STEM SI Workflow

In STEM, the electron beam is focused into a small probe and rastered to acquire spatial information in a serial manner (X, Y). In this acquisition mode, spectrum imaging (SI) is performed when you acquire a parallel spectrum at each pixel position, hence build up the spectrum image on a spectrum-by-spectrum basis (STEM SI). A typical workflow includes:

  1. Set up the microscope in scanning mode and in the appropriate state for the spectrometer you wish to use.

  2. Use the DigiScan™ system to acquire and assign a Survey Image.

  3. Create a spectrum image survey region of interest on the Survey Image.

  4. View the spectra from the region you wish to acquire the spectrum image from.

  5. In the SI Setup dialog, specify which spectrum signals you want to acquire.

  6. If artifact corrections are required, enable them and/or create the artifact correction survey regions of interest.

  7. In the Spectrum Image dialog, specify the pixel dwell time you want to use and size of the spectrum image in pixels.

  8. Select Start on the Spectrum Imaging dialog.

Here are detailed STEM spectrum imaging workflows you can follow to better understand your sample when you use Gatan Microscopy Suite® software.


Microscope setup

Set up the microscope in scanning mode and in the appropriate state for the spectrometer you wish to use. Refer to your microscope manufacturer's user manual for details.

Enter and configure the STEM SI technique

STEM SI TechniqueWhen you configure your system for STEM spectrum imaging (SI) acquisition, you can control your STEM SI easily when you use Gatan Microscopy Suite® (GMS) software.

In the home Techniques Manager, select the STEM SI button to open the STEM SI technique palette, which contains everything that is required for the experiment:

  • Scan palette to control the DigiScan™ system

  • STEM SI palette to set up the STEM SI acquisition

  • Relevant signal acquisition palettes for each available signal

  • Additional data analysis palettes for the available signals

Note: All signal acquisition palettes hide their respective Capture functionality by default. You can expand the palettes using the plus icon to the right of the View button.

STEM SI palette

Setup and control of the SI acquisition is provided via the STEM SI palette. On the initial display, or if no STEM SI experiment is currently set up, the palette will be displayed in mode selection state. Brief descriptions of the dialog features are given below.

Signal selection

Signal selectionThe top section of the palette features a button for each available signal in the STEM SI experiment (EELS, EDS, CBED, and CL). The according signal acquisition palette will be automatically expanded or collapsed which may trigger requests to change the microscope state if required. At least one signal (in addition to the Scan button) needs to be selected or the SI experiment cannot be started.

Note: Some signals are mutually exclusive. Selecting such a signal will automatically deselect the other.

Mode selection

Mode selectionUse the bottom section of the palette to set up the spatial coordinates of the SI. Pressing a mode button (2D Array, Line Scan, Multi-Point, and Time Series) will automatically assign a suitable survey image and create a default SI survey ROI marker on it. If no suitable image is available, a Preview type acquisition is automatically started and assigned.

  • 2D Array – Regular 2D SI that consists of an array of equally spaced points in X and Y

  • Line Scan – 1D SI that includes an array of equally spaced points along a line

  • Multi-Point – Set of point spectra acquired with identical parameters from individual spatial positions collected into a 1D SI data container

  • Time Series – Set of spectra sequentially acquired with identical parameters from the identical sample area collected in a 1D SI data container

After choosing your signal and mode, a new palette will be available. The palette will include:

  • Capture button – Starts a new SI acquisition using the specified parameters

  • Pixel Time (s) – Specifies the pixel dwell time of the beam and will apply to all spectral acquisition with the permitted values being restricted to the minimum and maximum readout times of the selected detectors

Note: The acquisition time estimate is automatically updated when the dwell time is changed.

  • Live button – Activates (blue) or deactivates (gray) live data display during SI acquisition

  • Drift button – Activates (blue) or deactivates (gray) spatial drift correctionMode specifics

The bottom parameters (boxed in portion of the image) dictate the specific modes of capture per the selection made in the STEM SI palette. The options available are dependent on the mode that was selected there, the options include:

2D Array

Size – Specify the size of the SI in pixels by using the Width and Height fields

The size automatically updates if you change the SI survey ROI position on the active survey image. When you change the Width value, it will automatically adjust the sampling resolution while keeping the acquired region the same. A change in height will change the aspect ratio and hence the acquired region of the SI, while keeping the sampling resolution identical.

Note: The acquisition time estimate automatically updates when the size is changed.

Line Scan

Line ScanSize – Specify the Line Scan length in pixels

The sampling resolution automatically updates when the size is changed. Step displays the current sampling resolution in calibrated units. The SI size in pixels updates automatically when the sampling resolution is changed, keeping the acquired scan length identical.

Average dose checkbox – Enables the averaged line scan functionality and is not available for all systems and settings


Multi-PointNo. positions – Allows you to specify the SI size in number of individual positions

The number of survey region of interest (ROI) point markers is updated accordingly.

