Optima AUC - Chromatic Aberration Calibration:

The Optima AUC is capable to perform multi-wavelength UV/visible detection experiments. This means that each scan of a particular cell or channel can be performed at a different wavelength. In UltraScan, up to 100 different wavelengths can be measured in a single experiment. When data are acquired at different wavelengths, the point on the photomultiplier tube where the radial image will be reorded will be affected by the refractive variation that is dependent on the wavelength. The problem of shifting radial positions is worse when the incidence angle of the light source with respect to the cell window is not perpendicular (either due to optical misalignment or rotor precession due to imbalance). A perfectly aligned optical system and a perfectly balanced rotor will produce a pattern that is essentially invariant with wavelength, but typically, some wavelength dependent deviations are observed in the Optima AUC, causing the radial position where a particular point in the cell is imaged to shift by 50-100 microns over the entire wavelength range accessible in the Optima AUC (190-800 nm). This effect is readily observed when the meniscus position in the cell is monitored at different wavelengths. Of course, the meniscus position should be constant (unless the cell is leaking) and the radial position of the meniscus peak should be invariant for the same channel. However, it rarely is. The deviation is very reproducible and can be observed from each cell with a similar deviation as a function of wavelength.

To correct for this deviation, a chromatic aberration profile can be determined for each instrument and uploaded to the LIMS database, so collected data can be radially corrected to remove the discrepancies between wavelengths. In order to obtain a chromatic aberration profile, a calibration run should be performed. You will need 4 cells fitted with quartz windows, which should be exquisitely clean to make sure there is no absorbance even at short wavelengths (where everything absorbs).

  1. Perform a radial calibration on the instrument at 3,000 rpm with an empty cell across from the counterbalance. The Optima AUC records calibration data at 250 nm. Run with a well-balanced rotor (less than 0.1 g difference between counterbalance and opposing cell, to minimize rotor precession).
  2. Fill each channel equally high with ddH2O water using pipette tips that have been soaked and rinsed in degassed ddH2O or MilliQ water to remove any contaminants from the tips. Each channel will be filled with ~ 0.30 ml of degassed ddH2O. You should weigh each cell and adjust the volume loaded (in both channels equally to avoid hydrostatic pressure differences across the septum) to balance all cells to within less than 0.1 g (including seals and fill hole screws). This will assure there is no rotor precession and provide a near perpendicular light path.
  3. Once the instrument has been radially calibrated, remove the counterbalance and place all four cells into the rotor (holes 1-4 in the An60Ti rotor, or holes 2, 4, 6, 8 in the An50 Ti rotor).
  4. Set up a protocol in the UltraScan data acquisition program to scan the entire wavelength range in 10 nm increments (190, 200, 210, ..., 800), 5 times at each wavelength, at 14,600 rpm, scanning each of the 4 cells. Since you are scanning 61 wavelength, and the system will perform intensity checks at 6.5 cm for each wavelength, and for each channel, the time until the first scan is measured will be quite long (>30 mins). Don't stop the machine thinking the protocol is not working, just be patient, eventually all cells, channels and wavelengths will be calibrated for the right PMT voltage, and the scanning will commence.
  5. Once the data are collected, use the "Utilities:View Raw Optima Data" function to load the calibration data, and zoom over each meniscus (which should be pointing downward since you will be looking at intensity data). Between each channel, unzoom and zoom again before clicking "Next" to get to the next channel and cell. Since there are two channels per cell, you will zoom 8 times. Each time you zoom, a file will be written for this channel that contains the radial positions of the meniscus minima. Be sure to zoom very close to the actual meniscus artifact, avoiding to pick up other minima that may be from noisy data or other spurious noise signals.
  6. In your $HOME/ultrascan/imports folder will be a directory with the run name. In this directory, you will find 8 new files:
    1. 1A.wavelen.radpos.dat
    2. 1A.wavelen.speeds-meniscus.dat
    3. 1B.wavelen.radpos.dat
    4. 1B.wavelen.speeds-meniscus.dat
    5. 2A.wavelen.radpos.dat
    6. 2A.wavelen.speeds-meniscus.dat
    7. ...
  7. Import all .wavelen.radpos.dat files generated in the $HOME/ultrascan/imports folder of your calibration run, one for each channel, into a spreadsheet program and average all observations from a single wavelength.
  8. Find the value of the offset at 250 nm and subtract it from all values, such that radial positions less than that value are negative, the value at 250 nm is zero, and all others are positive values.
  9. Fit this profile with an appropriate parametrization function that describes the chromatic aberration of your optical system.
  10. Export the wavelengths and corrected values of this parameterizing function from 190-800 nm in 1 nm increments to a datafile (using this ASCII format - see example here).
  11. In UltraScan, and as user with admin privileges (userlevel=3), load this file into the instrument record. (Edit:Preferences:Instrument Preferences:Change:Edit Current Entry:Load Chromatic Abberration Array) and set the Radial Calibration Wavelength to 250 nm.

Please note that this procedure does not care about the absolute values recorded for the meniscus positions, it only needs the relative differences between measurements made at different wavelengths to generate this file. Therefore, the absolute rotor stretch value here is not relevant (just in case you wondered).

IMPORTANT:
This procedure must be repeated every time there have been changes performed on the optical system by Beckman service. A new profile needs to be uploaded to the LIMS for all new data acquired after the change. Retain older chromatic aberration files for uploading and correcting when retrieving older data before the optical system change so older data can still be corrected with the original profile.

After uploading the chromatic aberration profile, test it out by re-importing the chromatic aberration profile data and allowing it to be corrected using the new chromatic aberration profile stored in the database. Repeat the radial zooming of the meniscus positions and plot the contents of the .wavelen.radpos.dat files. These should provide horizontal values for all wavelengths. The next time you run a multiwavelength SV experiment, the meniscus positions from all wavelengths will be aligned properly and only a single wavelength needs to be fitted to determine the boundary conditions of your velocity experiment. The fitted value can then be applied to all wavelength triples from the same channel.

FOR NEW MACHINES:
The UltraScan data acquisition module requires a chromatic aberration profile to be installed in order to perform data acquisition experiments. If you don't have a chromatic aberration profile available, you can retrieve this file with zero correction entries as an initial correction profile (it will not modify your measured values) as a blank until you have a measured chromatic aberration profile available.