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

larry_arnold@med.unc.edu larry_arnold at med.unc.edu
Mon Jan 21 21:36:46 EST 2008


Mario

Thanks for pointing this out to everyone.  You fail to mention that going to a 561nm laser will only mean you have a similar problem with PE as you do for FITC when using the 532nm laser.  You will still have to narrow the PE NI filter some (or shift it some - will depend on what you are trying to exclude on the high side of PE - presumably PE-TxRed)  to exclude the 561nm laser light.

We are going to put a 594nm laser (200mW -MPB Communications fiber laser)  (as well as a 532nm laser) on the Reflection we have just received. Our rationale  is that the 594nm laser excites mChery fluorescent protein as well as 561nm and better than a 532nm laser power for power (Bill Telford Nature paper) and can be used to excite  Texas Red as well as Alexa 594.   Many use a 575/25 NI filter for PE and this should exclude the 594nm light from the PE channel as well as the FITC 515/20NI excludes the 532nm laser from the FITC channel.  B-D as well is producing LSRIIs with the 594nm laser (in combination with the 532nm)  (Brent Wood has the most experience with this I'm told).  

I'm sure most will not be mislead by your inadvertent use of "watts" when meaning "milliwatts". 

Also where do you get the 200mW 532nm laser?  I thought you were using the Coherent Compass laser that I thought only came in 150mW.  Do they now have a 200mW version or is this another company's laser?

Regards, 

Larry

----- Original Message -----
From: Mario Roederer <roederer at drmr.com>
Date: Monday, January 21, 2008 5:23 pm
Subject: Green Lasers and Fluorescein (or: Get rid of that 530/30!)
To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>

> 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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