Knowledge Base

Problem

A user recording a side view of a fish tank needs to calibrate the arena correctly in EthoVision, but is concerned that glass and water refraction will distort the apparent scale and position measurements.

Details

Applies to any EthoVision XT setup where the camera views subjects through water and glass (e.g., zebrafish, fish behavioral assays with a side-view camera). Refraction at the glass-water and air-glass interfaces causes the apparent position and distances to differ from actual physical dimensions, making calibration against the outer tank wall inaccurate.

Solution

1. Do not calibrate using the outer tank wall dimensions alone, as refraction will introduce scale error.
2. Place a physical reference object (ruler, calibration rod, or grid) inside the tank, submerged at approximately the same depth plane where the fish will swim. This ensures the calibration reference experiences the same optical distortion as the subject.
3. In Arena Settings, use 'Draw Scale to Calibrate' and draw the calibration line between two known points on the in-tank reference object.
4. Enter the known real-world distance between those two points to set the scale.
5. Position the coordinate origin using the Calibration Axes tool (e.g., set the origin to the lower-left corner of the visible tank interior) so that reported XY coordinates are intuitive and consistent.
6. After calibration, verify by measuring a known distance in the arena image (using the scale tool) and confirming it matches the physical measurement.
7. Keep camera position, zoom, and focus fixed throughout the experiment; any change will invalidate the calibration and require recalibration.