Optical microscope image as background (map) for scan areas

The maximum scan area for the JPK SPM scan head is 100×100 µm. This is indicated by a red square outline in the Data Viewer. Usually the scanned area for an SPM image is much smaller. An optical image from the CCD camera can act as a "map" to easily find interesting areas to scan in detail with the SPM scan head.

There is a procedure to align the optical microscope image to the SPM scan areas, here is a description how to do this.

 

To start the alignment procedure, there is an entry under dorpdown menu "Accessories" called "DirectOverlay Optical Calibration"

 

This entry will bring up this window. The calibration area should usually be 100×100 µm, same as the maximum scan area.
The Grid Geometry should be 3×3, i.e nine calibration images, more than necessary, but that is good since some calibration images will not be working, shown further down.
The data directory where the nine optical images are stored.
"Automatic Image Acquisition" should always be active.

Make sure you have a well focused optical image with good, even illumination in the microscope image window (usually JUnicam).
Also make sure you can see the cantilever in the image with reasonable sharpness. It has to be quite close to the sample surface. But it must NOT be engaged, i.e NOT in contact with the sample surface. The cantilever will be moved to nine position evenly distributed across the scan area, and will damage the sample if in contact.

Then click "Next" to acquire the nine calibration images.

 

Here the calibration images are acquired and the coordinates of the cantilever listed. You can click on each entry and inspect the images, that they all look reasonable.
The sample is a microscope slide with some marks from a permanent ink pen.
Then click "Next" to proceed.

 

The laser that is illuminating the backside of the cantilever is leaking into the microscope objective. There is a special filter to completely block out the laser light. This is necessary in order to get good calibration images. Above image is without the laser blocking filter. As you can see there are several very bright maxima of the laser beam which would disturb the calibration.

 

The laser blocking filter is located on the right side of the microscope base, it does not have a handle, just a black metal piece that slides sideways.

Be sure to slide the filter into its snap positions, otherwise you might partly block the field of view.

 

Next step is to select one "reference" image. Select one with a high contrast between the cantilever tip and the background.

 

In the optical image chosen for reference, indicate the position of the cantilever tip by point and click. The default is to use a circular region, the radius of 50 pixels might have to be adjusted. When you have indicated the tip position, the pixel coordinates are displayed for the chosen reference image.
The click "Calibrate" for the next step.

 

The software tries to match (correlate) the circular region in the reference image with the same feature in the other images, i.e. tries to find the tip. This does not always work, as can be seen here for image #7.
Click on each image entry to check if the tip has been correctly found. If not (as in this case) un-tick the the tick mark in the "Use" column. These images will be excluded in the calibration computation.

Please note! You can also correct the images where the tip has not been correctly found. Just point and click at the tip of the cantilever and the "Pixel X" and "Pixel Y" values are updated. Be sure to activate the corrected image by ticking it in the "Use" column.

You can also reduce the mismatch between optical background image and the AFM scans by indicating the real position of the tip on the cantilever. The tip is usually located near the center between the apexes of the cantilever shape.

More detailed knowledge of the tip location on different cantilevers can be found at the manufacturers websites, often it is enough to do an web search for a model number of a specific cantilever.

 

Finally the aligned image is shown. The "Median" image is a combination of all the calibration images where the tip has been filtered out.
You can also use the calibration image, if the cantilever does not hide any important part.
Or you can load a previously acquired image.
Or take a snapshot, you should move the cantilever to a unobtrusive position first.
Click "Finish" to put the image as background in the Data Viewer(s).

 

Here the aligned optical image is shown in the Data Viewer. The maximum JPK SPM scan area is outlined in green.

 

Here a SPM-scan is started, overlaying the optical image.

Please note! The overlayed optical image could be displayed "on top", hiding the ongoing SPM scanning. This is controlled in the "Image Data" stack, displayed to the far right on the right hand screen.

 

The ongoing SPM scan is always shown at the top of the data stack. But it is not necessarily shown in the Data Viewer. The entry outlined in blue is the one shown, here "Scan 6", the onging scan. Please check this if you do not see any scan being displayed in the Data Viewer.

 

Here is a zoomed in display of the ongoing scan on top of the optical background image. Currently there seems to be a mismatch of about 10 µm between SPM scan and the optical image. This is probably due to the calibration, which is done using the outer edge of the cantilever, not the position of the actual tip.

 

Here are several SPM scan acquired. Please note the good match between consecutive SPM scan areas, the one outlined in blue matches very well with the underlying one.

Anders Liljeborg Albanova Nanolab, KTH, SU.