Quantification of the surface density of a fluorescent label with the optical microscope

Fluorescence microscopy can offer unique advantages for biomaterials characterization. Like spectroscopy or radioactivity, it can be used to quantify specific binding to surfaces, but it can also assess surface homogeneity at the micron scale or detect protein aggregation. To fully utilize the potential of this technique, there must be a way to calibrate the microscope in terms of the moles of a fluorophore per unit area. The method we propose involves the following steps: fluorescent labeling of erythrocytes and quantification of the label by flow cytometry; flattening of fluorescent erythrocytes for microscopic observation; imaging and digital analysis to relate the gray level intensities to the fluorophore density; and using this procedure to characterize a different, more easily obtainable, standard. The latter can be a 50% solution of Na fluorescein that yields a highly reproducible and uniform fluorescence. Concentrated fluorescein solution can also be used to correct images for the spatial nonuniformity of illumination and detection (shading correction). By applying this method to study the binding of IgG and fibrinogen to glass or amidated glass, we showed that protein adsorption to glass may result in protein aggregation that may affect the biological activity of the adsorbed protein.