Fig. 4.

Time-lapse, 2D and 3D SMLM of lysosomes in live HeLa cells using PFF-1. a Time-lapse SMLM of lysosomes in a live HeLa cell applying a single photoactivation pulse (405 nm, 2.6 W cm⁻², 20 ms). The green, dotted square in the bright-field image indicates the area imaged during the SMLM experiment. The bottom left corner of the image at 0 s displays the diffraction-limited image for comparison. b Time-lapse SMLM of lysosomes in a live HeLa cell applying photoactivation pulses (405 nm, 2.6 W cm⁻², 20 ms) every 10 min. The green, dotted square in the bright-field image indicates the area imaged during the SMLM experiment. The bottom left corner of the image at 0 s displays the diffraction-limited image for comparison. c Number of emissive molecules per frame over the entire acquisition employing probe PFF-1 (500 nM) with an initial, single-photoactivation pulse (405 nm, 2.6 W cm⁻², 20 ms, red triangles) or with the same pulse applied every 10 min (blue squares). d Single lysosome moving laterally within the focal plane in a live HeLa cell. e Bright-field image of a live HeLa cell. The green, dotted square indicates the area displayed in panel (f). f Snapshot at 0 s of a 3D, time-lapse, SMLM experiment, color-coded according to depth in a 1 μm-thick imaging volume. The magenta, solid-line square indicates the area displayed in panel (g). g Snapshots at different time points of a single lysosome diffusing in the axial direction, color-coded according to the scale depicted in panel (f). Scale bars from panels (a, b) 10 μm (bright-field) and 1 μm (diffraction-limited and SMLM), respectively; (d) 500 nm; (e) 10 μm; (f) 5 μm; (g) 200 nm