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

Amor, Rumelo and McDonald, Alison and Trägårdh, Johanna and Robb, Gillian and Wilson, Louise and Abdul Rahman, Nor Zaihana and Dempster, John and Amos, William Bradshaw and Bushell, Trevor J. and McConnell, Gail (2016) Widefield two-photon excitation without scanning : live cell microscopy with high time resolution and low photo-bleaching. PLOS One, 11 (1). ISSN 1932-6203

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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.