Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy

Fluorescence imaging is the most widely used method for unveiling the molecular composition of biological specimens. However, the weak optical emission of fluorescent probes and the trade-off between imaging speed and sensitivity(1) are problematic for acquiring blur-free images of fast phenomena, such as sub-millisecond biochemical dynamics in live cells and tissues(2), and cells flowing at high speed(3). Here, we report a technique that achieves real-time pixel readout rates that are one order of magnitude faster than a modern electron multiplier charge-coupled device-the gold standard in high-speed fluorescence imaging technology(4). Termed fluorescence imaging using radiofrequency-tagged emission (FIRE), this approach maps the image into the radiofrequency spectrum using the beating of digitally synthesized optical fields. We demonstrate diffraction-limited confocal fluorescence imaging of stationary cells at a frame rate of 4.4 kHz, and fluorescence microscopy in flow at a velocity of 1 m s(-1), corresponding to a throughput of approximately 50,000 cells per second.