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High‐Speed Large‐Field Multifocal Illumination Fluorescence Microscopy
Author(s) -
Chen Zhenyue,
Mc Larney Benedict,
Rebling Johannes,
DeánBen Xosé Luis,
Zhou Quanyu,
Gottschalk Sven,
Razansky Daniel
Publication year - 2020
Publication title -
laser and photonics reviews
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.778
H-Index - 116
eISSN - 1863-8899
pISSN - 1863-8880
DOI - 10.1002/lpor.201900070
Subject(s) - microscopy , optics , microscope , light sheet fluorescence microscopy , fluorescence microscope , field of view , materials science , fluorescence lifetime imaging microscopy , optical microscope , depth of field , confocal microscopy , image resolution , scanning confocal electron microscopy , fluorescence , physics , scanning electron microscope
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.