Abstract
Three-dimensional, fluorescence imaging methods with ~1 MHz frame rates are needed for high-speed, blur-free flow cytometry and capturing volumetric neuronal activity. The frame rates of current imaging methods are limited to kHz by the photon budget, slow camera readout, and/or slow laser beam scanners. Here, we present line excitation array detection (LEAD) fluorescence microscopy, a high-speed imaging method capable of providing 0.8 million frames per second. The method performs 0.8 MHz line-scanning of an 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. We implement LEAD microscopy as a blur-free flow cytometer for Caenorhabditis elegans moving at 1 m s−1 with 3.5-µm resolution and signal-to-background ratios >200. Signal-to-noise measurements indicate future LEAD fluorescence microscopes can reach higher resolutions and pixels per frame without compromising frame rates.
Highlights
Three-dimensional, fluorescence imaging methods with ~1 MHz frame rates are needed for high-speed, blur-free flow cytometry and capturing volumetric neuronal activity
For blur-free imaging at 1 m s−1, there is a need for a microscopic imaging method at ~1 MHz frame rates, which has been achieved for 2D brightfield cytometry[15], but not 3D fluorescence cytometry
line excitation array detection (LEAD) microscopy overcomes the limitations of imaging speed and photon budget by implementing the fastest beam scanning method using a longitudinal acousto-optic deflectors (AODs) in chirped mode, and a fast, sensitive, and parallel detection scheme using a linear Photomultiplier tubes (PMTs) array (Fig. 1)
Summary
Three-dimensional, fluorescence imaging methods with ~1 MHz frame rates are needed for high-speed, blur-free flow cytometry and capturing volumetric neuronal activity. We present line excitation array detection (LEAD) fluorescence microscopy, a high-speed imaging method capable of providing 0.8 million frames per second. Camera-based lightsheet microscopy methods can only reach maximum frame rates of a few kHz for fluorescence imaging of biologically relevant samples[13,14,17,18,19,20,21,22,23,24,25,26,27,28,29], which is too slow to avoid motion blur in 3D flow cytometry. We image mouse brain slices with the sensitivity to resolve single neurons distributed in 3D, demonstrating the potential of LEAD microscopy for future high-speed, time-lapse imaging
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