Effect of camera digitization in filter-based fluorescence spectral microscopy

Abstract

Image digitization of optical images was predicted to limit the accuracy of spectral estimates obtained in filter-based fluorescence spectral microscopy. We investigate experimentally the issue using two 3-CCD cameras - an 8-bit Hitachi HV-C20 and a 12-bit Hamamatsu ORCA C7780-20. Spectral measurement is achieved using a weighted superposition of basis spectra that are derived from a spectral library. Multi-channel detection using three independent camera channels and color filters allows the calculation of coefficients for the basis spectra. The emission spectra of yellow-green and orange fluorescent microspheres were measured. More accurate estimates are derived using the 12-bit camera compared to the 8-bit CCD camera.