Scattering Correction through Fourier-Domain Open-Channel Coupling in Two-Photon Microscopy

Light penetration depth in biological tissue is limited by tissue scattering. Correcting scattering becomes particularly challenging in scenarios with limited photon availability and when access to the transmission side of the scattering tissue is not possible. Here, we introduce a new two-photon microscopy system with Fourier-domain open-channel coupling for scattering correction (2P-FOCUS). 2P-FOCUS corrects scattering by utilizing the nonlinearity of multiple-beam interference and two-photon excitation, eliminating the need for a guide star, iterative optimization, or measuring transmission or reflection matrices. We demonstrate that 2P-FOCUS significantly enhances two-photon fluorescence signals by several tens of folds when focusing through a bone sample, compared to cases without scattering correction at equivalent excitation power. 2P-FOCUS can also correct tissue scattering over large volumes through subregion correction. This is demonstrated by imaging neurons and cerebral blood vessels in a 230×230×500 μ m ³ volume in the mouse brain ex vivo. 2P-FOCUS could serve as a powerful tool for deep tissue imaging in bulky organisms or live animals.