Recently, there has been a large expansion in the usage of optical microscopes for obtaining quantitative information from biological samples in order to determine fundamental biological information such as molecular kinetics and interaction, and heterogeneity within cell populations. Consequently, we built a highly stable, uniform, isotropically emitting and convenient-to-use light source, and designed image analysis procedures for calibrating the emission light path of optical microscopes. We used the source and procedures to analyse the quantitative imaging properties of a widely used model of laser scanning confocal microscope. Results showed that the overall performance was as high as could be expected given the inherent limitations of the optical components and photomultiplier tubes. We observed that the photon detection efficiency did not vary with photomultiplier tube gain and that the highest dynamic range was achieved with relatively low gain and 12-bit digitization. Practical applications of the light source for checking the transmission of optical components in the emission light path are presented.