Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy
Authors: de Vito G., Ricci P., Turrini L., Gavryusev V., Mullenbroich C., Tiso N., Vanzi F., Silvestri L., Pavone FS.
Autors Affiliation: Univ Florence, Dept Neurosci Psychol Drug Res & Child Hlth, Viale Pieraccini 6, I-50139 Florence, FI, Italy; European Lab Nonlinear Spect, Via Nello Carrara 1, I-50019 Sesto Fiorentino, FI, Italy; Univ Florence, Dept Phys & Astron, Via Sansone 1, I-50019 Sesto Fiorentino, FI, Italy; Univ Glasgow, Sch Phys & Astron, Kelvin Bldg, Glasgow G12 8QQ, Lanark, Scotland; CNR, Natl Inst Opt, Via Nello Carrara 1, I-50019 Sesto Fiorentino, FI, Italy; Univ Padua, Dept Biol, Via Ugo Bassi 58-B, I-35131 Padua, PD, Italy; Univ Florence, Dept Biol, Via Madonna del Piano 6, I-50019 Sesto Fiorentino, FI, Italy
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. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Journal/Review: BIOMEDICAL OPTICS EXPRESS
Volume: 11 (8) Pages from: 4651 to: 4665