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<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=306092215-23082007>I
think a couple of points regarding the use of pseudo-absorbance were
not sufficiently brought out by either Bo Demeler or Peter
Schuck:</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=306092215-23082007></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=306092215-23082007>(1)
With pseudo-absorbance there are vertical shifts of the scans from one scan
to another, somewhat similar to the TIN or 'jitter' in interference data.
Treating this systematic noise as a true constant vertical displacement
certainly helps, but if you look closely at the structure of these shifts they
do not appear to be truly constant across the cell. One source of this effect is
clearly the adjustment of the photomultiplier voltage based on the light levels
at 6.5 cm, as Peter mentioned, and thus the shifts tend to be largest as
the boundary moves past 6.5 cm.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=306092215-23082007></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=306092215-23082007>(2)
I've seen pseudo-absorbance data gathered by a number of users of
DCDT+ and that strongly suggests that this approach really only works well when
the absorbance in both sectors is pretty low. At higher ODs the
systematic errors associated with (1) and other effects appear to get
much worse. Exactly how high an OD is reasonable to use depends on the quality
of the data you need, so it is hard to give a general cut-off, but I would
probably stay below ~0.5 OD. Anyone planning to use pseudoabsorbance should do
their own testing to see what errors are acceptable for their particular
application.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=306092215-23082007></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=306092215-23082007>With
respect to (1) it would be useful for Beckman to provide an option that
suppresses the photomultiplier voltage adjustment after the first scan of each
cell, and that also drops the assumption that the voltage should be initially
optimized for measuring sample - reference.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=306092215-23082007></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=306092215-23082007>Best
regards,</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=306092215-23082007></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=306092215-23082007>John</SPAN></FONT></DIV>
<DIV></DIV>
<DIV><FONT face=Tahoma size=2>-----Original Message-----<BR><B>From:</B>
rasmb-bounces@rasmb.bbri.org [mailto:rasmb-bounces@rasmb.bbri.org] <B>On Behalf
Of </B>Peter Schuck<BR><B>Sent:</B> Thursday, August 23, 2007 5:10
AM<BR><B>To:</B> rasmb@server1.bbri.org<BR><B>Subject:</B> Re: [RASMB] XL/I
Absorbance Problem (follow-up)<BR><BR></DIV></FONT>
<BLOCKQUOTE dir=ltr style="MARGIN-RIGHT: 0px">Hi All, <BR><BR>we've used the
intensity data acquisition for a long time now after we discovered that the
noise structure of log(raw intensity) data is similar to that of interference
optical data and can be treated with the same algebraic noise elimination
technique. However, this also introduces the same disadvantages that we
have in interference optical detection from the lack of an optical reference
baseline. Therefore, we only use it for doubling the rotor capacity if
necessary. One should mention one particular downside of the use of
different samples in the two sectors, which is that one can't have a high
absorbance in the reference sector at 6.5 cm, because that's a reference point
for the photomultiplier voltage adjustment. More details are in the
original publication Anal. Biochem (2000) 285:135-142<BR><A
class=moz-txt-link-freetext
href="http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45FK4WR-4X&_user=10&_coverDate=10%2F01%2F2000&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=9f4421898d4498cb5245fdeaf9931f41">http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45FK4WR-4X&_user=10&_coverDate=10%2F01%2F2000&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=9f4421898d4498cb5245fdeaf9931f41</A><BR><BR>So
far I did not notice a marked improvement of signal/noise ratio when using
pseudo-absorbance instead of absorbance data. Marc Lewis has rigorously
analyzed in detail the statistical properties of intensity versus absorbance
data (see Methods Enzymology 2004, 384:232-242). As I see it, the
problem is more that introduced by taking the logarithm of the photomultiplier
counts, which causes nonlinearities in the error propagation, but I think this
is more of an issue in sedimentation equilibrium analysis, where the question
of statistical weights for the data points can have a higher impact.
<BR><BR>If you use TI noise subtraction from the regular absorbance data, you
can get excellent signal/noise ratio, as well. I'm puzzled by the 0.3 OD
example, which is actually well in the range of OD values that can be very
conveniently studied by regular absorbance, usually with or without TI
noise. In fact, velocity data down to 0.03 or less absorbance units can
be quite fine for c(s) analysis in SEDFIT, as repeatedly shown over the years
by a number of different labs.<BR><BR>Peter <BR><BR><BR><BR>Borries Demeler
wrote:
<BLOCKQUOTE cite=mid:200708230015.l7N0FZeG007451@biochem.uthscsa.edu
type="cite">
<BLOCKQUOTE type="cite"><PRE wrap="">Although many people record the lamp wavelength/intensity profile from
time to time e.g. to check wavelength calibration, I would recommend
also taking every so often a radial scan (collect intensity data) on an
empty hole at your favourite wavelength. This will rapidly show up any
developing problems in the lamp/monochromator/PMT system and help to
distinguish them from cell-associated problems. I'm quite happy to
compare "before and after" scans with anyone who thinks they have a
similar problem, just email me.
</PRE></BLOCKQUOTE><PRE wrap=""><!---->
Hi Andrew,
I think this is very good advice. In fact, the majority of our velocity
absorbance data are now actually collected in intensity mode. For
velocity data, time invariant noise that will always show up much more
dramatically in intensity mode (when no reference data is subtracted)
can be used for determining if the machine is working properly in the
way Andrew suggested and simultaneously can be cleaned up by TI noise
removal procedures (in ultrascan while doing a 2-dimensional spectrum
analysis) so the data can actually be used.
I would like to add one more point:
The results you can get from the intensity acquisition are remarkably
good (and after TI noise removal actually better than absorbance data)
because the stochastic noise from the reference data acquisition is not
convoluted with the stochastic noise that was acquired with the sample
channel. This buys an approximate square-root of 2 factor improvement
in random noise. As a result, you can measure at lower ODs and still
get acceptable data without too much noise. Another benefit is that
you can load 2 different samples in each cell and double your capacity.
I also recommend to do a water scan to get an intensity variation trace
that can be used to get improved I_0 values for the pseudo-absorbance
conversion. A routine is included in UltraScan to help with that.
I am attaching an example of a BSA run with just 0.3 OD absorbance before
and after TI noise subtraction using the 2-dimensional spectrum analysis
in UltraScan. You can see that even at 0.3 OD the data have remarkably
small random noise contributions and are quite suitable for routine
analysis after TI noise subtraction. The later versions of UltraScan now
also include modules for pre-processing intensity data and converting
it to pseudo-absorbance data.
Regards, -borries
</PRE><PRE wrap=""><HR width="90%" SIZE=4>
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