Chris writes- >I've got to drag my UV laser out of semi-retirement and I am wondering >just what the >latest word on alignment beads is. I need to align both my 488 and 325 >UV, lasers. Before aligning a 325 nm (I assume helium-cadmium) laser which hasn't been used for a while, you may want to measure the noise on the laser. Leaving a He-Cd idle for a few months almost always results in helium build-up in the tube, which will give you really high noise levels (power output fluctuations of greater than 50%). Since the fluctuations in laser power are not compensated for by the electronics in any commercial flow cytometer, they will translate into atrocious CV's no matter how well your laser is aligned. This not only makes it impossible to align the laser, it makes it impractical to use it (even ratiometric calcium measurements get ratty when the noise is really high, and you can just plain forget Hoechst, DAPI, etc.). Polysciences 2 um yellow-green beads, which are convenient alignment standards for 488 nm excitation, also excite at 325 nm; they're not great, but they probably will let you know if your CV is 25% as opposed to 2.5%. -Howard
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:55:33 EST