One of the key attributes we wanted in our new LSRII was an excitation source in yellow-green (Y-G) spectrum. In this regard, there are basically 2 options. The first option is the 532nm Diode-Pumped Solid State (DPSS) laser, which has been in use for many years in flow cytometry, and the newly "usable" 561nm DPSS laser, which has gained popularity in the past 2 or so years. The benefits of any green/yellow laser is the ability to excite fluorochromes not excited by the standard 488nm or 633nm lasers commonly found on flow cytometers. There has been this huge gap between blue and red that has been neglected on mainstream analyzers for years. On sorters, people have used gigantic gas or dye lasers to get lines like the 532nm or 594nm which was ok for sorters, but those lasers don't really fit in the footprint of the bench-top analyzer. These green/yellow lines are needed for excitation of fluorochromes like Texas Red, mCherry (and the rest of the fluorescent proteins), and even Phycoerythrin (PE). What? PE? Yep, you read correctly. You may be thinking, but PE is my brightest fluorochrome, surely it is excited well by the 488nm laser. That may be somewhat true, but if you look at the actual absorption spectrum (http://www.bdbiosciences.com/spectra/) of R-PE, you'll see that at 488nm, R-PE is excited at only about 50% of its max absorption. At 532nm, it increases to about 80% of max, and at 561nm it's nearly 100% of max absorption. So, as you can see, the green/yellow lasers excite PE up to 2x better than a 488nm laser. What this means, is that not only will you be able to see fluorochromes like Texas Red and mCherry, but all your PE and PE-tandems will appear brighter off the 561nm laser than they would on a comparably powered 488nm laser.
And, if that's not enough to make you jump for joy, listen to this. Now that we're exciting PE off of the YG laser, and FITC off the blue laser, we have a built in temporal and spatial separation between the FITC emission and the PE emission. What does this mean??? NO COMPENSATION BETWEEN FITC AND PE!!!! That's right, the spillover of FITC emission into the PE channel is close to 0%. We all know that spillover reducing resolution of dim populations, so pairing better excitation of PE with no spillover from FITC means super resolution of dim PE stained populations. This is where the power of YG comes into play!
So, if you haven't figured it out yet, we went with the 561nm laser instead of the 532 laser. Here's a couple of reasons. 1. the 532nm laser interferes (to some degree) with the emission of FITC, so you'll need to use a notch filter to make sure you exclude the green 532nm laser light from the green FITC emission. 2. With the longer yellow-green emission, we can better excite PE, Texas Red, and a whole host of fluorochromes people want to use. 3. Laser powers are increasing for the 561nm. It use to be that you got the 532nm because the 561nm only gave you 20mW of power or so, whereas you could get 500mW of 532nm. There are now 75mW 561nm lasers available, bringing us closer to the coveted 100mW mark for lasers on flow cytometers, a point where we should be maxed out on excitation. 4. I actually tested the 532nm versus the 561nm on an LSRII at BD, and thought the excitation and "brightness" was pretty comparable even though the 532nm was at 200mW and the 561nm was at 50mW. Given comparable PE "brightness" we went with the longer wavelength laser in order to hit more fluorochromes our users are interested in.
Got more questions about this feature of the new LSRII #2, shoot us an email @ firstname.lastname@example.org
Monday, February 18, 2008
Thursday, February 14, 2008
In the coming weeks, the Flow Cytometry Facility will be getting its second BD LSRII. This instrument will have some additional lasers and fluorescence detectors compared to our current instrument. We're really excited about the potential of this instrument. The laser choices are what makes this instrument unique amongst all our analyzers. In the next few posts, I will be familiarizing you with the capabilities of this new instrument so you can prepare your experiments to take full advantage of what it has to offer. Some of the items put forth won't be unique to this instrument, but will go over things pertinent to the DiVa system in general. I'll try to point that out as each topic presents itself.