A standard for calibration and shading correction of a fluorescence microscope

BACKGROUND: Numerous applications of fluorescence microscopy require quantitation of signal intensity in reproducible units. Two problems must be overcome to achieve this goal. First, due to various instrumental factors, the same sample imaged on two microscopes or even on the same microscope at different times may produce highly divergent readings. Second, because of shading, some areas within the same field may appear brighter than others despite the same amount of fluorophore. The first type of variability requires calibration using a sample of reproducible fluorescence yield; to correct for shading, a uniform fluorescent field is needed. METHODS: Standard slides were prepared by placing several microliters of 10%-50% w/v fluorescein or rhodamine between a coverglass and a slide. They were used to perform shading correction and normalization under a variety of imaging conditions. RESULTS: Concentrated fluorophores produced a uniform fluorescent field of moderate and reproducible brightness. By expressing the staining of a biological object in the units of standard slides, identical results were obtained irrespective of the imaging conditions or the microscope used. We compared shading correction based on concentrated fluorescein with two other standards. Concentrated fluorescein resulted in the best equalization of the field. CONCLUSIONS: Standardization of fluorescent images can be achieved by normalizing them to the image of a concentrated solution of a fluorophore. Due to its simplicity and efficiency, this method can be used in clinical analysis as well as in routine laboratory practice.