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 sensitivity1 are problematic for acquiring blur-free images of fast phenomena, such as sub-millisecond biochemical dynamics in live cells and tissues2, and cells flowing at high speed3. 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 technology4. 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.

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Figure 1: Implementation of FIRE microscopy.
Figure 2: Illustration of the radiofrequency tagging of fluorescent emission in FIRE.
Figure 3: Comparison of FIRE microscopy and wide-field fluorescence imaging.
Figure 4: High-speed imaging flow cytometry.


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The authors thank D. Di Carlo (UCLA) for use of his laboratory's cell culture facilities. The authors acknowledge the Broad Stem Cell Research Center at UCLA 2012 Innovation Award for financial support. The authors also thank L. Bentolila for assistance with the EMCCD imaging, which was performed at the California NanoSystems Institute Advanced Light Microscopy/Spectroscopy Shared Facility at UCLA.

Author information

E.D.D. conceived of the beat frequency multiplexing approach, built the FIRE microscope, and collected the data. B.W.B. conceived of and implemented the demodulation algorithms, generated the phase engineered excitation frequency combs, and performed image processing. D.R.G. cultured and stained the biological samples, and fabricated microfluidic channels. B.J. conceived of the use of DDS and other communication techniques for FIRE, and supervised the project. E.D.D. wrote the first draft of the manuscript, and all authors contributed to subsequent revisions.

Correspondence to Eric D. Diebold.

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The authors declare no competing financial interests.

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Diebold, E., Buckley, B., Gossett, D. et al. Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy. Nature Photon 7, 806–810 (2013).

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