From: James Slattery (SLATTERY@BIOVAX.TN.CORNELL.EDU)
Date: Mon Feb 15 1993 - 09:28:00 EST
>I would appreciate hearing from anyone that has done bivariate
>flow karyotyping of chromosomes using Chromomycin and Hoechst with
>either an EPICS Elite or EPICS 752. In particular I am interested
>in exactly how the "gated amp" trigger should be set up to acheive
>the best results. FALS seems to be un unreliable trigger source
>for this application and there will always be fluorescent emmision
>from both the UV and 458nm beam at nearly the same wavelengths.
>The gate trigger will only work properly on a "clean" signal that
>is unique to the top beam. So my question is : what is the best
>signal to use for this application?
I have done a fair amount of work with tomato chromosomes using the
Chromomycin/Hoechst staining on a 753 configured to a 752 and the
MDADSII. As you said the emission of the two stains is similar so
we got around that using long pass filters and a single PMT detector.
The signal from that PMT was split using a BNC T-connector so that
the split signal was going into the two different amplifier circuits
e.g. PMT1 and PMT2. In this way the PMT voltage was set on PMT1 and
gains could be independently adjusted using both PMT1 and PMT2 circuits.
Using the Auxilary circuit is not a viable option since the pulse
generated is a "PINT" i.e. a mixture of peak and integral. The problem
that generates is that the gated amp circuit just does not handle
the mixed pulses very well.
I know that a "clean" signal should be used for signalling.
In fact, you can use the above configuration and trigger on the
UV generated signal which we set to be the first beam encountered
and was sent to "PMT1". The delay was 'on' for PMT1 and 'off'
for PMT2 which processed the chromomycin derived signal. The one
thing that caused me the greatest problem in the beginning was
without really thinking it through I had set the gains fairly
high (10 or 20). This generates enough noise that the triggering
was not very clean. This was solved by dropping the gains
down to 2 or 5 and increasing the PMT voltage. ( A gain of 1
is not acceptable since the ADCs saturate at ~9V on that setting).
The trigger gain should match that of the signal being used to
generate the trigger.
When you are setting this up use very slow flow rates
(~200 events/sec). After it is set up you can decide how fast
you can get analyse given what your needs are but the
triggering is touchy and behaves better at the low flow
rate.
I hope this helps. If you have any questions feel free
to give me a call.
Jim Slattery
Cornell University Biotechnology Program
Flow Cytometry and Imaging Facility
607-254-4862
slattery@biovax.tn.cornell.edu
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