Fig. 1

Characterization of the lysosomal probes in live cells. a A co-localization study employing LysoTracker Green as the standard lysosomal marker. Live U2OS cells were simultaneously stained with Lysosome-565 (red) and LysoTracker Green (green, 50 nM) for 30 min and imaged by confocal microscopy. b Dual-color SIM images of lysosomes in live U2OS cells stained with both Lysosome-647 (magenta) and LysoTracker Red (red, 50 nM). c Enlarged time-lapse images from the boxed regions shown in b. d SIM images of live U2OS cells after incubation with Lysosome-488 (blue) or Lysosome-565 (red). e Enlarged super-resolution SIM (upper panel) and diffraction-limited (lower panel) images from the boxed region shown in d. f Average signal (lysosomes) to background (cytosol) ratios (SBR) of LysoTracker Red (SBR from n = 24 areas), Lysosome-488 (SBR from n = 41 areas), Lysosome-565 (SBR from n = 40 areas), and Lysosome-647 (SBR from n = 58 areas) in three SIM images (mean ± s.d.; ^** P < 0.01, ^*** P < 0.0001, two-tailed t-test; statistics were performed using SPSS 19.0 software package (IBM Co.)). g Time-lapse images illustrating the process of lysosomal fission-fusion. Each SIM frame was acquired over 270 ms (i.e., a raw data exposure time of 30 ms). For the time-lapse images, consecutive SIM frames spaced at 6-s intervals were obtained; representative images of consecutive SIM frames are displayed (more frames are shown in Supplementary Movie 1). Scale bars, a 5 μm, b, d 4 μm, and c, e, g 1 μm