Wednesday, July 23, 2008

The trouble with Filters

We've recently gone through some filter issues on the new LSRII-B. It turns out 2 of the filters pre-installed on our LSRII were bad. The two in question are the Alexa 700 filter and the Pac Orange. The Alexa 700 filter (720/40) was a non-standard diameter filter (probably 12mm diameter or so), and when the filter was in place, it would tip forward a bit since it was not snug in the filter holder. This allowed stray laser light or ambient light to get in and swamp the detector. The solution? Simply putting in a standard, 1 inch filter at 730/45. This should now allow for true, 3-color detection off the red laser. The Pac Orange filter was just a bad filter...sometimes you get a bad coating. We received 3 filters to try in its replacement; a 560/40, 550/40, and a 565/30. The winner will be chosen empirically by looking at not just how much PacOrange it collects, but how much Qdot 605 and PacBlue is excludes. If you want to give the different filters a try yourself, send me a note and we'll take a look together.

Next up to tackle on the LSRII-B...PE optimization. The problem with PE on the LSRII-B is the fact that the laser line exciting PE, 561nm, is so close to the emission of PE that there's a propensity to swamp the detector with laser light. I'll be testing a few different filters to see where we can get optimal PE detection without an increase in background from laser light. The PE-tandems, on the other hand, are absolutely fabulous off that laser line. No increase in background, and screaming bright fluorescence... Enjoy!