Extended Depth-of-focus in a Laser Scanning System Employing a Synthesized Difference-of-Gaussians Pupil

Abstract

Traditional laser scanning systems, such as those used for microscopy, typically image objects of finite thickness. If the depth-of-focus of such systems is low, as is the case when a simple clear pupil is used, the object must be very thin or the image will be distorted. Several methods have been developed to deal with this problem. A microscope with a thin annular pupil has a very high depth-of-focus and can image the entire thickness of a sample, but most of the laser light is blocked, and the image shows poor contrast and high noise. In confocal laser microscopy, the depth-of-focus problem is eliminated by using a small aperture to discard information from all but one thin plane of the sample. However, such a system requires scanning passes at many different depths to yield an image of the entire thickness of the sample, which is a time-consuming process and is highly sensitive to registration errors.

In this thesis, a novel type of scanning system is considered. The sample is simultaneously scanned with a combination of two Gaussian laser beams of different widths and slightly different temporal frequencies. Information from scanning with the two beams is recorded with a photodetector, separated electronically, and processed to form an image. This image is similar to one formed by a system using a difference-of-Gaussians pupil, except no light has been blocked or wasted. Also, the entire sample can be scanned in one pass. The depth-of-focus characteristics of this synthesized difference-of-Gaussians pupil are examined and compared with those of well-known