The Shuttle & Find (S&F) workflow can be described in the following steps:

Image Acquisition on the Light Microscope (LM)

Before acquiring an image with the light microscope and using it for correlative microscopy, it is necessary to set up the system correctly e.g. stage calibration, camera orientation, calibrating objectives and setting the correct scaling. Note that we do not describe theses topics within this guide as we focus on the Shuttle & Find workflow only.

  • Step 1: Calibrating the Holder
    After starting the software, you first need to calibrate the correlative holder for the LM system to setup the correlative coordinate system. Note that the holder calibration must be done twice on both systems the LM and the SEM. For the calibration you have to use the S&F Holder Calibration Workbench. To learn how to calibrate a correlative holder, read the chapter Calibrating the S&F Holder.
  • Step 2: Acquiring the LM image
    Now you can perform the image acquisition on the LM. Note that you can easily move the stage by double-clicking on the live image. The double-clicked position is then moved to the center of the image area.
    To learn more about image acquisition, read the corresponding topics of the Online Help.
  • Step 3: Drawing ROIs/POIs
    In this step you can draw in regions or points of interests onto your sample images. Theses are usually the positions you want to investigate further on the other (SEM) system. For drawing in the ROIs/POIs use the S&F ROI/POI Drawing Workbench.
  • Step 4: "Shuttling" the Sample to the SEM
    Now you can bring your sample to the SEM system. At this point do not remove the sample from the sample holder.
    For transferring the image data we recommend to use the Archive functionality of the software. If both system PCs have access to a network, image data can be easily exchanged in that way. If there is no network connection, you must transfer the image data via a storage device (USB stick or external hard disc) and open the file via the Load File workbench.

Image Acquisition on the Scanning Electron Microscope (SEM)

  • Step 1: Calibrating the Holder
    After bringing the image data and the sample holder including the sample to the SEM, again you first need to calibrate the correlative holder for the SEM system. For the calibration you have to again use the S&F Holder Calibration Workbench.
  • Step 2: "Finding" the Sample Positions on the SEM
    After calibration you can now start and relocate the sample positions with the S&F Find Tool (in the Acquisition workbenches). Of course you should also bring the image data from the LM acquisition to recognize your drawn in ROIs/POIs. If you have loaded the LM image, you will see the ROIs/POIs in a list and can move the scanning stage to theses positions by one click with the mouse.
    Alternatively, you can use the S&F Find (List) Tool to relocate the sample positions if you have a list of your positions in form of a .csv file.
  • Step 3: Acquiring the SEM image
    Now you can perform the image acquisition on the SEM.
  • Step 4: Generating the Overlay Image
    After having acquired the SEM image from the same ROIs/POIs as on the LM system, you now can combine both (or more) images together and generate an overlay image. Use the S&F Overlay Workbench for this.