A New Spatiotemporal Scanning Technique for Two-Photon Fluorescence

Two-photon laser scanning microscope (TPLSM) provides outstanding optical three dimension section properties and it has been widely used in fundamental science and biomedical application. However, Current 3D two-photon fluorescence (TPF) imaging techniques usually overlook the spatiotemporal evolution of TPF ellipsoid along the axial direction, which might contain fine dynamical information of imaged targets. Here, we develop a spatiotemporal scanning technique and realize the measurement of spatiotemporal scanning of TPF ellipsoid with a semiconducting CsPbBr₃ nanosheet. Results have shown that axial size of TPF ellipsoid present linear growth as a function of excitation fluence by using spatial scanning. Furthermore, we have observed that axial size of TPF ellipsoid exhibits inhomogeneous linear growth with time delay by introducing spatiotemporal scanning technique. We attribute this phenomenon to the fact that surface and bulk region of CsPbBr₃ nanosheet have inhomogeneous timescale on TPF decay lifetime. Our results not only provide new insights for spatiotemporal resolving of TPF ellipsoid, but also helpful to promote the development of fluorescence lifetime microscopy technology.