Performance of a confocal laser scanning microscope

Abstract

Confocal Microscopy is a technique that allows three-dimensional (3D) resolution as well as increased the contrast ofmicroscope images. By using a point source and a pinhole at the detector, the microscope can filter the signals coming from the out-of-focus planes and thus achieving resolution along the optical axis ofthe microscope. Early designs of confocal microscopes renders a two-dimensional image within the focal plane by scanning of the sample using translational stages in the x- and y- direction. This result to a stable optical system that is stationary and allows the resolution to be maintained throughout the image. The speed of stage scanning, however, is limited to the mechanical properties of translation stages and therefore result to slow acquisition of the image. Stage scanning would take about two hours to acquire a 128x128 image with pixel dwell time of 0.5s, and therefore not a practical method to observe time-critical samples such as fluorescence from biological tissues.
An alternative approach to speed up the image acquisition is by scanning the laser beam that serves as the illumination source using scanning mirrors or by acousto-optic beam deflectors. This allows shorter pixel dwell time which is in the millisecond range and therefore enables acquisition of the images in less than a minute. Real-time acquisition of confocal images is also possible by distributing the excitation beam into separate smaller beams (multi-channel) that pass through a rotating Nipkow disk, which contains lenslets and pinhole arrays. The rotation of the disk effectively scans the location of multiple focal points in the sample and therefore enables acquisition of the image in real-time.
Although a multi-channel approach provides real-time observation, the excitation power is distributed into smaller beams and may not be appropriate in microscopy techniques where power of the excitation source is critical. The beam-scanning method is a good compromise that provides full use of the excitation power and at the same time render relatively fast image acquisition. Fully integrated confocal laser scanning microscopes (CLSM) from Carl-Zeiss, BioRad, Olympus, etc. that employ beam scanning designs provide efficient performance and post image processing software for contrast enhancement and 3D rendering. However, aside from the high cost considerations of these microscopes, flexibility and further manipulation of the optical system to improve the imaging performance is unrealizable.
This paper describes the implementation and performance rating of a CLSM that is based on beam scanning using galvanometer mirrors. Initial investigation on the imaging performance is done on reflectance mode. The axial point spread function is evaluated using a perfectly reflecting mirror. Microscopic images of an exposed area of a semiconductor memory (EPROM) are acquired by scanning a beam of Argon-ion laser through an objective lens with numerical aperture, NA=0.5.