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

Mario Roederer roederer at drmr.com
Wed Jan 23 10:00:05 EST 2008


Clare (and others)--

I went back to my primary data on the fluorescence spectra of antibody  
conjugates (now that I've returned from my trip -- I composed the  
original email on the airplane. I should know better...)

The peak of Fluorescein emission is 517 nm.  In an optical system by  
themselves, a  515/20 filter will capture about 85% of the emission  
light as a 530/30.  So the two filters will perform roughly the  
same...  I was mistaken when I wrote the 515/20 captures more than the  
530/30.

However, I should note that for our instrument without a green laser,  
we chose a 525/50 filter for fluorescein, which captures twice the  
emission as either the 530/30 or the 515/20.  Thus, there is no reason  
to use a 530/30 -- either use a 515/20 (with a green laser) or a  
525/50 (without a green laser).

I believe all the reactive forms of FITC will give the same emission  
spectrum.

mr

(Oh, and thanks to Howard for pointing out the millimistake... in my  
youthful exhuberance, I was increasing our laser power by three orders  
of magnitude.  Or maybe all those "laser enhancement" products really  
do work!)



On Jan 22, 2008, at 10:11 AM, Clare Rogers wrote:

> 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: Cytometry Mailing List
> 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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