Alignment and focusing unit for dual-laser excitation in the fluorescence-activated cell sorter

The two laser beams in a dual-laser fluorescence-activated cell sorter FACS-II can be aligned and focused independently on the sample stream with an additional unit, which can be fitted easily on the optical bench of the FACS. The unit consists of two spherical lenses, which have been mounted in separate holders and can be moved in three directions by way of micrometer gauges. The lenses, which have different focal lengths, have been cut off on one side so each laser beam only passes one lens. The setup has been tested using the flow analysis of a suspension of double-stained chicken red blood cells. The histograms of both fluorescence signals showed normal distributions with a coefficient of variation of approximately 6%. After willful interference with the adjustments, the laser beams could be readily readjusted within five minutes.