Two-Photon Excitation and Selective Plane Illumination Microscopy: A Combination to Minimize Scattering Effects While Imaging Thick Samples

Abstract

Imaging thick samples in optical fluorescence microscopy still represents an open challenge because sample-induced aberrations can lead to a worsening of the imaging quality and a wrong interpretation of the data collected. Two-photon excitation microscopy is a suitable tool to enhance the penetration depth capabilities of a microscopy system but may still be affected by scattering effects, generating an out-of-focus fluorescence, which can result in a shift of the intensity excitation distribution (1). Selective Plane Illumination Microscopy represents an optimal tool to perform imaging of large samples, and recently it has been combined with two photon excitation (2) in order to improve the performances of such a system while imaging deep into a scattering sample. The aim of this work is to characterize how relevant scattering effects are in distorting the shape of a light sheet. A comparison between single and two-photon excitation light sheet has been performed measuring the excitation distribution profiles in fluorescent immobile phantom samples mimicking the optical properties of some biological tissues. Results show that two photon excitation is able to preserve the shape of the light sheet even in strong scattering samples, while single photon light sheet shows a strong shift in the intensity excitation distribution as penetration depth and scattering strength increase. To show the performances of the imaging of the system, a 3D reconstruction of a tumor spheroid, a suitable model for a thick scattering sample, is reported.1. Theer, P. Denk, W. “On the fundamental imaging-depth limit in two-photon microscopy”. J Opt Soc Am A, 23 (12), (2006).2. Truong, T. et al. “Deep and fast live imaging with two-photon scanned light-sheet microscopy.” Nat Methods, 8 (9) (2011)