High‐speed photodamage cell selection using a frequency‐doubled argon ion laser

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Abstract
A flow cytometer was developed for the high‐speed “sorting” of desired cells by selectively irradiating (zapping) the undesired cells from a population. After previous efforts to photoinactivate cells with photosensitizers had failed, it was decided to exploit the photosensitivity of the cell's DNA at 257 nm. It was shown that a 257 nm laser output power of 20–100 mW was sufficient to induce a 4.5 log cell kill after the cells were processed through a focused 257 nm laser beam. Experiments proved that the photodamage flow cytometer (ZAPPER) could selectively photoinactivate cells at rates over 22,000 events/s, and selection purities ranged from 81% to 100%. The yields of the desired cells depended on the selection mode. In the Enrichment mode, the zap laser was not aimed at the jet, and only undesired cells were exposed to a brief ultraviolet (UV) pulse after modulation of the UV laser beam. The yields of desired cells ranged from 95% to 105%. In the Purge mode, the zap laser beam was aimed onto the jet, and only desired cells were allowed to pass after deflection of the UV laser beam; the yields of desired cells ranged from 12% to 52%. The cause of the reduced yields in the PURGE mode was traced to the fact that the Electro‐Optic Modulator was used to modulate the zap laser proved too slow for the intended application. The lifetime of the frequency‐doubling crystal used for the generation of the 257 nm beam was found to be limited to several days. These technical limitations could theoretically be overcome by the application of large argon ion lasers capable of emissions at 257–275 nm and an Acousto‐Optic Modulator to deflect the zap laser.