The study of fluorescent probes by quantitative video intensification microscopy (QVIM)

In this paper we describe a system for the quantitation and display of fluorescence at the cellular level. It uses a low light level video camera which is interfaced to a fluorescence microscope and to a microprocessor-controlled video digitizing system. With the use of a light pen entry system one can specify areas of the field for measurement. The data obtainable are the area and perimeter of the delimited zone, the distribution of pixel intensities within this zone over a 16-level gray scale, and a value for total fluorescence intensity. Statistical outputs for repeated measurements are also obtained. The system responds linearly to light input, has a high degree of reproducibility, and provides good spatial resolution. Using the DNA-specific dye, Hoechst 33248, in diploid fibroblasts as test material, the system is shown to be able to reproduce expected distributions for amounts of DNA per cell. The capabilities and advantages of pseudocolor display are also demonstrated. We conclude that, in conjunction with appropriate fluorescent probes, systems such as the one described make it possible to do quantitative histochemistry of living cells and to measure substances not previously amenable to study.