A scanning confocal microscope for transmission and reflection imaging

Abstract

IN a confocal scanning laser microscope^1,2 (CSLM), the detector pinhole is confocal with the illuminated spot on the sample, and rejects light reflected from objects that are not in the focal plane. This results in images that contain only sharp or empty areas, as opposed to non-confocal images which contain sharp and blurry areas, and allows the CSLM to perform optical sectioning. Individual optical sections can be used to produce a true three-dimensional image or can be added together to produce an extended-depth-of-focus image. For biological specimens, the intensity of the reflected-light (or fluorescence) signal decreases with increasing penetration into the sample, and self-shadowing also limits the information in the image. Moreover, many biological specimens are only weak reflectors, but produce transmission images with good contrast. The only confocal transmission images reported previously have used scanning-stage microscopes^3,4, although Goldstein⁵ has reported a confocal scanning-beam micro-scope that should in principle work in transmission. Here we describe a microscope that produces true confocal images in trans-mission, and also forms a reflected-light image from both the top and bottom of the specimen. The optical slices produced in trans-mission do not change in average intensity with depth in the specimen, and as the microscope can also examine the specimen from the bottom in reflected light, loss of data by self-shadowing is minimized. All of the original contrast modes of the CSLM (reflected light, fluorescence, differential phase contrast and so on) are also available using this new microscope.