Design and application of a versatile triple-laser cell and chromosome sorter

A high-resolution triple-laser sorter was designed and constructed to provide flexible switchover and high-resolution sorting of cells or chromosomes with any combination of one, two, or three lasers. These features provide a central facility instrument that currently serves multiple users and analyzes different stain combinations with minimal switchover effort between experiments. Improved optics and mounts that focus the three laser beams independently are able to resolve beads and chromosomes better than our previously reported dual-laser sorter. An improved signal collection unit with electronically controlled reference positions can be focused more quickly and precisely for any signal combination. A removable dye laser extends the range of usable fluorochrome labels. A rapid sheath switchover permits sorting of sterile cells and sterile chromosomes sequentially without additional sterilization or reservoir sheath change. Improved dual-laser chromosome resolution is at least as good, analyzing 8,000 chromosomes/s, compared to the previous dual-laser bench at 2,000/s. Stimulated and unstimulated peripheral blood lymphocytes were analyzed according to simultaneous measurements of cell surface receptors labeled with a fluoresceinated neuropeptide and a Texas red-labeled antibody as well as DNA content during the cell cycle. These results demonstrate the broad range of potential applications of this triple-laser system.