Journal Article

Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy

Buckley, Brandon W., Jalali, Bahram, Gossett, Daniel R., Diebold, Eric D.

2013

Overview

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. A confocal fluorescence microscopy scheme that maps the image to the radiofrequency spectrum by beating together two optical fields offers enhanced read-out speeds at kilohertz frame rates. It provides a new way for observing dynamic phenomena in cells.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/328/1978 / 639/624/1107/328/1978 / Applied and Technical Physics / Beat frequencies / Confocal / Emission / Emissions / Fluorescence / Fluorescence microscopy / Fourier transforms / High speed / Imaging / letter / Microscopy / Optics / Photonics / Physics / Quantum Physics