Software Autofocus¶

The Inscoper I.S. software autofocus mode keeps samples in focus throughout the acquisition sequence.

Configuring Software Autofocus¶

1. Specify the number of autofocus attempts per position (this defines how many Z-planes the algorithm analyzes).
2. Define the step size (in µm) between these focus iterations.
3. Choose the active optical channel used to evaluate focus.
4. Select the active camera and define the sensor exposure time for the autofocus capture frames.

5. Choose the autofocus algorithm:

   • Maximal Intensity: Optimal for bright fluorescent signals.
   • Sharpness: Optimal for dense morphological structures (e.g., brightfield/phase contrast), where the algorithm detects the highest high-frequency contrast.

6. Add Condition: Define precisely when the autofocus sequence triggers based on the acquisition dimensions (e.g., execute autofocus at every position but only every 3 time points).

7. Open Advanced Parameters to configure hardware integration:

   • Specify which mechanical device drives the autofocus routine (e.g., a fast piezo stage vs. the microscope's internal Z-drive).
   • (Optional) Apply a discrete Z-offset if the optimal focal plane differs from the plane of maximum signal/contrast.

8. Test the designed autofocus protocol on a live sample before initiating the complete sequence.

9. Click Confirm to lock in the settings.