[RASMB] Interference Optics - HELP!

[Kristian Schilling]

Lisa,

the approach of realigning the camera is absolutely correct. If fringes are 
tilted in empty holes, the camera has to be rotated; the image is just 
projected upon the CCD chip with an azimuthe offset. And you need not be 
concerned, subtracting a blank scan is an absolutely legitimate approach to 
cure this problem as long as the camera has not been aligned.

BTW, aligning the camera is easy and you could easily do it yourself. The 
camera need just be turned on its socket, but probably you would infringe 
your warranty.

So blank scans are OK but if the blank scanning does not work then this is 
the actual problem. I think the blank scan option in Beckman software is 
unfavorable, because I can not see the effect of the correction and cannot 
undo it. As we *always* have to correct for fringe errors (as we use 
sapphire windows) we do it another way.

For each run, we acquire a "blank" scan at the beginning of the experiment. 
It has turned out that blank corrections only work if acquired in the same 
run. We do blank subtraction only if feasible and use our own software for 
it. Often, we restrict b. s. to a range of interest in order not to 
introduce errors elsewhere for the benefit of removing them in one place. 
Sometimes, it is not possible to acquire a blank scan due to instantaneous 
sedimentation. In such cases, we use a statistical procedure, adding all 
scans so that the errors, always at the same position, add up to a 
signifiant error profile that we subtract.

But if your fringes are just tilted, you can have it an easier way. Just 
get the derivative of your data. Your tilt shows up as a vertical offset in 
all scans. Subtract the average offset from all scans and reintegrate. You 
need no integration constant as interference datasets always start with zero.

Hope this helps,
Kristian

At 20:21 16.03.2003 -0700, you wrote:
>Hello All,
>We have recently purchased an XLI and I have some questions regarding the 
>interference optics that I am hoping some of you can help me with. My 
>problem is basically non-linearity of the scans, and correction of that 
>non-linearity using a blank scan correction.
>
>Firstly, when scanning an empty rotor hole the slope of the best fit line 
>to the resulting data is about 0.2 finges/cm (well outside Beckman s specs 
>of 0.04 fringes/cm). Scan's of an empty cell are comparable.  Beckman is 
>not terribly concerned about this and will have a service rep align the 
>camera to try and improve, they suggest that if I just do the appropriate 
>blank scan subtraction everything will be OK (confidence that I do not 
>share!).
>
>Which brings me to my next, more troubling, concern - blank scan 
>subtractions. Subtraction of a blank scan taken of the same empty rotor 
>hole, empty cell or water cell does not always give me a flat baseline, 
>particularly when I use the option of subtracting a previously saved scan. 
>When dealing with only one cell the result is fine, however when I do 
>several cells in one scan only the one whose baseline was scanned last is 
>flat, all the rest have a significant deviation from zero. I think what s 
>happening is that even though all cells have a blank file (blank.ipx) all 
>the subtractions are using the last file scanned. We have version 4.5 of 
>the data acquisition software and it doesn t have an option to select an 
>actual scan so I m assuming that it should just use whatever the last 
>blank.ipx is for the relevant cell. I have tried deleting some of these 
>blank files and the software is looking for them apparently just not using 
>them. Can anyone shed any light on this?
>
>While I agree that I need not worry about this non-linearity for our g*(s) 
>analysis, I am trying to get good equilibrium data for a system that we 
>have to use interference for (buffer contains nucleotide) and I am sure 
>this is causing a problem. Any suggestions or information would be appreciated.
>
>Thanks
>
>Lisa
>
>
>----------
>Lisa Joss                                                            (801) 
>585-3919
>Postdoctoral Fellow                                           email: 
><mailto:ljoss at biochem.utah.edu>ljoss at biochem.utah.edu
>
>UNIVERSITY OF UTAH
>BIOCHEMISTRY
>20 N 1900 E RM 211
>SALT LAKE CITY UT 84132-3201
>
>----------
>
>



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