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

Clare Rogers crogers at accuricytometers.com
Tue Jan 22 10:11:29 EST 2008


Mario - Would you please clarify a couple of things about your post,
particularly the suggestion that a 530 BP is not optimal for detecting
FITC fluorescence?  

 

-Are you saying that irregardless of using a blue or yellow-green laser
that a 512 BP is always better to detect FITC?

 

-Would you also address this comment from one of our engineers:
Interestingly, if you try out a 515/20 filter in the BD fluorescence
spectrum viewer, it only captures 35.1% of the FITC emission vs. 47.2%
for the 530/30.  Additionally, the viewer shows the peak ~520nm.  

 

-And lastly, I believe there are 2 forms of "FITC" available for
conjugating - do you know what they are & if they have slightly
different fluorescent characteristics?

 

Thanks for any clarification you can give -

 

Clare Rogers

Applications Scientist

173 Parkland Plaza

Ann Arbor, MI 48103

734-994-8000 ext. 120

734-994-8002 Fax

734-776-5508 Cell

 

crogers at AccuriCytometers.com

 

www.AccuriCytometers.com

 

-----Original Message-----
From: Mario Roederer [mailto:roederer at drmr.com] 
Sent: Sunday, January 20, 2008 5:09 PM
To: cyto-inbox
Subject: Green Lasers and Fluorescein (or: Get rid of that 530/30!)

 

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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