Fig. 4: Time-lapse volumetric live-cell super-resolution imaging with 4Pi-SIM.

a, 4Pi-SIM image (maximum intensity projection) of the mitochondrial outer membrane (TOM20-oxStayGold and oxStayGold-OMP25) in a live HeLa cell (Supplementary Video 9). b, An oblique view (–7 degrees along the z axis) of the boxed region denoted ‘b’ in a. The x–z and y–z cross-sections were along the dashed color lines in a. c, Time-lapse magnified views of the boxed region denoted ‘c’ in a, showing that a mitochondrion transitioned from rod-shaped to concave-disc-shaped. Right at each time point, corresponding x–z and y–z cross-sections along the colored dashed line. d, Left: time-lapse magnified views of the boxed region denoted ‘d’ in a, showing the mitochondria generating nanotunnels from both ends (dashed outline). Right: corresponding x–z cross-sections at positions labeled with colored arrowheads. e, 4Pi-SIM image (maximum intensity projection) of the microtubules (oxStayGold-Ensconsin) in a live COS-7 cell (Supplementary Video 10). f, Left: 3D rendering of the boxed region labeled ‘f’ in e. Right: time-lapse x–y and y–z cross-sections (along the microtubule denoted by the arrowheads), showing the growth of a microtubule whose tip is labeled with arrowheads. g, 4Pi-SIM image (maximum intensity projection) of the actin filaments (Lifeact-mStayGold) in a live COS-7 cell (Supplementary Video 11). h, Time-lapse magnified views of the boxed region labeled ‘h’ in g, showing that actin puncta structures that formed (colored arrowheads) and disappeared (dashed circles). i, Top: time-lapse magnified views of the boxed region labeled ‘i’ in g, showing that the bundle of actin filaments in the filopodia is redistributed and spread. Bottom: the x–z cross-sections along the dashed line. j, Left: time-lapse magnified views of the boxed region denoted ‘j’ in g, showing the retraction and merging of the actin filaments. Right: the y–z cross-sections at positions denoted by the arrowheads. Colors represent depth, denoted by the color bars. Scale bars: 2 μm (a,b,e,g); 1 μm (c,f,h,i); 1 μm (left) and 500 nm (right) (d,j). The thickness of all cross-section slices is 60 nm in f and 30 nm in all other panels. Representative results from four to six independent experiments are shown.