How do I perform a 3D Calibration?

This documentation explains the procedure for performing a 3D calibration of your microscope using the microCAL software. The procedure described here can be used for the 3D calibration of an AFM/SPM, a confocal microscope, or a 3D-capable SEM.

Preparation and procedure

For successful 3D calibration, you will need:

• a marker-based 3D calibration standard
• the reference file associated with the calibration sample
• the microCAL software

1. Capture a measurement file of the 3D calibration standard using your microscope.
2. Save it in a format that microCAL can read. You can find an overview of supported file formats here: Supported Data file Formats.

Please note the following:

• All reference markers belonging to an array or a single pyramid must be included in the dataset. Only if all reference markers are included in the dataset — and can therefore be recognized by the microCAL software — can the correct position of the calibration structure relative to the measuring device be determined and the calibration be completed successfully.
• The resolution of your measuring device must be high enough to clearly represent the circular reference marks in the measurement data set. As a general rule, under optimal measurement conditions, a resolution of 512 × 512 measurement points is sufficient for a single pyramid; for the entire array, the resolution should be 1024 x 1024. If the resolution is insufficient for detecting the reference marks, please increase the resolution.
• To calibrate your microscope using microCal, you will always need the reference file that corresponds to your 3D calibration structure. This file contains all the reference information for your 3D calibration structure. The software will prompt you to load this file at the appropriate point. Follow the software’s instructions. Please note that each 3D calibration structure has its own reference file, which must be used exclusively with that structure. Using the ID number listed on the delivery slip for your 3D calibration structure ensures a unique assignment.

After capturing the image, you can begin analyzing the data set as described below and determine the device parameters of the microscope you are using. The result includes 6 linear parameters: the scales in all three spatial directions and the shears between the three spatial coordinate axes. This allows you to calibrate your microscope both laterally and vertically in a single step. If desired, you can generate a report and save the calibration results as correction factors in a file. Save all results to the project file.

You can use the determined calibration parameters either to adjust your microscope or to correct your microscope's measurement data.

For more information on the available parameters and parameter sets:

• Supported Parameters
• User Interface / Command Reference

Analysis with microCAL

Project preparation

For calibration, you will need:

• The measurement file that you recorded from the calibration structure using your measuring device.
• The reference file that was supplied with the calibration structure.

1. Create a project folder on your PC.
2. Copy the measurement file and the reference file to this folder.

Project settings in microCAL

1. Launch the microCAL software.
2. Click the New button in the "Project File" group to create a new calibration project.
   
   
3. Set a name for the new calibration project.
4. Select the following settings from the Calibration menu to set up the new calibration project:
   • Dimension: 3D
   • Type: Calibration parameter "AffinShearXYZ3D" (default)
   • Detection Settings: Settings for measuring the calibration markers "SPM"
   • Parameter Settings: Settings for parameter calculation "No outlier detection"
   • Date: Date of the last calibration
   • Device Name: Name of the measuring device
   • Comment: Additional information (e.g., name of the operator).
   • Name: Name for the calibration project
5. Click the Save button to save the prepared project to the project folder.

Loading the measurement file

1. Click the (+) icon in the Measurement Data Files section.
   
   
2. Open the measurement file from the project directory.
   The file name appears in the list of measurement files, and a view of the measurement data appears in the "Data View" section. If the data set contains multiple channels, you can customize the view using the pull-down menu.
   See also: Supported Data File Formats

Loading the reference file

Click the folder icon next to the measurement file entry in the list to load the reference file located in the project directory. The name of the reference file is displayed to the right of the measurement file name. This ensures that the measurement and reference data are correctly matched. If all preparations were successful, the control element with the Run icon in the toolbar will be activated.

Running the calibration

Run the calibration by clicking the control button with the Run icon. Now wait for the calculation to complete. Please note that this involves a large number of computationally intensive individual tasks:

• Detection and assignment of reference markers
• Subpixel-accurate three-dimensional measurement of marker centers
• Calculation of calibration parameters based on the least squares method
• Statistical evaluation of the results

Analyzing the calibration results

Once the calculation has been successfully completed, you will see the following results:

• A list of the determined calibration parameters in the results area
• Statistical results in the results area
• A graphical display of the measured calibration marks and error vectors in the display area

To assess the quality of the results, review the statistical values:

• The Number of measured points indicates how many markers were successfully measured and used to calculate the calibration parameters.
• The Number of outliers indicates how many erroneous measurements were detected during the transformation and excluded from the calculation.
• The Overall RMS allows you to assess the overall accuracy of the calibration. It represents the global residual error remaining after calibration (the mean of all mean errors of all measured 3D marker coordinates). The unit is set in the Results toolbar under "Settings" group.
• Overall RMS (Pixel) is the same value, but converted to pixels for easier interpretation.
• Mean RMS Image (Pixel) is the global residual error of the lateral marker centers that were successfully measured with subpixel accuracy (mean of the mean weighted unit errors of the pattern correlation, in pixels).

Comparing the residual errors from image processing with the global residual error of the parameter determination (in pixels) allows conclusions to be drawn about the accuracy of the calibration:

• If both values are close to each other, there are neither significant systematic nor random errors.
• If the two values differ significantly, there are either additional random errors (e.g., noise) or additional systematic errors (e.g., distortions). In this case, graphical analysis allows one to assess whether additional systematic errors are present or whether the errors are random.

Graphical analysis of the calibration results

In the Data View display area, you can analyze the extent to which the selected parameters can model the existing errors in your microscope. To do this, the measured values of the reference marks are transformed using the resulting parameters and superimposed on the reference data.

Select the View toolbar and check Error vectors to perform detailed analyses:

• Use Lateral Residuals to see the magnitude of the remaining deviations from an "ideal measurement" of the position, and use "Vertical Residuals" to see the remaining deviations in height.
• Use Scale to set a scale for displaying the residual errors.
• Use Rejected Points to display the points excluded by an outlier test.

Very high residual deviations are largely caused by nonlinear fluctuations that occur during measurement with your microscope, which cannot be accounted for by the parameter set used by microCAL.

If the residual deviations are not randomly distributed and a significant pattern of residual errors can be identified, there are likely additional nonlinear systematic errors in your microscope’s imaging system. To analyze these further, you can export all results — including the measured marker centers and their residual errors — and perform further analysis using third-party software.

Saving the calibration results and generating a report

To save the determined calibration parameters and generate reports, select the Results toolbar. Here, you can save the determined calibration parameters or export them to a CSV file to use them for correcting measurement data or adjusting the microscope.

If the project contains multiple measurement files, you can choose whether to save the results individually or to save the results of multiple selected measurement files consecutively. You can also use "Export Parameters for All" to export the calibration results of all selected files to a CSV file.

To generate a report, click the Create button and choose whether you want to save the report as an ASCII file (text file) or as a PDF.

Once calibration is complete, you should save the project. To do so, select the Calibration toolbar, add any descriptive entries as needed, and save the project file using Save or Save As.

You can reopen the saved project in microCAL and save, export, or generate a report of the calibration parameters. A generated report always includes the date of the original calculation, unless you perform a new calculation.