Extended depth of field microscopy for rapid volumetric two-photon imaging

Abstract

Two-photon fluorescence microscopy is an influential tool in biology, providing valuable information on the activity of cells deep inside the tissue. However, it is limited by its low speed for imaging volume samples. Here we present the design of a two-photon scanning microscope with an extended and adjustable depth of field, which improves the temporal resolution for sampling thick samples. Moreover, this method implies no loss of optical power and resolution, and can be easily integrated into most commercial laser-scanning microscopy systems. We demonstrate experimentally the gain in performance of the system by comparing volumetric scans of neuronal structures with a standard versus an extended depth of field system.