Microscope Phase Fluorometer for Determining the Fluorescence Lifetimes of Fluorochromes

Abstract

A microscope which can measure the mean fluorescence lifetimes of small fluorescent samples is described. Special emphasis is given to fluorochromes dissolved in biological solutions and bound to thin polymeric substances, such as the desoxyribonucleic acid complexes present in the nuclei of cells. Fundamentally, the method is similar to that described by physicists and physicochemists for solutions of fluorochrome only. A diffraction modulator is used to sinusoidally modulate the intensity of a high-pressure mercury-vapor lamp. The phase shift between the unabsorbed exciting light, passing through the microscopic sample, and the fluorescent light is related to the fluorescence lifetime by tanΔφ=ωτ. The phase shift Δφ is obtained differentially from the phase angles φ1 and φ2 of each microscopic light signal with respect to a third less noisy signal, the tracer signal. The principal effort has been directed toward studying the various sources of error in the fluorometer and variabilities in lifetime measurements. The data indicate that the mean fluorescence lifetime (2.7 msec) of proflavine, bound to nuclei of tumor cells and observed with a 130× oil immersion lens, can be determined with a precision of 4×10−10 sec.