. The position of the HMSiR cyclization equilibrium depends on both pH and the polarity of the environment. (b) Cartoon of an in vitro experiment to assess whether the HMSiR ON/OFF fraction is affected by environment polarity. HMSiR was immobilized to a glass surface via either a protein (upon reaction of HMSiR-CA (Supplementary Fig. 1b) with Halo-GST) or a liposome (upon reaction of HMSiR-Tz (Supplementary Fig. 1b) with Cer-TCO). (c) Super-resolution images of HMSiR immobilized as described in (b). Rainbow colored temporal look-up table. Scale bar: 200 nm. Laser intensity: 17.6 kW/cm2. (d) Plot illustrating the normalized number of localizations observed as a function of time when HMSiR was immobilized to a protein, within an aqueous environment, and within a liposome, a hydrophobic environment. τ values were calculated from a single exponential fit (mean ± standard deviation (SD) of data from 9 fields of view (protein) or 40 individual liposomes.
Figure 2
Figure 2
Long time-lapse, wide-field SMS imaging of endoplasmic reticulum (ER) dynamics using the HIDE probe Cer-HMSiR generated upon reaction of Cer-TCO and HMSiR-Tz. (a) Schematic illustration of the 2-step procedure employed to label the ER with Cer-HMSiR. (b) Super-resolution image of the ER in HeLa cells showing time-colored localizations. A plot of the average fluorescent signal as a function of position along the 4 profiles shown as yellow lines is shown as an insert. The solid yellow line shows the signal in the super-resolution image, while the white dashed line shows the signal in the diffraction-limited image. These profiles are characterized by FWHM values (mean ± SD, n = 4) of 50 ± 3 nm (super-resolution image) and 367 ± 38 nm (diffraction-limited image). The elongation of an ER tubule later in the recording is indicated by a green arrow (top right corner). A kymograph (signal along the purple line profile as a function of time) is shown in the right side panel. The time-dependent appearance of new ER tubules is indicated by yellow arrowheads. Laser intensity: 9.9 kW/cm2. Scale bar: 1 μm. (c–f) Super-resolution imaging of the ER in a live HeLa cell over 25 min. (c) One snap shot (t = 258 s) from a 25-min movie. The image was reconstructed from 800 frames recorded over 2 s and Kalman-filtered. s: sheet-like ER, t: tubular ER. Laser intensity: 9.9 kW/cm2. Scale bar: 1 μm. The full 25 min movie is available as Supplementary Movie 2. (d, e) Time-lapse images of ER dynamics. Each image was reconstructed from 800 frames, recorded over 2 s and Kalman-filtered. The full movie (1296 s) is available as Supplementary Movie 3. (d) Small ER regions are observed to bud off (yellow arrowhead), loop back (green arrowhead), and fuse (magenta and blue arrowheads). (e) ER sheets can reversibly transition into tube-like structures (white arrowheads). Scale bar: 500 nm. (f) Kymograph (line profile versus time) of the purple line in (c). (g) Comparison of the number of localizations per μm2 observed between HMSiR localized to a lipid (via Cer-HMSiR) and to a protein (via Sec61β-HMSiR) that resides within the ER. (h) τ values calculated from a single exponential fit to the photobleaching curves in g (mean ± SD, n = 3).
Figure 3
Figure 3
Long time-lapse, wide-field SMS imaging of mitochondria using the HIDE probe RhoB-HMSiR generated upon reaction of RhoB-TCO and HMSiR-Tz. (a) Chemical structure of RhoB-TCO and schematic illustration of the 2-step procedure employed to label mitochondria with RhoB-HMSiR. (b,c) Comparison between time-colored super-resolution images of mitochondria labeled with the protein probe Halo-OMP25-HMSiR and the HIDE probe RhoB-HMSiR. SMS images of HeLa cells expressing (b) Halo-OMP25 and treated with HMSiR-CA or (c) treated with RhoB-TCO and HMSiR-Tz. The diffraction-limited image obtained from the RhoB channel is marked by the dashed white line in (c). Kymographs of both images, derived from the time-dependent signal along each brown line vs. time, are shown on the right side of each panel. Laser intensity: 4.4 kW/cm2. Scale bar: 1 μm. The full 9 min movie is available as Supplementary Movie 7. (d) A plot of the fluorescent signal in both the SMS and diffraction-limited image as a function of position along the yellow line in (c). The signal from the SMS image is shown in teal; the signal from the diffraction-limited image is shown in black.
