Laser auto-alignment

When should you do a laser auto alignment?

A given batch of calibration spheres should give a peak at roughtly the same location on the x-axis (for a given gain or PMT setting). The peak should also be of the same width, as evidenced by the CV and HPCV. If the peak is wider than usual or not in the same location, first try/check the following (not in any particular order):

• make sure the sheath fluid has been filtered
• rinse once or twice
• flushe the system (In the "instrument" menu)
• use a fresh batch of calibration spheres

If none of the above work, the laser may be out of alignment.

Procedure

• Use calibration spheres diluted in sheath fluid. Our NPE rep uses the 3.8um spheres, but other spheres will also work;
• Go to Auto optical alignment in the Instrument menu;
• Click Start
• Make sure the rate is < 500 counts/sec
• Adjust the PMT setting (the "gain") of the fluorescent channel to get the peak between channel 150 and channel 250 (near the little red square on channel 200);
• Click on Laser alignment. (Note that "laser alignment" aligns both the objective focus and the position of the flow cell, while "focus alignment" only adjusts the objective focus.).
• With the 3.8um calibration spheres and the setup as described above, the best value should be >= 16.
• Note that after alignment, you may see a change in the position of the fluorescent peak e.g. before it might have been around channel 200, while after it was around channel 205.