Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy

TL;DR

This paper introduces FIRE, a radiofrequency-multiplexed fluorescence imaging technique that significantly accelerates imaging speed, enabling real-time, high-throughput microscopy of live cells and tissues with sub-millisecond dynamics.

Contribution

The authors develop a novel fluorescence imaging method using digitally synthesized optical fields to achieve unprecedented real-time pixel readout rates, surpassing traditional EMCCD technology.

Findings

Achieved 4.4 kHz frame rate for stationary cell imaging.

Enabled flow cytometry at approximately 50,000 cells per second.

Demonstrated diffraction-limited confocal fluorescence imaging.

Abstract

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.