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

Richard Grenfell rlg at mrc-lmb.cam.ac.uk
Tue Jan 22 11:13:49 EST 2008


I'd like to second this.

I use the 528nm line from an Argon ion on my Moflo to excite some of the 
red fluorescent proteins, alongside my usual 488nm line, and I routinely 
use a 510/21 bandpass to collect the FITC or GFP fluorescence at the 
same time.

Richard

Mario Roederer wrote:
> 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).

-- 
Richard Grenfell
Laboratory of Molecular Biology
Medical Research Council
Cambridge
rlg at mrc-lmb.cam.ac.uk 




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