Abstract: The invention pertains to a calibration layer comprising an optically transparent polymer containing an amount of photobleachable luminscent material present in such a way that the final polymer film contains less than 10 wt. % of luminophore and has an optical attenuation of less than 0.3 absorption units in the wavelength region of interest. The invention further is concerned with a method of calibration of an optical image device, preferably an optical or Raman microscope, by using the decrease in luminescence as the result of photobleaching between two consecutive images for calibration.