In-depth polarization resolved SHG microscopy in biological tissues using iterative wavefront optimization.

Polarized nonlinear microscopy has been extensively developed to study molecular organization in biological tissues, quantifying the response of nonlinear signals to a varying incident linear polarization. Polarization Second harmonic Generation (PSHG) in particular, is a powerful tool to decipher sub-microscopic modifications of fibrillar collagen organization in type I and III collagen-rich tissues. The quality of SHG imaging is however limited to about one scattering mean free path in depth (typically 100 micrometers in biological tissues), due to the loss of focus quality, induced by wavefront aberrations and scattering at even larger depths. In this work, we study how optical depth penetration in biological tissues affects the quality of polarization control, a crucial parameter for quantitative assessment of PSHG measurements. We apply wavefront shaping to correct for SHG signal quality in two regimes, adaptive optics for smooth aberration modes corrections at shallow depth, and wavefront shaping of higher spatial frequencies for optical focus correction at larger depths. Using nonlinear SHG active nanocrystals as guide stars, we quantify the capabilities of such optimization methods to recover a high quality linear polarization and investigate how this approach can be applied to in-depth PSHG imaging in tissues, namely tendon and mouse cranial bone. This article is protected by copyright. All rights reserved.