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

Marty Bigos mbigos at gladstone.ucsf.edu
Thu Jan 24 19:19:46 EST 2008


I don't know why Mario recommends against the 532nm notch filter.  
They are more expensive (~$900) than most filters but compared to the  
cost of an LSR...

We have a 150 mW 532nm laser on our LSR. For FITC (excited with a 20  
mW blue diode) we use the 525/50 along with the 532nm notch (obtsined  
from Asahi Optical). The measurements are better than the 530/30 with  
green laser off and 515/20. However there is also an increase in  
autofluorescence signal, so the separation improves, but not  
tremendously.

Regarding PE and it's tandems with green laser excitationr, our  
measurements show that	1) the high power green does significantly  
better than the high power blue, and 2) more green laser power (up to  
150 mW) yields better excitation. It is well known that with 488  
excitation, using low power and a long transit time through the laser  
spot gives the best signal from PE.. Higher 488 power sends PE into  
some unavailable state. With green excitation this doesn't happen. PE  
is works better with more power (up to the point I measured). I  
disclaim any expertise here, and would be happy to hear more, but  
I've been told that PE internally is made up of a number of energy  
transfer moieties (somewhat similar to the tandem dyes) which is why  
the excitation curve is so broad. Supposedly the blue absorbing  
moiety maxes out easily, but the green absorbing one doesn't.

The comments about the bandpass filter blocking the laser excitation  
line is very relevant. The HQ525/50 we bought from Chroma has an OD  
of 6 at 488nm. Because almost all cytometers have some scattered  
light from all the lasers, it is important that bandpass filters  
block other laser lines at OD 3.

DON'T LOOK AT LASER WITH REMAINING EYE! but your eye is a good	
detector for checking the filters out. Turn off the room lights and  
look through the bandpass a a brightly scatter spot of light from the  
laser. If you see the light through the filter then you will have  
problems using it. You need to make sure, though, that the bandpass  
is 90 degrees to the incident light. Tilting it some will change its  
properties.


Marty Bigos, Director
Gladstone Flow Core

mail:
J. David Gladstone Institutes
1650 Owens Street
San Frnacisco CA 94158

Phones:
(office) 415-734-4821
(mobile) 415-845-8450

fax:
415-355-0855



On Jan 20, 2008, at 2:09 PM, 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).




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