Note: At least 2 points have to be specified before SI acquisition can start.

Arrange position buttons – Click these buttons to arrange (selected) positions in various ways over the field of view

Time Series

Time SeriesRepeats – Allows you to specify the SI size in number of individual acquisitions

Note: At least 2 repeats have to be specified before SI acquisition will start.

Delay (s) – Where you specify a pause in between two successive acquisitions

During these pauses, signal controllers are stopped and the beam control is released to the default. The acquisition is then in an idle state similar to a paused acquisition, but a countdown is displayed at the bottom of the STEM SI palette. At the end of the countdown acquisition, the next spectrum is automatically started.

2D Scan – If left unchecked, the spectra will be acquired from a stationary position as indicated by an ROI point marker in the survey image

If checked, spectra are acquired while a specified area is rapidly and repeatedly scanned. The area is represented by an ROI rectangle in the survey image and sampling for this area is defined by the two fields below the checkbox. Enter values to change the area size or use the up and down arrows to change the sampling density of the area.

Survey Image

Each spectrum image acquisition requires a survey image. It provides a frame of reference for the acquisition and serves as a useful reference image of where the data was acquired. You can only assign one image at a time as the active survey image. This image should be recently acquired and of good quality. Images acquired with too short a pixel dwell time can result in inaccurate beam positioning and a mismatch of indicated and acquired spectrum image position. The survey image can be either a recently acquired (static) image, or a still continuously acquiring image. The former might be preferred for beam sensitive specimen; whereas the second can be beneficial to avoid charging induced specimen drift at high magnification and to provide the most accurate survey image at the start of spectrum image acquisition.

Collect and assign a survey image

  1. Locate the region of the sample you are interested in.

  2. Collect an image.

    Use a dwell time of 16 ms/pixel or longer. The long dwell time ensures an accurate microscope beam position in subsequent analysis. Make certain that the region you wish to acquire is smaller than the field of view, typically about one-third of the size. This allows the beam deflectors to correct for spatial drift and also ensures that the spectrum image (SI) region is well represented in the survey image.

  3. Select a SI mode.Select a SI mode on the STEM SI palette or use the survey region of interest (ROI) tool from the floating menus over the image.

    Either action will automatically assign the front more image as the survey image. The window title will change to remind you that this is now the current survey image.

  4. Refresh the survey image.

The current survey image is automatically de-assigned when a different survey image is selected. If you close an active survey image or remove all survey ROIs, the STEM SI palette will revert to the mode selection state.

When a SI is acquired, a copy of the survey image is created on the SI data workspace. This image acts as data reference, which causes the survey ROI to no longer be moved or resized. Only the survey image on the View workspace is actively linked to the STEM SI palette and interactive.

Select and Survey ROI

Survey region of interest (ROI)

For a spectrum image (SI) acquisition, it is necessary to allocate a region on the survey image to be used for acquisition. These regions are marked by ROI markers in green. The type of the SI markers depends on the SI mode and can be either point, line or rectangle ROIs. You can position, resize and remove the survey ROI like any other ROI marker.

  1. Add a survey ROI by selecting a SI mode.

If the STEM-SI palette is in mode selection state, selecting any mode will automatically add the appropriate survey ROI to the survey image which is also automatically assigned by this action. The position and size will be the same as the one used for the last SI acquisition of that type.

  1. Floating menuAdd a survey ROI by using the floating menu.

When you right-click a recent image on the View workspace, the SI ROI tool in the floating menu will appear. This dropdown menu allows you to select the SI type. With this tool, you can create the survey ROI directly on the image. Then, the image is automatically assigned as a survey image while the spectrum imaging mode is adjusted. Other, now obsolete survey ROIs are automatically removed.

Note: For Multi-Point, keep the Shift key pressed while adding a point to keep the tool active and allow multiple points to be added in a series of clicks.

  1. Remove a survey ROI.

Use the mouse to click the ROI marker while you press the Delete key on the keyboard. If all survey ROIs of the current SI mode are removed, the STEM-SI palette automatically reverts to mode selection state.

Survey ROI types

2D Array

A regular 2D SI consists of an array of equally spaced points in X and Y. It is represented as a single, rectangle ROI in the survey image.

Resizing the survey ROI will keep the total number of pixels and hence total acquisition time approximately constant. The SI size in pixels as well as the sampling resolution will change automatically when the ROI is resized.

The survey ROI will snap to positions of appropriate size and aspect ratio. The smaller the total number of pixels in the SI, the rougher those snaps may appear.

Line Scan

A 1D SI consisting of an array of equally spaced points along a line. It is represented as a single, line ROI in the survey image. A yellow cross indicates the start position of the scan. If the averaging option is enabled, the cross is replaced by a yellow line that indicates the averaging direction and width.

