High speed functional imaging with source localized multifocal two-photon microscopy

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Title: High speed functional imaging with source localized multifocal two-photon microscopy
Authors: Quicke, P
Reynolds, S
Neil, M
Knopfel, T
Schultz, S
Foust, AJ
Item Type: Journal Article
Abstract: Multifocal two-photon microscopy (MTPM) increases imaging speed over single-focus scanning by parallelizing fluorescence excitation. The imaged fluorescence’s susceptibility to crosstalk, however, severely degrades contrast in scattering tissue. Here we present a source-localized MTPM scheme optimized for high speed functional fluorescence imaging in scattering mammalian brain tissue. A rastered line array of beamlets excites fluorescence imaged with a complementary metal-oxide-semiconductor (CMOS) camera. We mitigate scattering-induced crosstalk by temporally oversampling the rastered image, generating grouped images with structured illumination, and applying Richardson-Lucy deconvolution to reassign scattered photons. Single images are then retrieved with a maximum intensity projection through the deconvolved image groups. This method increased image contrast at depths up to 112 μm in scattering brain tissue and reduced functional crosstalk between pixels during neuronal calcium imaging. Source-localization did not affect signal-to-noise ratio (SNR) in densely labeled tissue under our experimental conditions. SNR decreased at low frame rates in sparsely labeled tissue, with no effect at frame rates above 50 Hz. Our non-descanned source-localized MTPM system enables high SNR, 100 Hz capture of fluorescence transients in scattering brain, increasing the scope of MTPM to faster and smaller functional signals.
Issue Date: 12-Jul-2018
Date of Acceptance: 6-Jun-2018
ISSN: 2156-7085
Publisher: Optical Society of America
Start Page: 3678
End Page: 3693
Journal / Book Title: Biomedical Optics Express
Volume: 9
Issue: 8
Copyright Statement: © 2018 The Author(s). Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.
Sponsor/Funder: Commission of the European Communities
Biotechnology and Biological Sciences Research Council (BBSRC)
Engineering & Physical Science Research Council (E
Royal Academy Of Engineering
National Institutes of Health
Wellcome Trust
National Institutes of Health
Funder's Grant Number: 249867
UPMC: C15/0244
Publication Status: Published
Appears in Collections:Physics
Electrical and Electronic Engineering
Department of Medicine
Faculty of Medicine
Faculty of Natural Sciences

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