Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy

de Vito, G., Ricci, P., Turrini, L., Gavryusev, V., Müllenbroich, C. , Tiso, N., Vanzi, F., Silvestri, L. and Saverio Pavone, F. (2020) Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy. Biomedical Optics Express, 11(8), pp. 4651-4665. (doi: 10.1364/BOE.396388) (PMID:32923069) (PMCID:PMC7449752)

Abstract

Light-sheet microscopy (LSM) is a powerful imaging technique that uses a planar illumination oriented orthogonally to the detection axis. Two-photon (2P) LSM is a variant of LSM that exploits the 2P absorption effect for sample excitation. The light polarization state plays a significant, and often overlooked, role in 2P absorption processes. The scope of this work is to test whether using different polarization states for excitation light can affect the detected signal levels in 2P LSM imaging of typical biological samples with a spatially unordered dye population. Supported by a theoretical model, we compared the fluorescence signals obtained using different polarization states with various fluorophores (fluorescein, EGFP and GCaMP6s) and different samples (liquid solution and fixed or living zebrafish larvae). In all conditions, in agreement with our theoretical expectations, linear polarization oriented parallel to the detection plane provided the largest signal levels, while perpendicularly-oriented polarization gave low fluorescence signal with the biological samples, but a large signal for the fluorescein solution. Finally, circular polarization generally provided lower signal levels. These results highlight the importance of controlling the light polarization state in 2P LSM of biological samples. Furthermore, this characterization represents a useful guide to choose the best light polarization state when maximization of signal levels is needed, e.g. in high-speed 2P LSM.

• Item Type: Articles
• Additional Information: Funding: European Research Council (692943); H2020 Marie Skłodowska-Curie Actions (793849).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Muellenbroich, Dr Caroline
• Authors: de Vito, G., Ricci, P., Turrini, L., Gavryusev, V., Müllenbroich, C., Tiso, N., Vanzi, F., Silvestri, L., and Saverio Pavone, F.
• College/School: College of Science and Engineering > School of Physics and Astronomy
• Journal Name: Biomedical Optics Express
• Publisher: Optical Society of America
• ISSN: 2156-7085
• Published Online: 24 July 2020
• Copyright Holders: Copyright © 2020 Optical Society of America
• First Published: First published in Biomedical Optics Express 11(8): 4651-4665
• Publisher Policy: Reproduced in accordance with the publisher copyright policy