Manual

Editing Absorbance Velocity Data:

All experimental velocity data aquired on the XL-A require substantial editing and preliminary diagnostics before the data can be successfully analyzed with various analysis methods. UltraScan assists you in this process by handling most essential diagnostics and editing steps automatically, but permits you to intervene, where necessary. Editing is started by selecting

from the main menu of UltraScan. This will bring up the editing window with the plot area and the control panel.

At the bottom of the control panel is a "Wizard" that provides step-by-step instructions to guide you through the multi-step process of velocity data editing. Refer to it to determine which action is required next. Buttons are greyed out (disabled) until their functionality becomes active, so only applicable buttons can be clicked.

Step 1: Select a data directory that contains the data files acquired by the Beckman data acquisition program. Such a directory can generally be found as a subdirectory of the Beckman data acquisition software. The default installation for the Beckman software is C:\XLAWIN (under Windows). Under Unix, the drive with Windows will generally be mounted under the /dosc or /dos directory. You can always set the desired default location in the configuration panel so you can avoid lengthy directory traversals to find the desired location quickly.

The experimental data will then be located in a subdirectory of the form mmddyy/hhmmss (month/day/year/hour/min/sec), identifying the date and time on which data acquisition commenced for a particular run. A file dialog will allow you to specify the desired directory. If the directory contains files of the type 00001.ra1, 00002.ra1, ..., etc., then it is a proper data directory. If the program cannot find enough scans in the selected directory, a warning message will be printed on the screen.

Note:
Data directories retrieved from the database contain an additional file called "db_info.dat" which contains database index information that is automatically included in the edited data. The file is parsed during editing and parameters such as run identification, celltype, centerpiece type, rotor, buffer corrections, etc. are included in the edited data and later available during analysis.

Please note that UltraScan needs at least 3 scan files for each cell in order to identify the wavelengths used for the run. Once you have navigated through the directory tree to the desired data directory, simply press the OK button (there is no need to highlight or individually select scans in the directory, specifying the directory itself is sufficient). Next, the program will load the data in this directory and determine a number of important diagnostics from the scan files present in this directory:

1. The number and position of cells that were scanned in this run. If not enough datapoints are found in the selected directory, a warning message will be printed. If the sequence of the scan files is out of order, a warning message will be shown. If this message shows up, and you acquired the data at a single wavelengths, you can rescue your data by rebuilding the data sequence with the Scanfile Reordering Utility.
2. The number of wavelengths and their settings that each cell was scanned with
3. The number of scans that were taken for each cell and each wavelength
4. The average temperature over the entire run
5. The program will determine if the temperature varied for more than a pre-set tolerance value over the course of the run and warn the user if the tolerance was exceeded. In this case, a warning message is printed forcing the user to acknowledge the diagnostic before editing can resume.
6. The time correction in minutes and seconds accounting for the rotor acceleration period at the beginning of the run. Due to the near linear acceleration of the XL-A, this number will typically be about half of the time it took to reach final rotor speed. If you acquired your velocity data with an older version of the Beckman data acquisition program that failed to correctly record the times of the scans to file, you will get a warning message. In this case you may want to turn on the compensation switch in the configuration panel. This message can also be generated when there were more than usual initial scans made at a low speed. In that case it is safe to ignore the warning, since the delay is real. (Loading equilibrium data will often generate this message as well, of course, you shouldn't attempt to edit equilibrium data with this method.)
7. The length (duration) of the entire run in hours and minutes
8. The rotor speed at which the measurements were made
9. The identifications for each cell that were entered at the begin of the run.

The status bar will keep the user informed about the progress of the diagnostics. The length of time required to complete these diagnostics will depend on the number of files in the directory that have to be analyzed. Once the diagnostics are completed, the diagnostics window will be displayed that shows the details of the selected run. The top panel of the "Run Detail" Window will provide you with a profile of the temperature variation over the course of the run. Shown is the temperature value of each scan in the experiment. Ideally, the temperature should not vary more that a few tenth of a degree over the course of the run. If the temperature varies more than a pre-set tolerance value, the "Temperature Check" field will show a red flashing LCD, otherwise a continuous green LCD.

Clicking on the "Time Plot" button will present you with a plot of the time differences between scans. The "Time Plot" button will then change to a "Speed Plot" button. Clicking on the "Speed Plot" button will then show you a plot of the speed at which each scan was recorded. Finally, you can recall the temperature plot by clicking on the "Temp. Plot" button, and cycle through the different plots by repeatedly clicking on the same button.

The purpose of this window is to facilitate the identification of experiments from the rather cryptic information provided by the file- and directory names created by the Beckman data acquisition software. Information about various cells available for a particular run can be obtained by selecting the appropriate cells in the listbox (UltraScan allows for up to 8 cells to accommodate experiments performed with the 8-hole rotor AN-50 Ti, and allows for an unlimited number of scans to be analyzed for each dataset. The cell description will be updated for each cell that is selected in the listbox. If the description for a particular cell was not sufficient, it can now be updated and edited to accommodate changes. After editing is complete, you can also edit the description of each cell with the Cell ID Editing Utility for the edited data.

