scatterBrain Using the Plot Window

Owned by SAXS Cluster (Unlicensed)
Nov 09, 2021
9 min read

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The Plot Window of scatterBrain is used for displaying, using and exporting radially averaged, 1-D SAXS profiles. The plot window is typically used during an experiment for Q-range calibration and Normalisation as well as monitoring data as it is collected. After an experiment, it is typically used in the Analysis version of scatterBrain for checking data, background subtraction and exporting data as '.dat' files.

This window has three sections as outlined below:


  • Top Menu (running along the top of the Plot Window): has controls for normalisation, Q-range calibration, etc...
  • Plot Area (left side, takes up most of the Plot Window): SAXS profiles display and well as zoom in/out.
  • Profile Tree (right side): filenames of plotted data and background subtraction

Axis Scaling (Linear or Log Scales)

Depending on the analysis being undertaken it is useful to view SAXS profiles on either a linear or a log scale. The Plot Window of scatterBrain provides straightforward controls for auto-scaling the Plot Area and to toggle both axes between Linear and Log scales. When auto-scaling the axes scatterBrain will scale based upon all currently loaded data.

To Auto-Scale the axes of the Plot Area:

  • Right-click anywhere in the Plot Area. This will auto-scale to display all of the currently loaded SAXS profiles

To Toggle Between Linear and Log Scales:

  • Use the "X-axis" and "Y-axis" buttons located in the top-right of the Plot Window
    • Clicking these buttons will toggle the named axis between a linear and a log scale
    • Each axis is toggled separately


Zooming In/Out

The Plot Window provides a straightforward tool to zoom in on an area of interest and to zoom out.

Zoom In: Click and drag the mouse in the Plot Area to define a box around the area of interest (define the opposite corners of the box – drag diagonally). This will rescale the axes of the Plot Area to zoom in on the area of interest. Repeat protocol if further zoom is required.

Zoom Out: Right-clicking anywhere in the Plot Area to reset zoom.


Plot Highlighting

When a large number of profiles are displayed in the Plot Window it can become difficult to see which one is which. If a profile, or profiles, is selected in the Profile Tree the plots(s) will be highlighted with a thicker line style. This is a fast way to see which pattern is which in the Plot Area of the Plot Window when there are many pattern overlaid. It is also possible to move up or down in the Profile Tree using the arrow keys, which can be more convenient than using a mouse.


Subtracting Patterns

The Plot Window provides a straightforward utility for subtracting one SAXS profile from another (buffer/background subtraction from sample). Similarly, it is also possible to subtract a single background from multiple files. The Profile Tree is used to both perform and keep track of profile subtractions as detailed in the list and graphic below:

Subtract a background/buffer from sample: click and drag the sample name onto the background name (Profile Tree)

Subtract a background/buffer from multiple samples: Shift-select or Ctrl-select the sample names, then drag and drop them onto the background name (Profile Tree)

Please note:

  • By default the Plot Window will continue to display the background profile as well as the sample profiles
  • Individual profiles may be hidden using the Context Menu of the Plot Window
  • The background profile will remain at the top-level hierarchy of the Profile Tree and the sample profiles will move to the second level in the hierarchy (sample names slightly offset to the right, see image below)
  • The background name will now also show a "-" or "+" symbol which allows the sample profiles associated with each background to be shown or hidden in the Profile Tree


Undo a background subtraction to sample: click sample to be unsubtracted and drag it off the background profile to anywhere in the white area of the Profile Tree (unsubtracted sample profile will return to the top-level of the hierarchy in the Profile Tree). Sample is now ready to be subtracted against a new buffer/background.


Plot Window Context Menu

Context Menu is found within the Plot Window and provides a range of commands and functions to be used on the loaded profiles (see image below).


To use the Context Menu:

·       select the profile/s in Profile Tree (Shift+Left-Click or Ctrl+Left-Click to select multiple profiles)

·       Right-Click anywhere in the Profile Tree (Context Menu will pop up, as shown in the figure)

·       Select the command/function to be applied to the profile/s




The options within the context menu are explained in detail below:

Delete: removes the selected profile/s from the Profile Tree, Plot Area and from memory (original data is not deleted). The Delete All button above the Profile Tree will delete all profiles.

