A confocal laser scanning microscope for fluorescence and reflection at video rates
In confocal microscopy, the object is scanned by the coinciding focal points (confocal) of a point light source and a point detector both focused on a certain plane in the object. Only light coming from the focal point is detected and, even more important, out-of-focus light is rejected.This makes it possible to slice up optically the ‘volume of interest’ in the object by moving it axially while scanning the focused point light source (X-Y) laterally. The successive confocal sections can be stored in a computer and used to reconstruct the object in a 3D image display.The instrument described is able to scan the object laterally with an Ar ion laser (488 nm) at video rates. The image of one confocal section of an object can be displayed within 40 milliseconds (1000 х 1000 pixels). The time to record the total information within the ‘volume of interest’ normally depends on the number of slices needed to cover it, but rarely exceeds a few seconds.