Step 2: Before editing of the data can proceed, a unique run identification needs to be entered in the box labeled "Run Identification". This run identification should not contain any spaces and should help the user in identifying the run by name. Spaces can be replaced by underscores. Please note that unlike under Microsoft Windows, file names will follow Unix name conventions (case sensitive). For example, a run identification such as "Chromatin_pH8" is distinct from "chromatin_ph8". A practical way of naming runs is to use the logbook number of the run, and to append a "v" for velocity-, or an "e" for equilibrium runs. If the data was retrieved from the database, the run identification will be filled in automatically.

If you change your mind and do not want to edit these data, you can cancel the editing process at this point by clicking on "Cancel" and you can then select a different directory instead. However, if you do want to proceed, click on "Accept". If you forgot to enter a unique run identification, you will be reminded by an error message.

Step 3: Make sure that the cell descriptions are correct and adequate to identify the contents of each cell that contains data. If not, you can now edit the description for each cell by modifying the string in the editable text box. After entering a unique run identification, the window will close and you will return to the main editing window. The control panel will now show all fields updated with the proper information obtained during the diagnostics. If the data directory was retrieved from the database, the run identification is automatically set and all cell and centerpiece parameters should be automatically set to their proper values. Otherwise, adjust the centerpiece, cell and rotor settings now. Some data analysis methods rely on the geometry information included in these definitions.

The number of editable datasets will be displayed in the "Run Detail" box. A dataset is the collection of scans acquired for a particular wavelength and a single cell. For example, if the cell in position 3 has been measured at 260 nm, all scans measured at this wavelength for cell 3 are considered to be a single dataset. To load the first dataset for editing, click on the "Start Editing" button, which is now highlighted. All scans will then load into the editing plot window, and the status bar indicates the progress of data loading.

Step 4: After loading is completed, the data will be displayed in the editing window. Review the settings for rotor selection and centerpiece type, and adjust if needed. The selection of these items determines the method of editing as well as the cell dimensions for the finite element fit. If the data was loaded from a directory that was retrieved from the database, these settings should already be adjusted to their appropriate values.

In case the sample and reference channels were reversed during loading, you can click on the "Invert Sign" button to flip the data back into a standard display mode.

The first step in the editing of your data involves defining the meniscus. Find a point to the left of the meniscus and click on it, then find a point to the right of the meniscus. After defining a point to the left of the meniscus, the program will find the maximum absorbance between the two limits and define it to be the meniscus. A small crosshair will be placed at that position and the meniscus position will be marked.

Step 5: The next step requires that the data limits are chosen. Those are the limits between which the data contains useful information for data analysis. This region should exclude the meniscus and the bottom of the cell. First, the left limit is defined by dragging the mouse to a position near the right of the meniscus, and then clicking on it.

Step 6: Next, the right limit is chosen in a similar fashion. The data will be rescaled to the new limits, and the extraneous datapoints will be discarded.

Step 7 (optional): Now you will have the opportunity to edit scans that contain scratches, outliers or spikes in the data. This is accomplished by selecting a single scan at a time with the "Edit Single Scan" button. The procedure is explained here.

Step 8 (optional): Next, you may want to exclude one or more scans from the run. While not strictly necessary at this point, you can exclude a single scan by clicking on "Exclude Single Scan" or a range of scans by clicking on "Exclude Scan Range", after setting the scan number in the counter. ">>" and "<<" buttons move in steps of 10, while ">" and "<" move in single step mode. To select a range of scans for exclusion, select the scan at the beginning of the range with the counter of "Exclude Single Scan". Then use the counter from the "Exclude Scan Range" button to define the last scan of the range to be excluded. As you select scans, the about to be excluded scans will be highlighted in red. After clicking on one of the "Exclude..." buttons, the highlighted scans will be deleted from the dataset. Please also refer to the van Holde - Weischet tutorial's section on data editing.

Step 9: Now you have to define a plateau absorbance for all scans by clicking with the mouse at the appropriate point in the cell. Simply pick at a point that defines a stable plateau for most of the scans in the plot. Even if some scans clearly do not meet the criteria of a stable plateau, certain methods will give the appropriate diagnostics (van Holde - Weischet method) and allow you to exclude those scans from the analysis, while other methods (finite-element and other whole boundary fitting methods) can incorporate those scans without a stable plateau into the analysis. After clicking, the program will average a few points to the left and to the right of the point you clicked to arrive at a value for the plateau absorbance of each scan. Please also refer to the van Holde - Weischet tutorial's section on data editing.

Step 10: The last step in the analysis is the definition for the baseline. After defining the plateau absorbances for each scan, the last scan will be displayed, because it will have the most pronounced baseline. Find a point at which the baseline absorbance is constant and click on it. The program will average a few points to the left and to the right of the point you clicked to arrive at a value for the baseline absorbance.

Now the data for the first dataset is edited and the program will automatically cycle to the next dataset by returning to the main editing window and updating the progress bar to indicate the status of file loading. Repeat Steps 4 - 10 for all datasets in the run. When all datasets are edited, the program will return with a message window indicating that all scans have been edited and the data has been written to a binary file.