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?Optimizing fluorescence excitation and detection for intravital two-photon microscopy


Commercial two-photon microscope systems incorporating turnkey ultrafast lasers have made the technology more user-friendly and accessible to nonspecialized biology laboratories. This has been accompanied by the development of an exciting range of new fluorescent proteins and dyes such as near-infra-red fluorescent proteins and optical highlighters. However, the two-photon absorption properties of these fluorescent molecules are not widely available and cannot be reliably predicted from their single photon absorption spectra. Furthermore, the spectral characteristics of fluorescent proteins in vivo can be affected by the local environment and light scattering by deep tissue and can vary greatly from one laboratory to the next. Here, we describe a simple protocol for determining the two-photon excitation peaks of fluorescent reporters that can be tailored to the relevant tissue samples to suit the imaging goals of individual biological laboratories.

Type Journal
Authors Suan, D.; Hampton, H.R.; Tomura, M.; Kanagawa, O.; Chtanova, T,; Phan, T.G.;
Responsible Garvan Author Dr Daniel Suan
Publisher Name Methods in Cell Biology
Published Date 2013-01-16
Published Volume 113
Published Pages 311-323
Status Published in-print
DOI 10.1016/B978-0-12-407239-8.00014-8
URL link to publisher's version
OpenAccess link to author's accepted manuscript version