Figure 4
Figure 4
Long time-lapse, wide-field SMS imaging of the plasma membrane using the HIDE probes DiI-HMSiR and DiI-C6-HMSiR generated upon reaction of HMSiR-Tz with either DiI-TCO or DiI-C6-TCO. (a) Chemical structures of DiI-TCO and DiI-C6-TCO. (b) Super-resolution image of the plasma membrane visualized with the HIDE probe DiI-HMSiR. Laser intensity: 9.9 kW/cm2. Scale bar: 1 μm. The image in the green box is magnified for clarity to illustrate the line profile quantified in (c). The image in the pink box is viewed at 5 time points in (d). (c) A plot of the fluorescent signal in both the SMS and diffraction-limited image as a function of position along white line within the green box in (b). The signal from the SMS image is shown in teal (fit to a Gaussian equation in orange with a FWHM of 36 nm); the signal from the diffraction-limited image is shown in black. Adjacent filopodia can be distinguished when separated by distances as small as 160 nm. (d) Time-lapse images of filopodia dynamics over the course of 96 s, with notable changes highlighted by yellow arrows. Scale bar: 1 μm. The diffraction-limited image is shown for comparison. The full 7 min movie is available as Supplementary Movie 8. (e) Comparison of the number of localizations per μm2 as a function of time when cells were visualized using either DiI-HMSiR, DiI-C6-HMSiR, or Smo-HMSiR. Scale bar: 1 μm. (f) τ values calculated from a single exponential fit to the photobleaching curves in (e) (mean ± SD, n = 3–4 cells). The full 20 min movie obtained using DiI-C6-HMSiR is available as Supplementary Movie 9.
Figure 5
Figure 5
Two color SMS imaging of the ER in live cells. (a) SMS images of the ER in HeLa cells expressing the two ER markers KDELR-mEos3.2 (green) and Halo-Sec61β and treated with HMSiR-CA (red). An enlarged view of the image within the yellow box is shown at right. (b, top) A plot of the average fluorescent signal as a function of position along the 6 profiles shown as pink lines. These profiles are characterized by FWHM values (mean ± S.D., n = 6) of 53 ± 6 and 63 ± 12 nm in the green and red channels, respectively; (b, bottom) A plot of the intensity in both channels along the tubule boxed in blue. The red and green arrows identify regions where each probe is enriched. Laser intensities: 2.90 kW/cm2 at 560 nm (KDELR-mEos3.2), 9.42 kW/cm2 at 642 nm (Sec61β-HMSiR). (c) SMS images of COP1 vesicles and mitochondria in HeLa cells expressing the markers COPβ1-mEos3.2 (green) and Halo-OMP25 and treated with HMSiR-CA (red). An enlarged view of the image within the yellow box is shown beneath. Laser intensities: 3.08 kW/cm2 at 560 nm (COPβ1-mEos3.2), 6.30 kW/cm2 at 642 nm (OMP25-HMSiR). (d) SMS images of the COP1 vesicles and the Golgi in HeLa cells expressing the markers COPβ1-mEos3.2 (green) and treated with Cer-TCO and HMSiR-Tz at 20 °C (red). An enlarged view of the image within the yellow box is shown beneath. Laser intensities: 3.08 kW/cm2 at 560 nm (COPβ1-mEos3.2), 6.30 kW/cm2 at 642 nm (Cer-HMSiR). (e) SMS images of COP1 vesicles and the Golgi and ER in HeLa cells expressing the marker COPβ1-mEos3.2 (green) and treated with Cer-TCO and HMSiR-Tz at 37 °C (red). An enlarged view of the image within the yellow box is shown beneath. Laser intensities: 3.08 kW/cm2 at 560 nm (COPβ1-mEos3.2), 6.30 kW/cm2 at 642 nm (Cer-HMSiR). All scale bars: 1 μm.
Figure 6
Figure 6
3D long time-lapse, wide-field imaging the ER with the HIDE probe Cer-HMSiR. (a) Depth-colored snapshot of a 15 min movie of the ER in HeLa cells treated with Cer-TCO and HMSiR-Tz. Laser intensity 9.7 kW/cm2. The full movie is available as Supplementary Movie 11, rendered for 3D glasses as Supplementary Movie 12. (b) Perspective view of the snapshot shown in (a). (c) Top view of the magenta cube shown in (b) highlighting tubules t1–t4. (d) Front view of the magenta cube shown in (b) highlighting tubules t1–t4 and their connections c1–c3. (e) Cartoon representation of the three dimensional arrangement of tubules shown in the magenta cube. A multi-perspective 3D zoom-in view of this region is shown in Supplementary Movie 10. (f) 3D time-lapse images of HeLa cells treated with Cer-TCO and HMSiR-Tz; the region in the orange box is shown at 4 different time points. The full movie is available as Supplementary Movie 13. (g) Snapshot of the nucleus-proximal region of the ER of a HeLa cell labeled with Cer-TCO and HMSiR-Tz. (h) y,z-cross section of the red box in (g). (i) Line profile of the area in the brown box in (h) with FWHM values of 167 and 118 nm for the ER-derived channel and the ER structure associated with the nucleus. All images/movies were recorded at 9.7 kW/cm2.

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