Resizing the green survey ROI will adjust the sampling resolution, but keep the number of sampled points constant. Changing the dimensions of the yellow survey ROI will adjust the averaging width, but keep the number of sampling points along that direction constant. You can adjust the averaging direction angle by pressing the Ctrl key down while you drag the green handle of the yellow survey ROI. Deleting the yellow survey ROI will switch the averaging option off.


A set of point spectra acquired with identical parameters from individual spatial positions collected in a 1D SI data container. Each point is represented by a single, numbered point ROI in the survey image. This number specifies the spectrum position within the SI data container.

Points are most conveniently added using the SI ROI tool from the floating menu. Keeping the Shift key pressed allows multiple points to be added in a series of mouse clicks. You can remove individual points by selecting them and pressing the Delete key. The remaining points are automatically renumbered.

Click the Arrange points buttons to arrange the selected set of points or, if none are selected, all points. If a rectangular ROI is selected, points are arranged within this area, otherwise, they are arranged over the entire survey image.

Auto arrange points
Selection of 9 points and destination area (left). Randomize positions in the area where each point is given an arbitrary position within the area (right).

Auto arrange points
Arranged points along a regular grid (left). Arrange point along a regular grid, but randomize around this position (right).

Time Series

A set of spectra sequentially acquired with identical parameters from the identical sample area collected in a 1D SI data container. The sample position is either represented by a point ROI for stationary acquisition or by a rectangular ROI if the 2D Scan option is enabled.

Resizing the 2D Scan area is restricted by the number of sampling points of the scan, which in turn is limited by the SI Pixel Time. Slower scans allow higher sampling. To access larger scan areas, it can, therefore, be necessary to either reduce sampling with the spin controls for the 2D Scan fields and/or to increase the Pixel Time.

Flow Control


Once the spectrum image (SI) acquisition parameters and any artifact corrections have been set up, to start an acquisition hit the Capture button on the STEM-SI palette. If you wish to update the survey image before proceeding with acquisition, right-click on it and select Restart scan from the context menu.

Once started, the SI acquisition routine will take control of the microscope beam, and the acquisition will then begin. During acquisition, the Capture button will pulse in blue indicating that an acquisition is active and that a subsequent click of this button will halt acquisition. A new workspace is created and filled with a copy of the survey image and data containers for all acquired signals.

Note: During active SI acquisition, the spectrometers being used will not be available for single spectrum acquisition.

If spatial drift correction is enabled, then some preliminary measurements will be performed. Visual feedback on the measurements may be provided, depending on the preferences specified.

You can monitor how the acquisition is proceeding using the Visual Feedback provided. Each SI will be displayed as a new image window, which will fill as the acquisition proceeds. Live spectra may also be shown or hidden as desired. If at any point you wish to halt the acquisition, click on the pulsing Capture button. When the acquisition is halted or completes, the beam control is returned to the Beam Safe Point if one has been set up, or alternatively it is returned to the microscope.


During a SI acquisition, it may become necessary to perform a microscope adjustment that will temporarily degrade the data being collected by the spectrometer. At such times, you should pause the acquisition, carry out the adjustment, and resume after conditions have stabilized. To pause the acquisition at any time, click on the pulsing Capture button in the STEM-SI palette while keeping the Alt key pressed at the same time. The label of the Capture button changes to Resume.

When the acquisition is paused, the beam is either moved to the beam Safe Point if you have defined one or otherwise beam control is returned to the microscope. If the acquisition is paused and restarted, the software immediately carries out spatial drift corrections if Apply on Resume from Pause has been enabled, before continuing to acquire data from where it was before the pause.

To resume acquisition from the paused state, click on the Resume button. To stop acquisition from the paused state, click on the Resume button while keeping the Alt key pressed at the same time.

Note: Pausing is not available for hardware synchronized acquisitions. On systems which by default use hardware synchronized acquisition, you can force software synchronization via the Setup dialog.

After the acquisition

If the autosave functionality is enabled, all experimental data as saved immediately using the current SI group saving options. If the close column values after the acquisition option are enabled, the microscope column valves are closed (on systems which support column valve access). You can use the (still) active survey image on the View workspace to immediately restart an identical or similar acquisition after adjusting survey ROIs and parameters.

Visual Feedback

Spectrum image display

During spectrum image (SI) acquisition, each SI is displayed in its own image display. The spectrum image will fill as the spectral data is acquired and placed into position in the SI. You can use the 3D visualization tools to explore the spectral data while the acquisition is running (e.g., Slice tool), however, since there is a processing overhead for this that may introduce artifacts into your data acquisition, it is advised to do this after acquisition or while paused.

Spectrum image display

Live spectra

Live spectral feedback during acquisition can be enabled or disabled with the Live button in the STEM-SI palette. You can manipulate the live spectra using the standard tools for line plot display visualization.

