Correction for light absorption within the object for quantitative microfluorometry: a theoretical approach.
Quantitative microfluorescence measurements often involve the integration of the fluorescent light over the field of view of the microscope. This can easily be done by simply directing the light onto the cathode of a photomultiplier as long as all the emitted light leaves the specimen without being absorbed. However, if the light (exciting or fluorescent) is absorbed by the fluorescent dye, the intensity of the light output will become a nonlinear function of the amount of fluorescent material following the Lambert-Beer law. This makes the integration more complicated. A simple method is described which allows one to test whether significant absorption takes place and which gives formulas for correcting the error. The information about the absorption is obtained from measurements at different wavelengths and with different geometries of excitation. The method does not need complicated mechanical or optical equipment.
Ernst
D
D