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.
Highlights
Light-sheet (LS) fluorescence microscopy is a powerful optical imaging technique [1] based on the principle of a planar illumination oriented orthogonally with respect to the detection axis [2]
We observed similar fluorescence levels when employing vertically- or horizontallypolarized light, we revealed a small, albeit statistically significant, difference between the two polarization states: the signal in horizontal-polarization condition (544.7 A.U.; 95% C.I.: [539.2, 550.2] A.U.) is ∼3% larger with respect to the vertical-polarization condition (529.5 A.U.; 95% C.I.: [527.6, 531.5] A.U.). This observation indicates that even in a situation that favors high-level of molecular mobility a residual degree of spatial anisotropy induced by photoselection can be detected in fluorescence emission, with increased signal for horizontal polarization since it fulfills the orthogonal alignment condition between the excitation polarization direction and the imaging axis, as expected for the LS microscopy (LSM) detection geometry
For the fixed-condition, we measured the average fluorescence signal emitted by arbitrarily selected Region Of Interest (ROI), to what we did for the enhanced GFP (EGFP) experiments, and we show the results in Figs. 4(a) and 4(c)
Summary
Light-sheet (LS) fluorescence microscopy is a powerful optical imaging technique [1] based on the principle of a planar illumination oriented orthogonally with respect to the detection axis [2] It employs wide-field detectors that allow to parallelize the photon collection, offering a large increment in the acquisition speed. The excitation wavelengths used in 2P absorption are usually in the infrared region: a frequency range characterized by reduced scattering inside biological tissues compared to visible light [10] This effect, combined with the quadratic dependence of the absorption rate on the excitation light intensity, offers several additional advantages: a larger penetration depth in highly scattering samples [4], a reduction of the sample-induced aberrations, a better uniformity of the illumination distribution and an improved image contrast [11], without resorting to advanced illumination schemes [12,13,14]. It allows the study of neuronal responses in conditions where they can otherwise be affected by the visible excitation light used in one-photon imaging [5,15,16]
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