Green Lasers and Fluorescein (or: Get rid of that 530/30!)

Mario Roederer roederer at drmr.com
Sun Jan 20 17:09:20 EST 2008


This message is solely intended for those of you who are outfitting a  
new instrument, or retrofitting an existing one, and are thinking  
about green or yellow-green lasers.

I recently visited some laboratories that are acquiring new LSR II  
instruments, and was told that BD is recommending that they equip the  
instruments with "yellow-green" lasers (~560 nm) rather than green  
lasers (532 nm).  The stated reason for this is that the green laser  
has been observed to result in deteriorating fluorescein (FITC)  
measurements.

The big reason to use a green laser is because of the significantly  
better detection of PE and PE tandems.	This comes about for two  
reasons -- one is the increased excitation efficiency at 532 nm  
(compared to 488 nm), and the second is the availability of high-power	
lasers (200 watts).  In multicolor experiments, we find as much as 10- 
fold increased sensitivity on these channels using a high-power green  
laser compared to low-power blue laser.  (The benefit over a high- 
power blue laser is still significant, albeit less so).

It is possible that the green-yellow laser may provide this advantage  
as well; I'm not sure because we haven't done the comparison.	
Certainly, if you get a 560 nm laser at less than 200 watts, I would  
expect it to perform less well than the 200 watt 532 nm laser.

So why the recommendation for the switch to a the green-yellow laser?	
Because of the poor FITC results.  But changing lasers is not the  
solution -- changing filters is!  Most FITC measurements are made with	
a 530/30 nm filter -- a filter that is nearly centered on the green  
laser.	Thus, the high powered green laser provides all sorts of stray	
light that contaminates this measurement (off the blue laser), and  
leads to high background.  There are two solutions to this:  one is a  
"notch" filter that selectively blocks the 532 light; the other is to  
use a shorter bandpass filter.

First, why are manufacturers supplying a 530/30 filter for FITC?   
FITC's emission peak is ~512 nm -- the 530/30 is only collecting less  
than half of the tail of the FITC emission!  We should use filter that	
is much closer to the peak.  The only reason 530/30 filters are still  
supplied is purely historical inertia. Years ago, when filter  
technology was less advanced, a filter bandpass needed to be far from  
a laser line to block the laser light -- i.e., anything closer to the  
488 than a 530/30 let through some of the blue light.  But filter  
technology is vastly better these days; we can come much closer to the	
laser line and still block it out.

Currently, we use a 515/20 filter for FITC.  Not only does it  
efficiently block the 488 nm line, it also efficiently blocks the 532  
nm line.  AND... it collects more FITC fluorescence than the outdated  
530/30 filter.

As for the notch filter solution:  don't use it!  Why take less than  
half of the available fluorescence, and eliminate a large fraction of  
it, simply to block the green laser light!  It's a silly solution that	
costs much more than the relatively inexpensive 515/20 nm filter  
(which can be ordered from any filter company).

There are reasons to get a yellow-green laser (for example, to detect  
certain fluorochromes or fluorescent proteins).  But don't get it  
because the green laser makes FITC look worse -- that's not a valid  
reason.

Finally .... if you get a high power green laser (or even if you get a	
yellow-green laser), then you should use a low-power blue laser (NOT  
the high power blue laser).  The high-power blue laser helps for  
detecting PE and PE tandems, but it does not help with FITC, and it  
hurts significantly with PerCP.  If you use the green laser for PE  
etc., then get the cheaper low power blue laser: you will get as good  
(or better) results.

Regards,

mr

PS, the information about the green laser, PE tandems, and the use of  
alternative FITC filters is fully described in Perfetto et al.,  
Cytometry A, 71, 73-9 (2007).



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