Fluorescence spectra of DNA dyes measured in a flow cytometer

The spectral properties of fluorescent dyes often vary with experimental conditions. Spectral changes contain information about the particles to which the dye is bound and can have practical consequences for the design of flow cytometry experiments. It is therefore desirable to have spectral information obtained under conditions that approach those under which the measurements in a flow cytometer are performed. This report describes the measurement of fluorescence spectra in a flow cytometer. We have attached a monochromator to one of the detector ports of our flow cytometer. The monochromator scans continuously over a range of wavelengths. For each measured particle, the monochromator wavelength is recorded along with the fluorescence intensity at that wavelength. Other fluorescence and scatter signals can also be recorded to distinguish different particle populations within the sample. This method for measurement of fluorescence spectra has advantages over conventional fluorimetry in that it distinguishes between bound and unbound dye and that spectral properties of different particle populations within a single sample can be separately reconstructed. Fluorescence spectra of common DNA dyes bound to chromatin are presented. A shift to the red in Hoechst 33258 fluorescence with increasing dye concentration is demonstrated. The spectra of DNA-bound Hoechst and ethidium bromide are compared with those of the free dyes in solution. Energy transfer between several pairs of DNA dyes is demonstrated using spectral measurements. We also find slight spectral variations between the different human chromosomes dualstained with Hoechst and chromomycin.