3-D image analysis of serial focal sections from confocal scanning laser microscopy

Abstract

The Confocal Scanning Laser Microscope (CSLM) is used in two quite different imaging modes: reflection and transmission. Most instruments maintain their confocal optics only when operated in reflection mode, in which light reflected or emitted from points in the sample or on its surface, lying in the focal plane of the microscope, is detected to form an image. Point elevations can be measured by scanning through a range of focal distances. Recording in memory the maximum brightness at each pixel forms an image containing the “in-focus” information for the entire irregular surface or object (Figure 1). These are powerful capabilities and account for much of the current use of confocal microscopes for metrology of rough surfaces, and for use with fluorescent dyes.True transmission confocal imaging can be achieved by passing the light from the source through the specimen to a mirror, reflecting it back through the specimen again (maintaining the focus in the same plane), and thence to the detector. This is shown schematically in Figure 2. This is only possible with monochromatic light, because of sample-induced chromatic aberrations.