When you configure your system for EFTEM spectrum imaging (SI) acquisition, you can adjust the settings in the EFTEM SI Setup dialog when you use Gatan Microscopy Suite^® (GMS) software.

EFTEM SI has a basic version and an advanced version. Below, we will discuss the workflow for setting up the basic version.

Press Setup under the main EFTEM SI technique to access the EFTEM SI Setup dialog. This dialog box is divided into different groups of parameters.

• Energy Range – Determines the total energy range and energy sampling of the EFTEM SI acquisition

  • Range – Specifies the energy range over which the EFTEM SI will be collected

  • Slit width – Indicates the slit width to be used during EFTEM SI acquisition

  • Step – Determines the step size between successive planes

  • Link – When checked, the energy step size and the slit width will automatically be linked to the same value

• Detector – Contains the detector reconfiguration parameters for the EFTEM SI acquisition

• Options – Contains optional items to modify the data acquisition

  • Acquire high to low – Gives you the flexibility to change the direction of the SI acquisition

    • High to low is recommended to limit detector after-flow

  • Align ZLP – Choose this option to perform a ZLP alignment before acquiring the spectrum image

    • This is not recommended as is often results in ghosting of the zero-loss image in the EFTEM SI

Flow control

1. Locate the region of interest, and ensure the microscope and filter are correctly aligned.

2. Set up the EFTEM spectrum imaging (SI) acquisition parameters.

3. Click the Capture button to start EFTEM SI acquisition.

4. Complete pre-acquisition steps.

   1. Align the ZLP.

   2. Determine the auto-binning value.

   3. Set the initial auto-exposure value.

5. Perform EFTEM SI acquisition.

You can monitor how the acquisition is proceeding using the visual feedback features.

Visual feedback

• Zero Loss Peak Alignment – Invokes the EFTEM zero-loss alignment routine at the start of the acquisition

• EFTEM SI Spectrum Image display – Displays two images during data acquisition; the left-most image is the EFTEM spectrum image, while the right-most image is the last acquired image

• Live Spectra – Extracts a live electron energy loss spectrum from a desired region of the EFTEM SI being acquired
  • Place a spectrum picker region of interest (ROI) to the EFTEM SI – Right-click on the EFTEM SI data and select the Spectrum Picker tool
  • Move the added ROI to the desired area of the spectrum image
  • A live spectrum will then be displayed and will update as the acquisition proceeds

The EFTEM spectrum image (SI) will most likely suffer from x-ray hits and sample drift during the acquisition. The x-ray hits will appear as occasional extreme values in the image planes of the spectrum image. If the sample drifted during the experiment, then the image planes will not line up with each other and spectra drawn from the spectrum image will contain artifacts (e.g., sharp discontinuities). DigitalMicrograph^® software can help reduce or eliminate these problems.

Removing x-rays

1. Choose Remove X-rays from the Volume submenu.

2. The software will check each image plane of the spectrum image for spikes that lie more than a number of standard deviations above the local median (10 standard deviations by default).

3. Once complete, the routine will output the number of x-rays removed.

4. The number of standard deviations required before a pixel is considered to be anomalous, and also the maximum number of x-rays to remove, can be set by holding down the Alt key when selecting the menu item.

Spatial drift correction

Since the acquisition of a spectrum image can take a considerable period of time, sample drift can cause the planes of the spectrum image to be offset from one another. The relative amount of sample drift is dependent on a number of factors, including sample/microscope stability, exposure time and also magnification. You should measure and remove spatial drift, by utilizing one of the following options, before performing any subsequent analyses.

1. Image alignment tools – Allows you to measure this drift and realign the energy planes after the acquisition.

2. Measure Drift (automatic).

   1. Select the EFTEM SI dataset and then choose EFTEM | Measure Drift (Automatic) from the menu.

   2. Measures the spatial drift between all planes of the STEM SI with respect to the currently shown plane using cross-correlation and image filtering as specified in the EFTEM Mapping Preferences.

   3. Displays drift in a line plot display suitable as input for both the Image Alignment palette and the manual drift correction tool (see below).

   4. If any of the automatically determined measurements fail the minimum quality criteria, a dialog will be shown at the end of the measurement, which will offer to continue with manual measurement.

3. Measure Drift (manual).

   1. Select the EFTEM SI dataset and then choose EFTEM | Measure Drift (Manual) from the menu.

   2. This will launch the manual drift correction tool for 3D stacks.

   3. The tool offers the same options and functions as the one for image pairs but is extended:

      1. The image stack is shown next to the alignment overlay image.

      2. A line plot display shows all currently determined drifts.

      3. This display is suitable as input for the Image Alignment software.

   4. While the tool is active, it contains two regions of interest (ROI) markers that can be dragged to select the two image planes currently used by the alignment tool.

      1. The drift values are always respective to the reference plane (gray).

      2. You can change the active plane (green) with the two additional Plane buttons on the Image Alignment tool, or via the displayed plane of the EFTEM SI dataset using the slice tool.

Note: When you launch the measurement tool while a line plot of measured drift values already exists, these values will be used initially. Press Cancel on the tool to revert to these initial values; while OK replaces them permanently by the adjusted measurements.