Observations on 561 nm lasers

Telford, William (NIH/NCI) [E] telfordw at mail.nih.gov
Mon Jan 21 23:34:25 EST 2008

Hello everyone

I completely agree with Mario that a 532 nm green laser can certainly be compatible with FITC detection if the correct bandpass filter is used.  I’ve also talked with several LSR II users who think they see high backgrounds in their FITC channel when exciting PE with a 532 nm laser, and the cause is definitely a FITC filter that insufficiently excludes green laser light.  If you explain to the filter manufacturer that you will be using a green DPSS in your system, they can readily produce a short green filter with these characteristics.  

We’ve used both 532 and 561 nm lasers to excite PE, and both lasers are significantly superior to 488 nm.  A word of caution, however – if you are using a 561 nm laser to excite PE, the traditional 575/26 and 585/42 nm bandpass filters traditionally used for PE detection are too close to the 561 nm emission, and are not adequately blocked for this laser line.  You need to go to a slightly longer filter.  In our original Cytometry paper, we used a 610/20 nm bandpass to avoid the laser line – this has proven to be a less-than-optimal choice, as it is too far shifted off the PE emission spectrum.	We’ve since had several 590/20 nm filter made by Chroma and Semrock, and these are much more sensitive for PE while being compatible with 561 nm laser light.  I’m attaching data (Telford_561nm_laser.pdf) where you can see a comparison between filters ranging from 575 nm to 610 nm, using 488, 532 and 561 nm laser sources (power-matched at 50 mW).  The 590/20 nm and usual 575/25 nm and 585/42 nm filters all work pretty much equally well, the 610 nm filters less so.  As with the 532 nm laser, you need to make a small modification in your filters to accommodate the addition of this laser.

We’ve done some comparisons between 532 and 561 nm lasers for PE detection, which are also included in the linked document. 561 nm lasers at 50 mW improve PE sensitivity somewhat when compared to 532 nm at the same power level using our new 590/20 nm filter – this is not too surprising if you look at the PE excitation spectrum, which peaks around 556-560 nm. The 561 nm laser also likely excites less cellular autofluorescence, which will also improve the signal-to-noise.  But no one with an existing 532 nm install should panic and replace it with a 561 nm just for PE detection!	This comparison did not look at higher power levels, since (until recently) 50 mW was the highest power level available at 561 nm.  532 nm lasers applicable for flow can exceed 200 mW, which would presumably improve sensitivity.  But 561 nm lasers are now available at 100 mW, and higher power units are coming, so they should be comparable in power to the green very soon.	But also keep in mind that laser wavelength and power are only part of the equation – the laser must be properly integrated and aligned into your system, or you will get suboptimal performance even with optimal excitation.  And your filters have to be compatible with your lasers.

As Mario also points out, both 532 and 561 nm lasers are also useful for other things, especially fluorescent proteins.  Your applications beyond PE should also guide your laser choice.  The 532 nm does a great job with some of the shorter wavelength fruit FPs, including DsRed, mTomato, mRFP and mOrange.  The 561 nm laser will work for these too, although again you might have to use slightly longer filters.  We move to the 561 nm for better excitation of mStrawberry, mCherry and other mid-range red FPs.  And all the FPs definitely benefit from higher power levels.  We’ve used the 532 nm laser to excite rhodamine-based probes, and Texas Red and Alexa Fluor 568 and 594 with the 561 nm.
So when I’m asked if one should get a 532 or 561 nm laser, I’m always reluctant to answer – they are both very useful.  My ideal cytometer would have both of them, and with improvements in design and lower laser costs, this should soon be reasonable to achieve.



(Some info about the attached experiment – we labeled EL4 thymoma cells with PE-conjugated antibodies against CD3, CD25 and CD95.  Expression of all of these markers is low, so we could observe sensitivity at the low end of the detection scale.  All lasers were power-matched to 50 mW and beam profile-size matched.  The same trigon, filters and filter path lengths were used for all comparisons, and the detector gains were fixed throughout the experiment.  All samples were analyzed on the same day.  This is raw data and no sensitivity metric has been applied yet, but this can be done easily.)
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