Apply Parameters: recomputes the profile (as if loading it afresh) in order to apply any changes to the camera parameters. For example, if the beam centre or camera length is adjusted it is necessary to Apply Parameters to currently loaded data to reflect these changes.

Multiply: multiplication correction to a profile. The default setting is '1', i.e. no correction. If there are small subtraction errors in the data it may be possible to adjust the profile slightly. It may also be used to separate profiles for better visualisation. The correction is applied to the selected profile/s, although it is recommended to only correct one profile at a time. Any multiplication terms applied to the currently selected profile are displayed in the Plot Window, below the Profile Tree.

Offset: addition correction to a profile. The default setting is '0', i.e. no correction. If there are small subtraction errors in the data it may be possible to adjust the profile slightly. It may also be used to separate profiles for better visualisation. The correction is applied to the selected profile/s, although it is recommended to only correct one profile at a time. Any addition terms applied to the currently selected profile are displayed in the Plot Window, below the Profile Tree.

Fit Peak: applies a linear combination of a Gaussian peak and a linear background to the currently selected profile. A plot of the peak fit is shown as a dotted line in the Plot Area and should be used to check whether the fitting was successful. The basic properties of the peak fit, i.e. position, width and chi, are displayed at the bottom of the Plot Area. The fit is applied to the section of the profile currently shown in the Plot Area. For best results, it is recommended to Zoom In on the peak to be fitted prior to using the Fit Peak function.

Hide: remove profiles from the Plot Area while remaining in the Profile Tree and system memory. This is a useful tool when many profiles are displayed concurrently.

Show: undo Hide. It has no effect on currently plotted data.

Show <> Hide: profiles can be toggled between Hidden and shown in the Plot Area. This provides a one-step tool for returning currently Hidden profiles to the Plot Area while removing others.

Change Colour: opens a colour table selection box that is used to control the colour of all profiles.

Line Width: adjust the line width of the currently selected profile/s.

Opacity: adjust the opacity of the currently selected profile/s.

Protein Tools: Only for protein scattering data. Allows Autoporod program of the ATSAS analysis suite to be run on a single, selected profile. The fit to the data is displayed on screen and a temporary data file is written to the operating system's 'temporary' directory.


Top Menu of the Plot Window

Contains critical parameters to ensure an accurate data reduction. The key operations of Q-calibration, Normalisation, Scattering Calibration and Exporting Data are described in detail here. The last option in the Top Menu, 'Help', is not covered.

Exporting Data

scatterBrain provides tools for interpreting 2-D SAXS images, generating 1-D SAXS profiles and data reduction. Further data analysis using additional programs usually requires reduced SAXS data, i.e. after normalisation and background subtraction. Reduced data may be readily exported from the Plot Window of scatterBrain.

The Save Profiles Menu option offers three choices for saving data, each of which is described below:

Save ALL profiles to ONE large file

  • Saves all the currently plotted SAXS profiles into one, single file. This file contains three columns for each profile (q-vector, intensity and error in intensity). The filenames for each of the profiles is written above each set of data
  • Select directory and file name into which data is saved
  • Exported data will contain all normalisation, background subtraction and offset factors

 

Save EACH profile to individual file

  • Saves a separate file for each currently plotted SAXS profile (q-vector, intensity and error in intensity)
  • Select directory into which the data will be saved
  • Profiles will be saved into files with the same name as the profile in the Plot Window with a '.dat' file extension
  • Exported data will contain all normalisation, background subtraction and offset factors

 

Save current plot as image

  • Save an image of the current Plot Area (graphic for visualisation or publication)
  • Select directory, file name and file type that the image will have


Q-calibration

The Q Calibration menu option opens a new window (shown below) to set or calibrate the parameters used to determine the q-range. This includes setting the energy/wavelength for calibration or manual setting of the camera length. This is particularly useful for protein scattering experiments which typically use highly consistent camera lengths of: 959mm, 1576mm, 3349mm or 7200mm. If using the Q Calibration option to change the energy only use the keyboard to set the Calibrated Camera Length box to the same as the Current Camera Length.

