Fluorescence illumination optimization for three-dimensional microscopy

A fluorescence microscopy system and method allow selective and repeatable switching between Köhler illumination, providing a relatively large field of view, and Critical Illumination, providing a relatively higher axial resolution. This switching or toggle function may be facilitated by a feature associated with an alignment module that allows the focus of the fiber tip, for example, to be selectively and repeatably transitioned between the back aperture of the objective lens and the object plane without losing optical alignment.