Arnold Pizzey asked- >I see that the Nichia Corporation is producing 5mW 405nM laser diodes >(price ~$2000 with an expected MTBF of 15,000 hours) >Does anyone know if Hoechst would give a DNA signal if excited at this >wavelength?.... Yes, it will, but the CV won't be good because excitation is only about 5% of maximum (that's with a diode selected for emission near 395 nm; 405 nm is close to useless). Hoechst 34580 (available from Molecular Probes) excites well and may be usable for vital staining and sorting; there are questions about its stability. CV's are good (below 2%) for DAPI staining of isolated nuclei. The situation with Hoechst 33342 should improve using Nichia's 25 mW diode; I am now testing one. Results with the 4 mW version were presented at Montpellier and will be published in Cytometry, probably in the June 1 issue. >I am thinking of adding a green laser to my elite -I would be using it in >time gated mode so I'm not worried about crossover into the green channel- >the choices for (cheap)lasers seem to be between 532nM and 543.5nm -I guess >my question is; would the 532nM laser be good for much or would I have to >go for the (more expensive) 543nM unit A 532 nm green YAG laser that is cheaper than a 543.5 nm green HeNe is probably worthless due to high noise levels. A good single mode green YAG laser costs at least US$7,000, but this is a 10 to 25 mW laser, while the HeNe won't put out much more than 2 mW, and that makes the green YAG preferable for exciting, say, phycoerythrin and its tandems, even though 543.5 nm is closer to the excitation maximum. Autofluorescence of mammalian cells is much lower with 532 or 543.5 nm excitation than with 488 nm excitation, allowing you to resolve more dimly stained populations (in one of my systems, it is possible to gate reliably on cells bearing between 100 and 200 phycoerythrin molecules). -Howard
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