How to perform Q-calibration


  • Manually entering a known camera length in the Calibrated Camera Length Press the Apply Q Calibration button

or

  • Calibrating the camera length from a known peak of a calibration standard. Following instructions below:
    • Load a measured pattern from a calibration standard
    • Zoom in on a known peak
    • Use the Fit Peak command of the Context Menu
    • The Fitted Peak Position box will be updated from the fitting routine
    • Use the drop-down menu to select the standard and the peak used
    • Press the Apply Q Calibration button

Intensity Normalisation

Normalisation of the scattering intensity allows for a better comparison of the scattering profiles from different sample and different data collection times. Normalisation on the SAXS beamline is typically based upon the signal received from a photodiode housed in the beamstop. This provides a combined correction for both the power of the incident beam and the transmission of the sample. The signal received with nothing in the beam path, i.e. an air shot, is used as a baseline. Normalisation based on the beamstop signal is a relative correction only, it does not provide calibration of absolute scattering intensity of a sample.

Intensity normalisation protocol


  1. Ensure that the Q Calibration is correct
  2. Delete all data from the Plot Window ('Delete All' button above the Context Menu)
  3. Load an air shot (data collected with nothing in the beam path)
  4. Select the Intensity Normalisation option from the Top Menu (new pop-up menu)
  5. Left-click in the white space below the Profile Tree
  6. Select the air shot profile in the Profile Tree (updates the "New" I0, IBS and Ratio normalisation inputs)
  7. Click on the "Apply Normalisation to I0/Beamstop Counters" button. Sets the "New" normalisation inputs as the "Current" values. Confirm that both the "New" and "Current" values match
  8. Select the "Beamstop Intensity Normalisation" radio button. The "No Normalisation" button is selected by default and may be used to remove normalisation if required.
  9. Click the "Apply change of Normalisation button". Patterns are normalised for incident beam and transmitted flux variation using the signal received from the photodiode housed within the beamstop
  10. Close the Normalisation pop-up window


Absolute Scattering

Although Normalisation allows for a good comparison of scattering data from different samples the scattering profiles remain on an arbitrary plot of Intensity vs. Scattering Angle. To get the most from the data it is necessary to perform a Scattering Calibration that will allow data to be plotted as Absolute Scattering vs. Scattering Angle. This requires calibration using the scattering from a calibration sample of known scattering properties for a range of exposure times. This will allow a conversion from a number of counts on the detector to the Absolute Scattering intensity. On the SAXS beamline, a water-filled capillary is typically used as the calibration standard with 1, 2, 5 and 10 second exposures used. Note that 1, 2, 5 and 10 second background exposures of the empty capillary are also required. Absolute Scattering Calibration protocol:

  1. Perform an Intensity Normalisation as detailed above
  2. Delete all profiles
  3. Load the empty capillary and water filled capillary data (typically named 'mt_cap' and 'water_cap' respectively)
  4. Subtract the empty capillary scattering from each of the water exposures (ensure exposure time of empty capillary and water match before subtraction). When the background subtraction is complete the Plot Window should like:     
            
  5. Open the "Intensity and Normalisation Calibration" Screen and select the Intensity Normalisation
  6. Click in the white space below the Profile Tree in the Plot Window
  7. Zoom In on a flat region of the background subtracted water scattering profiles
  8. Use Ctrl-Left-Click to select all of the background subtracted water profiles (upon selection points are added to the Normalisation and Calibration Screen plot as well as two linear lines (see below).   
                                                                                                                                
  9. After profiles selection, click on the "Set Absolute Scaling" button at the bottom of the Normalisation and Calibration screen. Note: The check box next to "Use Absolute Calibration" should be automatically marked
  10. Close the Normalisation and Calibration screen
  11. Delete all currently plotted scattering profiles
  12. Check that the calibration is satisfactory
    1. Re-load the empty capillary and water scattering data
    2. Apply the correct background subtraction to each of the water scattering profiles
    3. Zoom In and check that the water scattering is scaled close to 0.0163 cm-1
  13. If the Calibration is not satisfactory it should be repeated from step 1.
  14. If the Calibration is satisfactory, save the configuration. First, save the configuration to RAM using the "Save" button in the top right of the Image Window of scatterBrain. Save the Experiment File to disk using the "File" tab of the Top Menu in the Image Window. Select the "Save Experiment" option to overwrite the current file or "Save As Experiment" to write a new file.