  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 tradeoff between imaging speed and
sensitivity is problematic for acquiring blur-free images of fast phenomena,
such as sub-millisecond biochemical dynamics in live cells and tissues, and
cells flowing at high speed. We report a solution that achieves real-time pixel
readout rates one order of magnitude faster than a modern electron multiplier
charge coupled device (EMCCD) - the gold standard in high-speed fluorescence
imaging technology. Deemed fluorescence imaging using
radiofrequency-multiplexed excitation (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, as well as fluorescence microscopy
in flow at a throughput of approximately 50,000 cells per second.
