A cassette for retaining a specimen of surgically exposed tissue from a patient in an orientation that facilitates optical sectioning of the tissue by a confocal microscopic or other optical imaging microscope. The cassette includes a base member having an optically transparent window upon which a tissue specimen is situated, a membrane locatable over a substantial portion of the base member including the window, and an upper member which can cover the base member to provide an enclosed cavity between the membrane and the window sealing the tissue specimen therein. The edges of the tissue specimen may then be positioned planar against the window and retained in that position by bonds formed between the membrane and window around the tissue specimen. The cassette may be part of a confocal imaging system to produce microscopic images of sections of the tissue specimen useful for Mohs surgery.