All biological images were recorded on a custom-built setup (Supplementary Fig. 6) based on a commercial microscope stand (Axio Observer D1, Carl Zeiss MicroImaging) with a 100×/1.46 NA oil immersion objective (alpha Plan-Apochromat 100×/1.46 Oil, Zeiss). The setup is equipped with lasers emitting at 405 nm (CrystaLaser, 50 mW), 568 nm (Coherent Innova 300, ~400 mW) and 642 nm (MPB Communications, 500 mW). Fluorescence was recorded by our sCMOS camera through the side port of the stand. All data was recorded at room temperature.
Fixed microtubule structures were imaged in a 128 × 128 pixel ROI for 40,000 frames at 1,600 fps (Fig. 1g–k), a 512 × 512 pixel ROI for 16,000 frames at 400 fps (Fig. 2a,b) and a 64 × 64 pixel ROI for 30,000 frames at 3,200 fps (Fig. 2e–h). The 642 nm laser was used at intensities of 18.4 kW/cm2, 5.5 kW/cm2, and 7.4 kW/cm2, respectively. Images were acquired using HCimage software (Hamamatsu). Focal adhesions were recorded in a 256 × 256 pixel ROI for 2,400 frames at 800 fps with the 642 nm laser intensity set to 9.2 kW/cm2. Clathrin data was recorded in a 256 × 256 pixel ROI for 50,000 frames at 600 fps with the 568 nm laser intensity set to 5.3 kW/cm2. Mitochondria were recorded in a 256 × 256 pixel ROI for 80,000 frames at 400 fps with the 568 nm laser intensity set to 5.3 kW/cm2. EB3 data was recorded in a 256 × 256 pixel ROI for 30,000 frames at 600 fps with the 568 nm laser intensity set to 5.3 kW/cm2. Peroxisomes were recorded in a 256 × 256 pixel ROI for 50,000 frames at 600 fps with the 568 nm laser intensity set to 5.3 kW/cm2. Transferrin data was recorded in a 128 × 128 pixel ROI at 1,600 fps with the 642 nm laser intensity set to 7.4 kW/cm2. During imaging, the intensity of the 405 nm activation laser was manually increased from 0 to 0.3 W/cm2 (for 512 × 512 pixel ROI) and 0 to 1.8 W/cm2 (for 256 × 256, 128 × 128 and 64 × 64 pixel ROI) to ensure optimal particle densities for either single emitter fitting or multi-emitter fitting31 (link).