Iterative multi-photon adaptive compensation technique for deep tissue two-photon fluorescence lifetime imaging

Fluorescence lifetime imaging can reveal the high-resolution structure of various biophysical and chemical parameters in a microenvironment quantitatively. However, the depth of imaging is generally limited to hundreds of micrometers due to aberration and light scattering in biological tissues. This paper introduces an iterative multi-photon adaptive compensation technique (IMPACT) into a two-photon fluorescence lifetime microscopy system to successfully overcome aberrations and multiple scattering problems in deep tissues. It shows that 400 correction modes can be achieved within 5 min, which was mainly limited by the frame rate of a spatial light modulator. This system was used for high-resolution imaging of mice brain tissue and live zebrafish, further verifying its superior performance in imaging quality and photon accumulation speed.