Tuesday, March 18, 2008

LSRII #2: Best Color Combos

Considering this LSRII will have some of the same lasers/filters as our other instruments, you may be thinking the color combinations will probably be the same. Well, you'd be right, but maybe not for the right reasons. Here, I'll explain the best color combos, and why.

Just for redundancy's sake, let's take a look at what's available. A 405nm with 3 PMTs, a 488nm with 2PMTs, a 561nm with 4 PMTs, and a 640nm with 3PMTs (4 lasers, 12 colors). So, let's say you want to do a 12 color experiment, which colors will you use.

Let's start with the blue. Off the 488nm laser you're options are going to be FITC, PerCP or PerCPCy5.5. Now, if you're going to use PECy5.5 off the YG laser, then you'll want to use PerCP off the Blue instead of PerCPCy5.5. If however, you will use PECy5 off the YG, then you'll want to use PerCPCy5.5 off the blue. Next, we'll tackle the red. Off the Red laser, your options are APC, APCCy5.5 or Alexa 700, and APCCy7 or APCAlexa750. We have a similar situation as before, you'll want to repeat "Cy5.5" as few times as possible. Additionally, you'll want to avoid repeating the same emission spectra off different lasers as much as possible. Cy5 and APC have the same emission, so you'd want to avoid using PECy5 and APC together. APCCy7 is not that great, so you probably want to opt for the APCAlexa750 option. Now, for the yellow-green (YG) line. Any of the PE and PETandems would be appropriate, so you'll have PE, PETexasRed, PECy5, PECy5.5, PECy7, PEAlexa610, etc... Again, pick emissions that you have not duplicated elsewhere. Finally, you have the violet. For the violet your choices are Pacific Blue, Pacific Orange, Qdots, or dyes like DAPI. Special care should be taken when choosing Qdots as most of the Qdots will be excited by the blue laser and maybe even the YG laser. They also have high quantum yields, so even if they get excited by a non-optimal laser line, they'll still be pretty bright. You should again try to use the Qdots in places where you have gaps in the emission of your other fluorochromes.

So, with all that said, let's pick a panel. I'm going to propose PacBlue, Qdot 565, Qdot 625, FITC, PerCP, PE, PEAlexa610, PECy5.5, PECy7, APC, Alexa700, APCAlexa750 for my 12 color assay. I chose the two Qdots because they fill the gap between FITC and PerCP, and are less likely to be excited by the YG or Red laser. This 12 color combination will offer the greatest sensitivity with the least amount of compensation requirements. If however, you need to look at fewer colors, you could envision a panel of maybe 6 or 7 colors requiring little or no compensation. Here's an example: Pacific Blue, Qdot 625, FITC, PerCP, PE, PECy5.5, APC. This 7 color panel would have little to no compensation necessary whatsoever. Pretty cool, eh?