Abstract: 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 (with or without an adhesion promoter) onto the support, and the solid surface layer including the fluorescent material located on the thin opaque mask.