High-Speed Large-Field Multifocal Illumination Fluorescence Microscopy

Abstract

Scanning optical microscopy techniques are commonly restricted to a sub-millimeter field-of-view (FOV) or otherwise employ slow mechanical translation, limiting their applicability for imaging fast biological dynamics occurring over large areas. A rapid scanning large-field multifocal illumination (LMI) fluorescence microscopy technique is devised based on a beam-splitting grating and an acousto-optic deflector synchronized with a high-speed camera to attain real-time fluorescence microscopy over a centimeter-scale FOV. Owing to its large depth of focus, the approach allows noninvasive visualization of perfusion across the entire mouse cerebral cortex, not achievable with conventional wide-field fluorescence microscopy methods. The new concept can readily be incorporated into conventional wide-field microscopes to mitigate image blur due to tissue scattering and attain optimal trade-off between spatial resolution and FOV. It further establishes a bridge between conventional wide-field macroscopy and laser scanning confocal microscopy, thus it is anticipated to find broad applicability in functional neuroimaging, in vivo cell tracking, and other applications looking at large-scale fluorescent-based biodynamics.