Design and operation of a dual laser chromosome sorter

We describe the design and operation of a dual laser fluorescence activated chromosome sorter that can analyze and sort chromosome suspensions stained with two complementary DNA stains. We used two 18-watt argon-ion lasers to provide maximal excitation of the DNA-specific stains Hoechst 33258 and chromomycin A3. The lasers are focused onto the sample stream by independent optics permitting optimal focusing of any pair of laser wavelengths. We incorporated beam expanders to decrease the energy density on the optics and improve the focal spot uniformity. Adjustable focusing lens holders are used to set the optimal focal spot size. The redesigned sorter separates suspensions of normal human fibroblast chromosomes into 21 fractions and can reliably sort more pure chromosome fractions than was previously possible.