LEAD fluorescence microscopy performing at 0.8 million frames per second for 3D imaging flow cytometry

Abstract

Three-dimensional, fluorescence imaging methods with ~1 MHz frame rates are needed for high-speed 3D imaging flow cytometry and volumetric imaging of neuronal activity. The frame rates of current fluorescence imaging methods are limited to kHz by the photon budget, slow camera readout, and/or slow laser beam scanners. I will present a new fluorescence imaging method called LEAD (line excitation array detection) microscopy, capable of providing 0.8 million frames per second. This method performs 0.8 MHz line-scanning of excitation laser beam using a chirped signal-driven longitudinal acousto-optic deflector to create a virtual light-sheet, and images the field-of-view with a linear photomultiplier tube array to generate a 66×14 pixel frame each scan cycle. I will also present an implementation of the LEAD microscopy as a blur-free 3D flow cytometer for Caenorhabditis elegans moving at 1 m/s with 3.5-micron resolution and signal-to-background ratios >200. Signal-to-noise measurements indicate LEAD fluorescence microscopy can reach higher resolutions and pixels per frame without compromising frame rates.