Flat-top illumination profile in an epifluorescence microscope by dual microlens arrays

Low uniformity in illumination across the image plane impairs the ability of a traditional epifluorescence microscope to quantify fluorescence intensities. Two microlens arrays (MLAs) were introduced into the illumination path of two different epifluorescence microscope systems to improve the uniformity of the illumination. Measurements of the uniformity of illumination were performed with a CCD camera in the focal plane and with fluorescent beads in the image plane. In semi critical alignment, a uniformity of illumination of 15-23% was found compared with 1-2% in the modified system. Coefficient of variation (CV) of fluorescent beads measured on the unmodified system was 20.4% ± 5.3% in semi critical alignment and 10.8% ± 1.3% in Koehler alignment. On the MLA systems, CV was 7.9% ± 2.0% and on a flow cytometer, the CV was 6.7% ± 0.7%. Implementation of MLAs in an epifluorescence microscope improves the uniformity of illumination, thereby reducing the variation in detection of fluorescent signals of the measured objects and becomes equivalent to that of flow cytometry.