[RASMB] XL/I Absorbance Problem (follow-up)

[Peter Schuck]

Hi All,

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
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

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. 

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.

Peter



Borries Demeler wrote:
>> 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.
>>     
>
> 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
>   
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