Live spectra

Beam position cursorBeam position cursor

During acquisition, an orange beam cursor marks the beam position on the survey image. This serves as an indicator of the acquisition progress, and its position may vary slightly from that of the beam. Note for particularly fast acquisitions, the cursor is displayed as a line or even completely disabled.

Pixels per second

Pixels per secondThe actual SI acquisition rate is posted to the DigitalMicrograph® status area at the bottom of the application. It is given in units of pixels per second. This information can be useful when you configure a spectrometer for optimal readout speed.

Time remainingTime remaining

The remaining acquisition time is displayed in real-time in the STEM-SI palette just above the Capture button. This time is based on the actual acquisition rate, excluding any pauses.


Spatial drift correction

Thermal effects and mechanical instabilities in the microscope can cause the sample to drift under the electron beam. Since the acquisition of a spectrum image (SI) can occur over a considerable period of time, this drift could easily cause features to be smeared in the SI. Indeed, SI acquired over a long time at high magnification may contain data from a much different region of the sample than desired. Spatial drift is worse at higher magnifications and will vary from one microscope to another and even from one microscope session to another.

Spatial drift correction entails a cross-correlation of a reference region with a new scan of the same region taken periodically throughout the acquisition of the SI. The cross-correlation process will work better if there is some unique spatial structure in this spatial drift region.

First, a reference scan of a user-defined region is obtained from the survey image. At the start of the SI acquisition, the first drift measurement scan is taken from the same region and any spatial drift is corrected. The scan resolution and pixel dwell time used for the spatial drift scans are the same as for the survey image. Drift is measured by cross-correlation, thus the correction is applied as an offset to the beam scan coordinates. When a measurement is performed, the result is posted to the DigitalMicrograph® Results window, with any shift quoted in calibrated units if the survey image is calibrated also. During acquisition, the drift measurement is then repeated periodically using the specified frequency. Once the acquisition is completed, you can view the drift measurement using the Show correction menu item in the SI menu.

Note: Since the drift image is acquired during the SI acquisition, the detector must not interfere with the spectrum acquisition when inserted.

To set up for spatial drift correction

  1. Ensure a survey image has been acquired and assigned for the SI acquisition.

  2. Enable the Drift button on the STEM-SI palette.

A rectangular ROI marker labeled Spatial Drift will be shown on the survey image, indicating the measurement area for drift correction.

  1. Adjust and accept the correction frequency settings.

Frequency optionsThe optimum frequency of corrections will depend on the magnitude of the drift. It is a compromise between keeping the total acquisition time short and getting clean SI data. When you choose a drift correction frequency based on spatial features (pixels, rows, frames), this will generally give a more pleasing result than the time-based settings.

  1. Adjust the Spatial Drift ROI.

Adjust the spatial drift ROIUse the mouse to resize and move the ROI to an appropriate location. The measurement area should be at least twice as big as the maximum drift expected in between two subsequent measurements. It should contain an easily recognizable feature of sufficient contrast. Ideally, the area should not overlap with the data acquisition area.

  1. Adjust the advanced drift correction options (if needed).

Click the Drift button while keeping the Alt key pressed to directly access the drift correction options page of the Spectrum Imaging Setup dialog.

Viewing point spectra

When configuring the various spectrometers for SI acquisition (e.g., determine the optimum pixel dwell time for acquisition), it is often desirable to acquire a continuous spectrum from a region in the Survey Image. The microscope beam can be placed anywhere within the survey image using the Spot functionality so you can view the spectrum from each spectrometer at that position.

Viewing point spectra

To view the spectrum at a particular point

  1. Acquire and assign a Survey image.

  2. Enable the Spot button the Scan palette.

A cursor labeled Beam will appear on the survey image. If the survey image is currently acquiring, the marker is labeled Park instead. In this case, stop the acquisition to get a stationary beam.

  1. Start a continuous acquisition from the spectrometer(s) of interest using the View button of the respective Spectral Acquisition palette – The spectrum acquisition will appear in a new line plot image display.

  2. Drag the beam cursor around to view the spectrum at various points.

The spectrum display continues to display data from the spectrometer as the spot marker tool is dragged around. Use this mode to establish the optimal acquisition parameters for SI acquisition.

Beam safe point

Beam safe pointWhen the acquisition is paused or has finished, an attempt is made to minimize possible beam damage to the specimen. The beam is either moved to the park point (if you have defined one) or the beam control is returned to the microscope.

Note: The point is labeled Park during an active scan. This label name changes to Beam while no scan is active and the beam is stationary at the indicated location.

To set up a safe point

  1. Ensure a survey image is assigned.

  2. Enable spot mode on the Scan palette, and position the Beam cursor at the safe point – A region in the vacuum is ideal.

  3. If the SI acquisition is paused, or when the acquisition terminates, the beam will be returned to this point.