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High speed functional imaging with source localized multifocal two-photon microscopy
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 optimiz...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Optical Society of America
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191622/ https://www.ncbi.nlm.nih.gov/pubmed/30338147 http://dx.doi.org/10.1364/BOE.9.003678 |
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author | Quicke, Peter Reynolds, Stephanie Neil, Mark Knöpfel, Thomas Schultz, Simon R. Foust, Amanda J. |
author_facet | Quicke, Peter Reynolds, Stephanie Neil, Mark Knöpfel, Thomas Schultz, Simon R. Foust, Amanda J. |
author_sort | Quicke, Peter |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6191622 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Optical Society of America |
record_format | MEDLINE/PubMed |
spelling | pubmed-61916222018-10-18 High speed functional imaging with source localized multifocal two-photon microscopy Quicke, Peter Reynolds, Stephanie Neil, Mark Knöpfel, Thomas Schultz, Simon R. Foust, Amanda J. Biomed Opt Express Article 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. Optical Society of America 2018-07-12 /pmc/articles/PMC6191622/ /pubmed/30338147 http://dx.doi.org/10.1364/BOE.9.003678 Text en 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. Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License (http://creativecommons.org/licenses/by/4.0/) . Further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation, and DOI. |
spellingShingle | Article Quicke, Peter Reynolds, Stephanie Neil, Mark Knöpfel, Thomas Schultz, Simon R. Foust, Amanda J. High speed functional imaging with source localized multifocal two-photon microscopy |
title | High speed functional imaging with source localized multifocal two-photon microscopy |
title_full | High speed functional imaging with source localized multifocal two-photon microscopy |
title_fullStr | High speed functional imaging with source localized multifocal two-photon microscopy |
title_full_unstemmed | High speed functional imaging with source localized multifocal two-photon microscopy |
title_short | High speed functional imaging with source localized multifocal two-photon microscopy |
title_sort | high speed functional imaging with source localized multifocal two-photon microscopy |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191622/ https://www.ncbi.nlm.nih.gov/pubmed/30338147 http://dx.doi.org/10.1364/BOE.9.003678 |
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