Dual laser beam focussing for flow cytometry through a single crossed cylindrical lens pair

Two laser beans (argon 488 nm blue, and krypton UV lines) have been focussed either to the same point or to two different points in a vertical plane in a multiparameter flow cytometer. The technique exploits the focal length shortening of the longer wavelength beam that occurs with spherical and astigmatic lens aberrations. Ethidium bromide-stained DNA is excited by both the argon 488 nm and krypton UV lines and the excitation intensities of both beams were set to give the same red fluorescence pulse height from ethidium bromide-stained nuclei. DNA histograms were obtained for sequential focussing of the two beams and these were virtually identical irrespective of which beam was the first excitor and irrespective of which beam the histogram was recorded from. Coincident focussing of the two exciting beams resulted in a histogram of double the emission intensity.