<!DOCTYPE html PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
<html>
<head>
<meta content="text/html;charset=ISO-8859-1" http-equiv="Content-Type">
</head>
<body bgcolor="#ffffff" text="#000000">
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 size="4" width="90%">
_______________________________________________
RASMB mailing list
<a class="moz-txt-link-abbreviated" href="mailto:RASMB@rasmb.bbri.org">RASMB@rasmb.bbri.org</a>
<a class="moz-txt-link-freetext" href="http://rasmb.bbri.org/mailman/listinfo/rasmb">http://rasmb.bbri.org/mailman/listinfo/rasmb</a>
</pre>
</blockquote>
</body>
</html>