Orthogonally-polarized excitation for improved two-photon and second-harmonic-generation microscopy, applied to neurotransmitter imaging with GPCR-based sensors.

Autor:	Pulin M; Institute for Synaptic Physiology, ZMNH, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany.; Research Group Synaptic Wiring and Information Processing, ZMNH, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany., Stockhausen KE; Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 22529 Hamburg, Germany., Masseck OA; Synthetic Biology, University of Bremen, Leobener Str. 5, 28359 Bremen, Germany., Kubitschke M; Synthetic Biology, University of Bremen, Leobener Str. 5, 28359 Bremen, Germany., Busse B; Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 22529 Hamburg, Germany.; Interdisciplinary Competence Center for Interface Research (ICCIR), University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany., Wiegert JS; Research Group Synaptic Wiring and Information Processing, ZMNH, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany., Oertner TG; Institute for Synaptic Physiology, ZMNH, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany.
Jazyk:	angličtina
Zdroj:	Biomedical optics express [Biomed Opt Express] 2022 Jan 14; Vol. 13 (2), pp. 777-790. Date of Electronic Publication: 2022 Jan 14 (Print Publication: 2022).
DOI:	10.1364/BOE.448760
Abstrakt:	Fluorescent proteins are excited by light that is polarized parallel to the dipole axis of the chromophore. In two-photon microscopy, polarized light is used for excitation. Here we reveal surprisingly strong polarization sensitivity in a class of genetically encoded, GPCR-based neurotransmitter sensors. In tubular structures such as dendrites, this effect led to a complete loss of membrane signal in dendrites running parallel to the polarization direction of the excitation beam. To reduce the sensitivity to dendritic orientation, we designed an optical device that generates interleaved pulse trains of orthogonal polarization. The passive device, which we inserted in the beam path of an existing two-photon microscope, removed the strong direction bias from fluorescence and second-harmonic (SHG) images. We conclude that for optical measurements of transmitter concentration with GPCR-based sensors, orthogonally polarized excitation is essential. Competing Interests: MP (P), TGO (P). (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)
Databáze:	MEDLINE
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