the present invention relates to calibration apparatuses , methods or tools used in microscopy . a calibration tool for fluorescent microscopy includes a support , a solid surface layer including a fluorescent material , and a thin opaque mask of non - fluorescent material defining reference feature openings having selected dimensions exposing portions of the surface layer . a first type of the calibration tool may include an adhesion promoter facilitating contact between the surface of the support and the solid surface layer including the fluorescent material , which is in contact with the thin opaque mask . a second type of the calibration tool may include the thin opaque mask fabricated onto the support , and the solid surface layer including the fluorescent material located on the thin opaque mask .