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 a rigid optically transparent window upon which a tissue specimen is situated, a pliable membrane locatable over a substantial portion of the base member including the window, and an upper member, having an aperture therethrough, 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 be positioned planar against the window and retained in that position by bonds formed between the membrane and window at multiple points or locations around the tissue specimen. The specimen retained in the cavity is imagible by a microscope through the window of the base member.