[RASMB] Photomultiplier tube disturbances?

smcbryan smcbryan at lamar.colostate.edu
Tue Oct 12 18:48:37 PDT 2010


Thank you all for your input. As you might recognize, the increase in
throughput via the INT->psuedo absorbance measurement regime is
significant. the machines are CSU are in nearly constant use as we assay
the saturation of nucleosome arrays, monitor mono-nucleosome homogeneity,
and many protein-p[rotein and protein-dna complexes. The increase in
resolution of solutes obtained via the conversion, time independent noise
subtraction,  and 2DSA, GA etc has proven to be very useful for examinining
many different types of systems, and has often resulted in MW
determinations with greater precision than equilibrium measurements of the
same samples (often even in the presence of contaminating solutes (unbound
DNA, etc). 
What this discussion comes down to for us is, where do we draw the line
between faulty experimental design and faulty instrumentation? The
internsity vs time scan as seen in the water sector ought to clarify this
aspect, no? Our field service technician for Beckman does not recognize the
intricacies of SV data, he only sees the output of the diagnostic scans he
has been trained to collect. It is therefore beneficial for the users to be
able to 'guide' the service technicians, so the correct parts and expertise
are brought to the repair session. 
Again, thank you all for your thoughful discussion.
steve


On Tue, 12 Oct 2010 19:31:08 -0500 (CDT), Borries Demeler
<demeler at biochem.uthscsa.edu> wrote:
>> 
>> Troy, have you read the past RASMB discussions of problems arising from
>> trying to run samples in the reference channel? Remember that the
>> instrument
>> is designed to scale the photomultiplier voltage and the programmable
>> gain
>> amplifier based on the intensity it sees at 6.5 cm before it starts
each
>> radial scan. 
> 
> John,
> 
> from what I see it is not entirely clear that this is the problem in
> this case.  UltraScan will give you a plot of the intensity over time by
> averaging the intensity for water scans. I have seen two cases now where
> the culprit was not absorbance in the reference channel, but clearly
> a problem with the intensity fluctuating wildly. A replacement data
> acquisition board fixed the problem in one case, a new monochromator in
> another. There could be other causes.
> 
>> It seems to me you are seeing exactly what has been discussed
previously:
>> when the DNA boundary in the reference channel sediments to near 6.5
cm,
>> the
>> instrument is readjusting the PMT voltage and/or the PGA gain, and this
>> produces a break in the pseudo-absorbance data for both channels of
that
>> cell. Basically pseudo-absorbance only works correctly if the
instrument
>> ends up not changing the PMT voltage or PGA setting for that cell at
all
>> during the run; if the instrument decides it needs to do so, either
>> because
>> something is sedimenting in the reference, or just because the lamp
>> intensity is drifting for some reason, you are screwed.
> 
> ...lamp intensity should of course not drift so much that it affects the

> quality of the data. In absorbance mode you are only caching the problem
> as stochastic noise increases with decreased intensity. This may be very
> hard
> to pick up, but looking at the intensity over time will reveal if some
> hardware
> problem exists, or if your buffer absorbs, adding to the absorbance of
> your sample.
> 
>> Whenever you run in pseudo-absorbance mode you are asking the
instrument
>> to
>> do something it wasn't ever intended to do. When the OD in the
reference
>> channel is quite low it usually works okay, but you are risking getting
>> low
>> quality or un-interpretable data. You also put yourself in a position
>> where
>> you can't complain to Beckman that something is wrong.
> 
> I must say that we have gotten consistently *excellent* results with
this
> approach
> and I haven't run a velocity run in absorbance mode in years. The
biggest
> benefit from doing what Troy does is a reduction of stochastic noise by
a 
> factor of 1.4, but also running a low concentration sample in the
reference
> channel is nice since you don't have to pay the price for the additional
> scan time and increase the capacity of the machine. So, yes, there is a
> risk
> that you screw up your data if your absorbance is too high, but that is 
> easily enough controlled, and the benefits far outweigh the downside.
> I'll attach an image for a side-by-side comparison of absorbance vs.
> intensity
> of the same dataset. Note the total OD and the difference in stochastic
> noise.
>  
>> I don't know if Tom Laue's AOS operating software can get into the
>> machine
>> internals and modify this re-scaling behavior. I tend to doubt it, but
>> if it
>> could that would help tremendously when running in pseudo-absorbance
>> mode.
> 
> As far as I understand it the AOS currently only works with the FDS from
> Aviv, not
> with the Beckman absorbance system.
> 
>> As Borries said, probably the only way to get around this issue is to
>> lower
>> the OD in the reference channel, or just run in normal absorbance mode
>> with
>> buffer in the reference.
> 
> ...or in intensity mode with water in the reference (provided your
machine
> works properly, if not, you should get it fixed anyway).
> 
> -b.

-- 
Steven McBryant, PhD
Director
Protein Production and Characterization Facility
Research Scientist/Scholar
Colorado State University
970-491-5586



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