Characterization of Scattering Effects in Phantom Samples using Single and Two-Photon Excitation Light Sheet Microscopy

Abstract

Scattering in thick biological samples plays a relevant role in imaging degradation and reduction of signal to noise ratio and contrast. Even if there are existing techniques that allow imaging of very thick samples, they can suffer from aberrations and distortions related to scattering properties of the sample. Recently, the capability of coupling light sheet techniques with Two Photon Excitation(TPE) (1,2,3) provided a powerful tool for deep imaging of biological samples. This has been proved to be a promising trend in thick-imaging microscopy, because it combines the advantages of light sheet techniques with the increased penetration depth intrinsic of TPE. For this reason, in this work a detailed characterization of the effects induced by scattering on the excitation profile of a light sheet based microscope is provided. In particular, different phantom samples, with different scattering coefficients mimicking the optical properties of various biological tissues, are used. Experiments were performed to investigate the shape distortions of the excitation intensity distribution provided by the light sheet, both in single and in two-photon excitation. This aspect represents a crucial point, since the effective light sheet intensity distribution is strictly related to the optical sectioning capability and image quality of the system. Results show that the light sheet intensity distribution is less affected by light-sample interactions and the shape is preserved in the case of TPE in comparison with single photon excitation regime. Furthermore, contrast measurements has been performed using fluorescent beads embedded in different scattering samples in both excitation modes. These experiments showed again the better performances of TPE compared to single photon excitation.(1) Cella Zanacchi et al. Proc. of SPIE, vol.7903 (2011)(2) Planchon et al. Nature Methods 8, 417-423, (2011)(3) Truong et al. Nature Methods 8,757-760, (2011)