Nonlinear microscopy (NLM) has emerged as a powerful technique for biological imaging in a label-free manner. Two-photon fluorescence imaging of neural activity, coherent Raman scattering imaging of lipid-rich assemblies, second-harmonic generation imaging of collagen structures and sensitive screening of pharmaceuticals, are just a few representative examples. Despite the tremendous impact of NLM, all of nonlinear imaging modalities can only image at shallow depths because deeper penetration imaging into biological specimens is hindered by scattering phenomena. In the first part of this presentation, and in order to present the current state-of-the-art nonlinear imaging capabilities, I will present a study of depolarization effects due to scattering on polarization-resolved coherent anti-Stokes Raman scattering imaging of opaque spinal cords myelin sheaths. The results highlight how scattering can lead to erratic conclusions and also how shallow nonlinear imaging is currently possible on biological specimens. I will then present how we used wavefront shaping (WS) technique to image through strongly scattering media in order to enhance the (imaging) penetration depth. After an explanation of WS fundamentals, I will show how we successfully imaged nonlinear sources of second-harmonic photons after strongly scattering medium, at a sub-diffraction limited resolution. These results demonstrate that nonlinear imaging deep in biological specimens is no longer limited to shallow depths.
