[RASMB] Photomultiplier tube disturbances?

[John Philo]

Borries,

I'm not sure one can really diagnose what is happening with the CSU
instrument based only on the graphs I have seen (and given that the OD is
clearly > 0.2 I'm not even sure whether those are for the sample or
reference channel). I agree that if there are similar discontinuities in a
channel containing only water then something other than the normal
gain/voltage re-scaling is happening.

It would help tremendously to diagnose these issues if Beckman would just
put the information about the PGA and PMT voltage for each scan into the
.log files the GUI generates. That file is supposed to be for diagnostic
purposes but it has very little useful information. But then it should also
be trivial for Beckman to give us a mode specifically for pseudo-absorbance
where the rescaling is suppressed. Also, my understanding from Tom Laue was
that AOS actually does have code to run the absorbance system (although
perhaps it is still hidden).

I understand fully the advantages of pseudo-absorbance for lower stochastic
noise and greater throughput. However to my knowledge no one has rigorously
tested the advantages and drawbacks of the two modes, over a range of total
OD, as you would do for qualifying and validating an analytical method in
the pharmaceutical world. Just getting good residuals is not a test for
either accuracy or precision. In particular, the lower stochastic noise is
clearly a much greater advantage when the absorbance is quite low, but one
suspects the linearity (which is never very good in velocity acquisition
modes) is even worse in pseudo-absorbance mode. If you run in the
conventional way with water in the reference, record in intensity mode, and
then analyze the data with 2DSA either as true absorbance, or as
pseudo-absorbance, do you actually get the same answers (same within the
estimated precision for each approach)? If the sample OD is near 1, do you
actually get the same total concentration (same total OD of sedimenting
species)? If the distribution details differ, which seems likely, how do you
know which one is correct? If you do this for three replicates of the same
sample what happens? If such studies have been published, I must have missed
it.

The original paper on the pseudo-absorbance approach (Kar et al. 2000) did
actually show differences in size distribution details [least-square g(s)]
when such tests were made on a few samples, most of which I believe were at
relatively high OD. 

When you use pseudo-absorbance rather than true absorbance you give up
something that may be important: zero absorbance has a unique physical
meaning. Gaining a factor of the square root of 2 in stochastic noise is a
good thing, but is that advantage ALWAYS dominant over the statistical
drawbacks of introducing 50-100 additional fitting parameters (one per scan)
to handle the RIN? Again, if this has been looked at systematically, I'd
love to see that.

John

-----Original Message-----
From: Borries Demeler [mailto:demeler at biochem.uthscsa.edu] 
Sent: Tuesday, October 12, 2010 5:31 PM
To: jphilo at mailway.com
Cc: rasmb at server1.bbri.org
Subject: Re: [RASMB] Photomultiplier tube disturbances?

> 
> 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.