Development of ultrafast camera-based imaging of single fluorescent molecules and live-cell PALM

The spatial resolution of fluorescence microscopy has recently been greatly improved. However, its temporal resolution has not been improved much, despite its importance for examining living cells. Here, by developing an ultrafast camera system, we achieved the world’s highest time resolutions for single fluorescent-molecule imaging of 33 (100) µs (multiple single molecules simultaneously) with a single-molecule localization precision of 34 (20) nm for Cy3 (best dye found), and for PALM data acquisition of a view-field of 640×640 pixels at 1 kHz with a single-molecule localization precision of 29 nm for mEos3.2. Both are considered the ultimate rates with available probes. This camera system (1) successfully detected fast hop diffusion of membrane molecules in the plasma membrane, detectable previously only by using less preferable 40-nm gold probes and bright-field microscopy, and (2) enabled PALM imaging of the entire live cell, while revealing meso-scale dynamics and structures, caveolae and paxillin islands in the focal adhesion, proving its usefulness for cell biology research. Summary An ultrafast camera developed by Fujiwara et al. allows single fluorescent-molecule imaging every 33 μs with a localization precision of 34 nm (every 100 μs; 20 nm), and enables ultrafast PALM imaging of whole live cells. ### Competing Interest Statement S.T. and Y.N. are employees of Photron Limited, a manufacturer of high-speed digital cameras for industrial and scientific applications. T.K. is an employee of Carl Zeiss Microscopy GmbH, a manufacturer of microscope systems for life sciences and materials research. Authors T.K.F., Z.K., T.A.T., K.I., K.P.R., K.G.N.S., and A.K. declare that they have no competing interests.