Dark value

Dark Signal Subtraction

Image processing in PyPOLAR includes the subtraction of the dark signal—the electronic noise produced by the camera sensor even when no photons are present. This value depends on your camera temperature, gain, and exposure time.

Automatic Calculation

By default, PyPOLAR estimates the dark signal using the following method:

1. The first image in the stack is tiled into 20x20 pixel cells.
2. The software identifies the cell with the lowest mean intensity.
3. The average of this cell across all polarization angles is defined as the Calculated dark value.

Manual Override

If your sample fills the entire Field of View (FOV), the automatic method will mistakenly treat the darkest part of your sample as "dark noise," leading to over-subtraction.

To set a manual value:

1. Go to the Advanced Options tab.
2. Activate the "Dark" toggle.
3. Enter your measured value (e.g., 480 for the Institut Fresnel spinning disk setup).

Caution

If your FOV is entirely filled with sample (e.g., tissue slices or dense monolayers), do not use the automatic calculation. You must provide a manual dark value to avoid losing real data signal.