Widefield two-photon excitation without scanning: live cell microscopy with high time resolution and low photo-bleaching

Rumelo Amor; Alison McDonald; Johanna Trägårdh; Gillian Robb; Louise Wilson; Nor Zaihana Abdul Rahman; John Dempster; William Bradshaw Amos; Trevor J. Bushell; Gail McConnell

doi:10.1371/journal.pone.0147115

Widefield two-photon excitation without scanning: live cell microscopy with high time resolution and low photo-bleaching

Rumelo Amor, Alison McDonald, Johanna Trägårdh, Gillian Robb, Louise Wilson, Nor Zaihana Abdul Rahman, John Dempster, William Bradshaw Amos, Trevor J. Bushell, Gail McConnell

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Abstract

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca2+ events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca2+ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

Original language	English
Article number	0147115
Number of pages	19
Journal	PLOS One
Volume	11
Issue number	1
DOIs	https://doi.org/10.1371/journal.pone.0147115
Publication status	Published - 29 Jan 2016

Keywords

two-photon
microscopy
femtosecond
Ca2+
hippocampal

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10.1371/journal.pone.0147115

Amor-etal-PLOS-2016-widefield-two-photon-excitation-without-scanningFinal published version, 2.81 MBLicence: CC BY 4.0

Cite this

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AU - Amor, Rumelo

AU - McDonald, Alison

AU - Trägårdh, Johanna

AU - Robb, Gillian

AU - Wilson, Louise

AU - Abdul Rahman, Nor Zaihana

AU - Dempster, John

AU - Amos, William Bradshaw

AU - Bushell, Trevor J.

AU - McConnell, Gail

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AB - We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca2+ events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca2+ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

KW - two-photon

KW - microscopy

KW - femtosecond

KW - Ca2+

KW - hippocampal

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JF - PLOS One

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ER -

Widefield two-photon excitation without scanning: live cell microscopy with high time resolution and low photo-bleaching

Abstract

Keywords

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Trevor Bushell

Gail McConnell

Multi-photon microscope without scanning for faster than video-rate fluorescence imaging of live cells