High-speed multifocal array scanning using refractive window tilting
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abstract
Confocal microscopy has several advantages over wide-field microscopy, such as out-of-focus light suppression, 3D sectioning, and compatibility with specialized detectors. While wide-field microscopy is a faster approach, multiplexed confocal schemes can be used to make confocal microscopy more suitable for high-throughput applications, such as high-content screening (HCS) commonly used in drug discovery. An increasingly powerful modality in HCS is fluorescence lifetime imaging microscopy (FLIM), which can be used to measure protein-protein interactions through Förster resonant energy transfer (FRET). FLIM-FRET for HCS combines the requirements of high throughput, high resolution and specialized time-resolving detectors, making it difficult to implement using wide-field and spinning disk confocal approaches. We developed a novel foci array scan method that can achieve uniform multiplex confocal acquisition using stationary lenslet arrays for high resolution and high throughput FLIM. Unlike traditional mirror galvanometers, which work in Fourier space between scan lenses, this scan method uses optical flats to steer a 2-dimension foci array through refraction. After integrating this scanning scheme in a multiplexing confocal FLIM system, we demonstrate it offers clear benefits over traditional mirror galvanometer scanners in scan linearity, uniformity, cost and complexity.
