Ultrafast PALM reveals the shape changes, migrations, and formation/disappearance of caveolae in 3 s in live cells. (A and B) Caveolin-1-mEos3.2 expressed in the basal PM of T24 cells at a density of 42 ± 5.2 caveolin-1-mEos3.2 molecules/caveola (n = 15; see the legend of Fig. 2 B) was imaged in both live (A) and fixed cells (B), using identical ultrafast PALM imaging conditions. Data acquisitions were performed at a rate of 1 kHz for 3 s (3,000 frames), and PALM images were reconstructed using the data acquired for every 1 s (= 1,000 frames; b) and every 0.33 s (= 333 frames; c). (a) Diffraction-limited and PALM images of caveolae in 10 × 10-µm2 observation areas, using a data acquisition period of 1 s. For the spatial resolution of these images, see Materials and methods. (b) Enlarged images of the purple-square regions in (a) showing time-dependent changes (every 1 s). The images in the middle column (top and middle rows) are the expanded images of the purple-square regions in a, for the data acquisition between 1 and 2 s. The regions surrounded by yellow squares in the middle row are magnified in the bottom row, and the localizations of single mEos3.2 molecules determined within each 1-s period are indicated by red dots. (c) The PALM image reconstruction performed every 0.33 s for the same caveola that is shown in the bottom row in b.
