Tutorial 3D - AyyerLab/Clement GitHub Wiki

Instructions for semi-automatic alignment of FM and EM images based on observable features are described below. It is recommended to read the 2D tutorial first!

  • Load the FM stack and the SEM image in the SEM tab.

  • Draw the grid squares and transform and align the images (as described for the 2D case above)

    Load images2

  • Go to the FIB tab on the EM side and load the FIB image. Transpose if necessary.

  • Specifiy the sigma angle for the FIB device

  • You can then calculate the grid square in the FIB projection now. It will most likely appear at a wrong position because of different field-of-views of the SEM and the FIB. You can roughly align the grid square by specifying the x- and y-shift.

    FM FIB

  • In order to do a FM-FIB correlation, one has to know the three-dimensional bead positions in the FM stack. Press 'Show peaks' on the FM side to calculate and show the bead positions. Uncheck the button if you like.

    Peaks

  • You can visually retrieve the z-position of the beads when you click the 'Z mapping' button. It will encode the z-position as different colors. Usually the tilt of the sample grid is larger than the height variations of the sample itself. This effect then appears as a color gradient throughout the image. You can correct for this by selecting the 'Remove tilt effect' check box.

    Z mapping

  • You can now click the 'Select points of interest button'. It is very important that, at this stage of the correlation procedure, you select beads in the FM image and do not click at random positions. Otherwise the z-position is not calculated correctly and the correlation will fail!

  • Select enough beads throughout the entire ROI of the FM but make sure that the beads are within the FOV of the FIB image (which is usually much smaller than the FOV of the FM image)

  • Move the points in the FIB image to the corresponding beads. It can happen that some beads in the FM are not visible in the FIB image. They can be below the surface or hidden by other objects. If so, delete the point find a better bead.

    Beads

  • (Optional) Specify the bead size in microns and click 'Auto-optimize'. This will fit circles with the correct bead size to the beads in the FIB image. For the majority of the beads this is expected to work well but for some the fitting can be a bit off. If so, adjust the positions manually.

    Fit beads

  • Once you are done, uncheck the 'Select points of interest button' and click 'Refinement'. This will recalculate the grid box the in FIB image. The refinement precision is shown on the FIB side. You can click 'Show error distribution' to have a look at the refinement errors for all the beads you selected before.

    Error dist

  • Once you are satisfied with the point-wise alignment, you can click 'Merge' on the FM side. This produces a popup window showing the merged image where the EM image is now shown as an extra color channel.