POWER AND LIMITS OF LASER-SCANNING CONFOCAL MICROSCOPY

In confocal microscopy, the object is illuminated and observed so as t o rid the resulting image of the light from out-of-focus planes. Imagi ng may be performed in the reflective or in the fluorescence mode. Con focal microscopy allows accurate and nondestructive optical sectioning in a plane perpendicular or parallel to the optical axis of the micro scope. Further digital three-dimensional treatments of the data may be performed so as to visualize the specimen from a variety of angles. S everal examples illustrating each of these possibilities are given. Th ree-dimensional reconstitution of nuclear components using a cubic rep resentation and a ray tracing based method are also given. Instrumenta l and experimental factors can introduce some bias into the acquisitio n of the 3-D data set: self-shadowing effects of thick specimens, sphe rical aberrations due to the sub-optimum use of the objective lenses a nd photobleaching processes. This last phenomenon is the one that most heavily hampers the quantitative analysis needed for 3-D reconstructi on. We delineate each of these problems and indicate to what extent th ey can be solved. Some tips are given for the practice of confocal mic roscope and image recovery: how to determine empirically the thickness of the optical slices, how to deal with extreme contrasts in an image , how to prevent artificial flattening of the specimens. Finally, futu re prospects in the field are outlined. Particular mention of the use of pulsed lasers is made as they may be an alternative to UV-lasers an d a possible means to attenuate photodamage to biological specimens.