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.

Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy / Giuseppe de Vito, Pietro Ricci, Lapo Turrini, Vladislav Gavryusev, Caroline Müllenbroich, Natascia Tiso, Francesco Vanzi, Ludovico Silvestri, Francesco Saverio Pavone. - In: BIOMEDICAL OPTICS EXPRESS. - ISSN 2156-7085. - ELETTRONICO. - 11:(2020), pp. 4651-4665. [10.1364/BOE.396388]

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

Giuseppe de Vito;Pietro Ricci;Lapo Turrini;Vladislav Gavryusev;Francesco Vanzi;Ludovico Silvestri;Francesco Saverio Pavone
2020

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.
2020
11
4651
4665
Giuseppe de Vito, Pietro Ricci, Lapo Turrini, Vladislav Gavryusev, Caroline Müllenbroich, Natascia Tiso, Francesco Vanzi, Ludovico Silvestri, Frances...espandi
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1202047
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