High-speed dual-beam, crossed line-scanning fluorescence microscope with a point confocal resolution

Abstract

Line-scanning microscopes are often used to overcome the limited scanning speed of conventional point-scanning confocal microscopes, at the cost, however, of spatial resolution. In this paper, we present a dual-beam fluorescence line-scanning microscope that can restore the original confocal resolution. This microscope forms two orthogonal line foci in the object plane with perpendicular scanning directions, which create two line-scan images of the same area. From these images, the real noise and confocal characteristics are analyzed. Based on this information, we developed an image restoration algorithm to produce a final image with spatial resolution comparable to that of a conventional point-scanning confocal microscope. This algorithm was derived with the total variation regularization, and the critical image restoration factor for the algorithm was determined via an iterative process. Our results indicate that the intrinsic resolution limit of line-scanning microscopes can be overcome without subsequent deterioration in spatial resolution.