[RASMB] AUC interference concentration limits

Wed Oct 21 01:41:48 PDT 2015

Hi John,

I agree that the reason for your missing data in the middle of the 
boundary is due to unresolved fringes in that region. This has been 
mentioned before, but here are some more considerations:

Your image appears much too bright for me. Some people think that an 
image as bright as possible and with sharp edge contrast between light 
and dark stripes is the optimum, but that is not true. The vertical 
course of light and dark follows a squared cosine function, so there are 
lots of grey transitions that are needed for the fourier transform. When 
the FT is performed on every pixel column, it can work the better, the 
more greyscale intermediates can stabilize the transform. When the image 
is too bright, the course of brightness towards the maximum within a 
white stripe cannot be used as the brightness is at its upper limit.

Brightness and contrast should be adjusted such that you can see as much 
grey as possible with only the center of the white stripes being pure 
white. Be aware that our eye will not judge the screen output in an 
objective way; it may be worthwhile to capture the image and analyse it 
with a graphics program to get the actual pixel values. If a pixel 
column apears like a wave with its maximum close to saturation, you are 
doing fine.

Another thing you might want to check is your camera's azimuth 
orientation. It looks fine when I look at your air-air region where the 
fringes appear perfectly horizontal. However, we have found that for 
high concentrations, the camera azimuth needs to be adjusted better than 
0.1°. This is also related to Fourier transform, and there are some 
pictures, calculations and a more thorough discussion in our recent 
paper (Schilling, Krause: "Analysis of Antibody Aggregate Content at 
Extremely High Concentrations Using Sedimentation Velocity with a Novel 
Interference Optics"; DOI:10.1371/journal.pone.0120820). Incidentally, 
there are also some magnifications in that paper, showing the interior 
of a boundary with steep fringes. They show that there may well be 
detectable fringes even if they are not perceptible by eye in the screen 
display.

So you might be able to increase the performance of your instrument by 
optimizing the camera's azimuth. As a matter of fact, we have found that 
the best way to do this is to run scans on a highly concentrated 
solution at high rpm, and to turn the camera slightly between scans 
until the non transformed region of the boundary is as narrow as possible.

With these two measures, you might be able to achieve the theoretical 
limit you are referring to.

As to Wiener skewing and the 2/3 plane:
It is crucial to focus on the 2/3 plane in order to make the third term 
of the Svensson equation vanish, so you can avoid the bending of the 
light path Borries mentioned. And I see no reason why that could not be 
accomplished. The Beckman technician will do this on every service of 
the interference system. It is important to do the focussing of the 
camera lens when the chamber is evacuated because the rotor will be 
slightly higher in that condition - which is the one we have during a 
measurement. And it is true that shorter centerpieces will need to be 
elevated within the housing so that their 2/3 plane is exactly at the 
same height as for other centerpieces. For this reason, we have 
manufactured housings for 3 and 1.5 mm centerpieces that guarantee this 
geometry. Please also have a look at Arthur's post on 3 mm centerpieces 
of Sep 19, 2006, where he demonstrates the influence of the third 
Svensson term in detail.

With 1.5 mm centerpieces, we have measured antibody solutions as high as 
150 g/l and achieved well resolved fringes (not with the Beckman setup, 
but with an improved CCD and slightly higher magnification, also 
described in the paper above). You will not get as far as that with the 
standard camera, but with the adjustments described above you should at 
least approach the theoretical limit you cited. BTW, 1.5 mm centerpieces 
are the thinnest we did because it becomes impossible to shoot a 1 mm 
filling hole into any thinner centerpiece.

However, there is another limit: though we get well resolved fringes at 
such high concentrations, they will not be as steep as they should. They 
do not exceed a steepness greater than 45 fringes/mm. This limitation is 
due to the optical resolution and can only be overcome by higher 
magnification.

Best wishes,
Kristian

Am 20.10.2015 um 21:32 schrieb John Sumida:
>
> Dear RASMB,
>
> I have been reviewing the thread posted on RASMB between September 1 
> 2008 and September 15 2008 under the subject heading “upper 
> concentration limit AUC”.
>
> We attempted to perform an SV measurement of a sample using 
> interference detection at a sample concentration of 17 mgs/ml.
>
> At this concentration however I observe a gap in the boundary data 
> that resulted from the spin (see bmp image attached); this behavior 
> was not observed at lower concentrations up to 2-5 mgs/ml.
>
> The gap in the data appears to be correlated with the absence of a 
> fringe pattern in the ccd image.  Adjustment of the duration setting 
> does not resolve the issue with the data, but it does improves the ccd 
> image so that I can make out the fringes in the region where I’m 
> observing the gap in the data.  Counting the fringes/mm in this 
> region, I estimate the fringe gradient to be approximately 50 
> fringes/mm. This is less than the 76 fringes/mm noted in the RASMB 
> thread but my count may be off.
>
> On a separate note, since I suspect that the artifact I’m seeing is 
> associated with the observed steep fringe gradient I had wondered if 
> our instrument was focused at the 2/3rds plane or not.  I have been 
> informed, by the vendor, that the process of focusing the interference 
> optics at the 2/3rds optical plane is not something that is possible 
> in the “real world” (their words, not mine), and that this is that 
> this is only something that is only “theoretically” possible.  This 
> appears to be in direct contradiction to the descriptions provided on 
> pages 311 and 312 of Yphantis et. al. Biochemistry 1964 vol.3  (see 
> attached) where concentrations of 12.5 and higher were measured (if my 
> reading is correct) where measurements were performed by using the 
> 2/3rds plane focusing.
>
> Questions.
>
> Is the displacement in the fringe data along the radial axis 
> consistent with a concentration gradient that is too steep?
>
> What is the best way to measure the fringe/mm value?
>
> Are concentrations up to 30mgs/ml possible in a 1.2 cm cell using 
> interference optics, and if so how should the optics be focused?
>
> Thank you in advance for your time, comments and your advice.
>

-- 
Nanolytics
Gesellschaft fuer Kolloidanalytik mbH
Dr. Kristian Schilling

Am Muehlenberg 11
D-14476 Potsdam
Tel: +49 331 5818360
Fax: +49 331 5818361
e-mail:	schilling at nanolytics.de
Internet:  www.nanolytics.de
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