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3.19. How to Derive Calibration Properties (µm/pixel) for Quantitative Analysis

How to Derive Calibration Properties (µm/pixel) for Quantitative Analysis

If you are performing quantitative analysis on image data, it is most accurate to retrieve the calibration properties from the acquisition system or re-acquire the data after calibrating with the specific objective lens.

Equation for Calibration Properties:

To calculate the spatial calibration (µm/pixel) for your image, use the following equation:

µm/pixel = Camera Cell Size / Total Magnification

Example Calculation:

For an image acquired using a Hamamatsu C9100-13 camera and a 40X objective lens (no intermediate magnification):

• Camera Cell Size (from the Hamamatsu C9100-13 specification sheet): 16 µm x 16 µm
• Magnification: 40
• Calculation:
  16 µm ÷ 40 = 0.4 µm/pixel

Notes:

• Magnification includes the objective lens magnification, but may also factor in:
  • C-mount magnification
  • Magnification introduced by other intermediate lenses (e.g., an Optovar).

Accurate calibration ensures reliable quantitative analysis of your image data.