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

[Borries Demeler]

> 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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