[Cytometry] Colour compensation

Cris Bare flowmail at verizon.net
Fri Apr 19 11:18:46 EDT 2013


Mario's first line statement is true even with the imprecise nature of diode based blue laser wavelength. Whether the laser emits slightly off of 488 my manufacture specs, or with what Bob Hoffman calls ASE side band emissions, the primary scattered light will far overwhelm the teensy bit of fluorescence signal.

To address Mike's last line, instruments that have hard-wired compensation circuits (Calibur) won't be able to compensate FSC and SSC, but third party offline analysis software will. Or at least almost any currently available analysis package. I'm not sure if Rabab is running CXP, RXP or whatever the FC500 currently ship with, but when I used RXP it saved a hybrid file format that allows post acquisition adjustment of the compensation matrix (not that you should if you use proper controls!).

-cbb
http://www.linkedin.com/profile/view?id=28573199


Apr 19, 2013 07:16:51 AM, roederer at drmr.com wrote:

===========================================

Fluorescence always involves loss of energy.  Thus, if you excite at 488 nm, then emission will be higher wavelength than 488 (usually well far enough to be out of the range of the narrow SSC filter).  Even if the SSC filter is too broad (most are +/- 5 nm) and does pick up some fluorescence, the amount of light from scatter is orders of magnitude higher than fluorescence, so you would never see it.

You might use SSC to "compensate" fluorescence signal if you want to remove a size-dependent component of the signal (e.g., if fluorescence is linearly related to SSC, such as big cells have more fluorescence and you want to know the fluorescence density rather than magnitude). This is more commonly done to "correct" for autofluorescence ("autofluorescence compensation") but really only works decently in highly autofluorescent cells (big cell lines, macrophages).  It's not really compensation, but the math is sufficiently similar that the compensation algorithms will work.

That being said, I'm guessing Rabab's query was not to use SSC as a compensation parameter, but rather with some automated set up that the FC500 software uses.

BTW, I applaud the use of log scaling for SSC.  You get much better visualization of cell distributions in log SSC (with linear FSC)!

mr

On Apr 18, 2013, at 1:39 PM, Michael Waring wrote:

> Actually, I've heard that you might need to compensate the scatters, if you
> have something like GFP--the scatter detectors are measuring LIGHT, just
> like the other channels, so any emission in their range of detection will
> be measured--GFP actually has emission about 20% of maximum at 488, so that
> light would "spill over" into the FSC and SSC detectors and make the GFP+
> cells seem larger than the GFP negative cells... That said, I dont know of
> any instruments that actually allow for this correction?
> 
> Mike Waring
> Ragon Institute
> Massachusetts General Hospital
> 
> 
> On Thu, Apr 18, 2013 at 6:11 AM, Vivek Tanavde  wrote:
> 
>> Hello Rabab,Why would you want to do compensation on side scatter? As far
>> as I know compensation is necessary for bleed through of a fluorescent dye
>> in other PMTs detecting fluorescence. Since the SSC detector does not
>> detect fluorescence, it is not necessary to compensate the signal on this
>> detector.Regards,
>> 
>> 
>> Vivek Tanavde
>> 
>> Bioinformatics Institute
>> Singapore
>> 
>> From: rababhaider3 at hotmail.com
>> To: cytometry at lists.purdue.edu
>> Date: Tue, 16 Apr 2013 19:18:33 +0400
>> Subject: [Cytometry] Colour compensation
>> 
>> Hi everyone!
>> I'm using FC500.
>> Is there anyone uses FC500 and knows how to do the automated compensation
>> on SS Log scale. All the options from auto setup schedule in the software
>> uses FS Lin/ SS Lin.
>> Thank you,
>> Regards,
>> Rabab Al lawati
>> Royal Hospital
>> Oman
>> 
>> 
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