OPTIMIZING THE PERFORMANCE OF CONFOCAL POINT SCANNING LASER MICROSCOPES OVER THE FULL-FIELD OF VIEW

To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view three s imple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent ma terial and a rectangular grid with a readily defined centre. Using the se specimens the adjustment of any combination of confocal scanning la ser visualization system and light microscope can be examined througho ut the field of view. The effects of misalignment of the various subco mponents of a confocal scanning laser microscope on both the axial spr ead function of a plane and the shading pattern oner the image field a re described. Finally, where the design of the confocal optics permits , the three specimens can be used to facilitate the alignment of the v arious components to the optimal level achievable.