As a general case, knowing the emission spectrum of the dye that you want to
detect is a good start. (Emission spectral shifts after conjugating to
protein occur, but for a first approximation you can ignore the shift if you
have only the dye spectrum as excited by your laser line.) There are lots
of sources of this information (Molecular Probes, Chroma Technologies, the
java app that Joe Trotter did, etc.)
Having said that, you always have a limited number of candidate filters for
a given dye, so simply try each one and use the filter that gives you the
best siganl. On ocassion, I've been surprised that the "best" filter is not
the one you might think . (But, I have a hard time believing that a 575/26
bandpass filter is really good for Texas Red since the dye's emission peak
when attached to protein is around 619nm and is skewed toward the red, if I
recall correctly.)
Where experience contradicts theory (that's when science gets fun, afterall)
you might wonder why?
Consider the entire light path from laser/stream intercept to the detector
that reports the dye fluorescence.
1. How many filters are there? Remember that even a good filter attenuates
about 10% of the light. So after a few good filters, you've lost half of the
signal. Do you need all the filters? If you are not using all detectors,
you may be able to remove some dichroics.
2. Are the dichroic mirrors really optimal? How do you know? Do you have
transmission spectra of the filters? The manufacturer often supplies them
(you kept the sheets and put them in a binder when you received the filters,
right?) Note that the region of reflection or transmittance, may not be best
for your laser/fluorochrome combination. Moreover, depending on filter
design, there can be lots of light coming from outside the region of
interest. In addition, the laminations in filters can degrade over time.
It's fairly easy to run a transmission spectrum of you filters and determine
what the detector is really seeing.
3. Lastly, as Dave McFarland noted below, is your system really aligned
well? Very small shifts in dichroic position can give 10-fold differences in
sensitivity. A regular calibration check of your instrument is the only way
to know.
As a final remark, the optimal filter configuration will depend on the
amount of fluorescence emission overlap that exists among the dyes that
excite with the same laser. A narrower bandpass centered about the emission
peak may exclude more of the emission of other dyes and give you a better
signal (desired emission) to noise (undesired emissions) ratio. Hence, you
may have to give up some desired fluorescence to discriminate among dyes.
The empirical approach will win here as well.
Dave
========================================
David M. Coder, Ph.D.
Consultant in Cytometry
email: d_coder@msn.com or dcoder1@hotmail.com
tel./messages: 206-499-3446
----- Original Message -----
From: David.C.McFarland@gsk.com
To: cyto-inbox
Sent: Wednesday, May 29, 2002 6:30 AM
Subject: Re: How to enhance PE signal?
Jeff,
Pardon me if this is too obvious, but I would also suggest tweaking the PE
signal by slightly adjusting the dichroic between FITC (FL1) and PE (FL2).
My thought is that it MUST be something pretty obvious though, because PE
(FL2) is one signal that I've never had a problem with! And as far as I
know, the 575/26 has always been in place.
And Joe, FYI the 575/26 is listed as the standard PE (FL2) filter for all
configurations listed in Appendix B (pp308-312) of the FACSDiVa User's
Guide. I for one had no idea that this was intended for Texas Red and that
a 585/42 was suggested for PE. (I just took a quick look and I do indeed
have a 585/42).
Cheers,
Dave
David McFarland
GlaxoSmithKline
----- Forwarded by David C McFarland/DEV/PHRD/SB_PLC on 29-May-2002
08:46 -----
JTrotter@PharMingen.com
24-May-2002 13:11
To: "Cytometry Mailing List"
cc:
Subject: Re: How to enhance PE signal?
Jeff,
Usually, when this occurs, the Vantage user has PE band pass filter
designed for use with Texas Red in the FL2 channel (a 575/26) , and the
Calibur uses a 585/42. Have a look and replace the filter if necessary, you
should also have a 585/42 for the Vantage in your kit that will let in a
lot more PE signal. You didn't mention what laser power you use, 100 - 200
mW on the sorter?
Joe
"Jeffrey Rice" <rice@aecom.yu.edu> on 05/23/2002 07:05:47 PM
Please respond to rice@aecom.yu.edu
To: cyto-inbox
Subject: How to enhance PE signal?
Hello,
I'm running into a problem here with my sorting. I've got a
peptide-tetramer
using biotinylated
peptide with SA-PE. On our Calibur, I get between half a log and a log
difference
between my negatives
and positives, but on our Vantage+DiVa I lose most of that. I really need
to enhance
the PE signal from
my PE positives to improve my sort.
Now, I know things like Oregon Green labeled anti-FITC exist. Do any
FL2-labeled anti-PE
reagents exist? Or, FL4-labeled anti-APC would be OK too.
I've tried switching fluorochromes... I get good labeling on the
Calibur with
a direct labeling of the
protein using Alexa Fluor 647 (FL4), but again I lose the separation
between my
populations when I try to
sort on the Vantage. I could use a brighter SA-fluorochrome conjugate for
my tetramers,
or a brigher
fluorochome to label the protein. But I would think that using SA-PE or
SA-APC for
tetramers, or Alexa
Fluor 647 for direct labeling, are among the brightest available.
Any suggestions would be greatly appreciated, as I'm fairly well stuck
at
the moment.
Jeff
----
Jeffrey Rice
rice@aecom.yu.edu
Diamond Lab
Microbiology and Immunology
Albert Einstein College of Medicine
Bronx, NY
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:26:11 EST