How to Change the Spatial Calibration of a Dataset in Volocity

Proper spatial calibration is essential for accurate quantitative analysis in Volocity. Each objective lens on the acquisition system should be calibrated using a stage graticule, which ensures that the measurements correspond to real-world dimensions. For detailed instructions on performing this calibration with Volocity Acquisition, refer to page 151 of the Volocity User Guide.

However, if the correct calibration values (microns per pixel) are already known, these can also be manually entered.

Steps to Change the Spatial Calibration:

1. Select the Image Sequence:

   • Open the dataset for which you want to adjust the spatial calibration.

2. Access Image Properties:

   • Go to the Edit menu and select Properties.

3. Modify Calibration Values:

   • Adjust the following fields as needed:
     • µm/pixel (X): Represents the pixel size in the X-axis.
     • µm/pixel (Y): Represents the pixel size in the Y-axis.
     • µm/pixel (Z): By default, this is set to the physical step size during acquisition but can be modified to account for spherical aberration.

Calculating µm/pixel for X and Y:

If the spatial calibration has not been performed using a stage graticule, you can calculate the µm/pixel value manually using the following equation:

µm/pixel = Camera Pixel Size (µm) / (Lens Magnification × Intermediate Magnification)

Example Calculation:

Scenario:

• Camera: Hamamatsu Orca AG
  • Pixel size: 6.45 µm (X and Y)
• Objective Lens: 63x magnification
• Intermediate Magnification: 0.63x reduction C-mount

Calculation:
µm/pixel = 6.45 / (63 × 0.63)
µm/pixel = 0.163

Result:
The spatial calibration values for X and Y would be 0.163 µm/pixel, which can be entered into the µm/pixel (X) and µm/pixel (Y) fields in the Properties dialog.

Best Practice: Calibrate Using a Stage Graticule

While manual calculations can provide approximate calibration values, it is best practice to use a stage graticule for calibration. This ensures the calibration values are as accurate as possible, accounting for all variations in optical components and camera setup.