Laser scanning two-photon fluorescence microscope with a virtual pinhole

Abstract

We construct and evaluate the performance of a two-photon fluorescence (2PF) microscope with a virtual pinhole. Instead of using a single-channel detector to render a 2PF image, we detect a wide-field image using multi channel pixelated array detector. As proof-of-principle, we use a conventional digital CCD camera to function as the wide-field detector. The virtual pinhole is implemented by selecting a region of interest in the wide field digital image and setting the pixel values outside the region to zero. The virtual pinhole removes scattered signals resulting to improve contrast and the visibility of details masked by scattered signals. The optical sectioning capability of the system was also shown.