Fig. 1. mSTED structure identification. (a) Schematic diagram of fluorescence lifetime multiplexing, where spectral multiplexing contains only a limited number of spectral channels, whereas lifetime multiplexing by phasor analysis allows simultaneous observation of all the different structures. (b) Fluorescent probe selection strategy, four live-cell STED probes were selected at 560 nm and 640 nm excitation wavelength each and the difference in fluorescence lifetimes between them was measured. (c) Comparison of mSTED with confocal and STED, the greyscale image represents the intensity map obtained by Confocal and STED imaging, while the colored image demonstrates that mSTED can identify mitochondria and microtubules after phase analysis. The phasor points are projected onto the fitted line and further classified, ultimately leading to the segmentation of the intensity image. Live U2OS cells were labeled with Atto 647N (magenta; mitochondria) and Tubulin Deep Red (cyan; microtubules). The two structures were simultaneously excited by a beam at a wavelength of 640 nm. The clusters indicated by magenta and cyan arrows in the phasor plot correspond to mitochondria and microtubules, respectively. (d–g) Comparison of mSTED imaging versus confocal imaging of segmented mitochondria and microtubules, corresponding to the regions marked by the white boxes in (c). (h, i) Line profiles along the dotted lines in (f, g) which show that STED is able to separate two microtubules in close proximity, revealing details unobservable by confocal images. The presence of sharp peaks in the profile suggests that the mSTED image exhibits a resolution of 60 nm. (j–l) Identification of DNA and microtubules using mSTED in the 560-nm excitation channel. Live U2OS cells were labeled with DNA Live 560 (yellow; DNA) and Tubulin Live 560 (cyan; microtubules). Scale bars: (c) 2 μm; (d, e) 500 nm; (j) 5 μm.

Fig. 2. Photo-bleaching and photo-cytotoxicity of mSTED. (a) Traditional dual-color STED imaging of mitochondria and microtubules in live-cell, labeled with Tubulin Deep Red (red; microtubules) and Mito Tracker Red (yellow; mitochondria) and excited alternately with two beams at wavelengths of 640 nm and 561 nm. (b) Dual-color mSTED imaging of mitochondria and microtubules in live-cell, labeled with Tubulin Deep Red (red; microtubules) and Mito Tracker Red (yellow; mitochondria) and excited simultaneously with a beam at wavelength 640 nm. For the time-lapse imaging, the acquire time was 10 μs for each single dual-color image in traditional STED and 10 μs for dual-color mSTED, and the interval time between each image was set as 14 s. Representative frames were displayed. (c) magnified views of the white-boxed regions in (a, b). (d) Photo-bleaching curves of the same probe Tubulin Deep Red under dual-color STED and mSTED imaging (mean value ± standard deviation). Scale bars: 2 μm.

Fig. 3. Multi-color live-cell mSTED using a single excitation beam. (a) Three-color imaging result of mSTED. Live U2OS cells were labeled with DNA Live 560 (blue; DNA), Tubulin Live 560 (green; microtubules), and PK Mito Orange (orange; mitochondria). (b, c) Cellular activity in the regions marked by the white boxes in (a). (d, e) Live U2OS cells were labeled with Tubulin Deep Red (cyan; microtubules) and Atto 647N (magenta; mitochondria) and imaged with mSTED (d) before and (e) after the addition of nocodazole. Scale bars: (a, d, e) 5 μm; (b, c) 2 μm.

Fig. 4. Extending mSTED through both spectra and fluorescence lifetime property separation. (a–c) Five-color live-cell mSTED imaging of live U2OS cells stained with DNA Live 560 (magenta; DNA), ER Tracker Red (green; Endoplasmic reticulum), Atto 647N (yellow; mitochondria), Tubulin Deep Red (white; microtubules), and Actin Live 610 (cyan; F-actin). (d–f) Four-color live-cell imaging results, Images of fluorescent probes excited at (b, e) 640-nm and (c, f) 560-nm laser wavelength. (g–i) Cellular activity in the boxed regions in (d) over a 38-minute period, with (h) and (i) demonstrating the interaction of multiple structures at different time points. Scale bars: (a–g) 5 μm; (h, i) 2 μm.