Series of multiple confocal or STED images at different pinhole size were acquired using the frame-sequential acquisition17 (link). The values of pinhole size were set as specified. The excitation power was kept constant unless specified otherwise. The number of line averaging was kept constant unless specified otherwise.
Confocal images were acquired on a Leica TCS SP8 confocal microscope, using an HCX PL APO CS2 63 × 1.40 NA oil immersion objective lens (Leica Microsystems, Mannheim, Germany). Tetraspeck fluorescent spheres with a size of 200 nm (TetraSpeck Fluorescent Microspheres Size Kit, ThermoFisher) were excited at 488 nm and their fluorescence emission was detected at 500–550 nm. TO-PRO-3 was excited at 633 nm and its fluorescence emission was detected at 640–700 nm using a hybrid detector (Leica Microsystems). CellMask Orange was excited at 561 nm and its fluorescence emission was detected at 565–650 nm using a hybrid detector (Leica Microsystems).
STED images were acquired on a Leica Stellaris 8 Tau-STED microscope, using an HC PL APO CS2 100 × 1.40 NA oil immersion objective lens (Leica Microsystems, Mannheim, Germany). Stimulated emission depletion was accomplished with a 775 nm STED laser. A white light laser provided excitation at the desired wavelength for each sample. Excitation wavelengths/emission bandwidths were the following: Atto647N (646, 651-720). 1024 × 1024 pixel images were acquired with a pixel size of 19 nm.
Confocal images were acquired on a Leica TCS SP8 confocal microscope, using an HCX PL APO CS2 63 × 1.40 NA oil immersion objective lens (Leica Microsystems, Mannheim, Germany). Tetraspeck fluorescent spheres with a size of 200 nm (TetraSpeck Fluorescent Microspheres Size Kit, ThermoFisher) were excited at 488 nm and their fluorescence emission was detected at 500–550 nm. TO-PRO-3 was excited at 633 nm and its fluorescence emission was detected at 640–700 nm using a hybrid detector (Leica Microsystems). CellMask Orange was excited at 561 nm and its fluorescence emission was detected at 565–650 nm using a hybrid detector (Leica Microsystems).
STED images were acquired on a Leica Stellaris 8 Tau-STED microscope, using an HC PL APO CS2 100 × 1.40 NA oil immersion objective lens (Leica Microsystems, Mannheim, Germany). Stimulated emission depletion was accomplished with a 775 nm STED laser. A white light laser provided excitation at the desired wavelength for each sample. Excitation wavelengths/emission bandwidths were the following: Atto647N (646, 651-720). 1024 × 1024 pixel images were acquired with a pixel size of 19 nm.
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