Zebrafish Flash Stimuli Calibration

The Zebrafish Assay panel provides the user with the option to configure a video recording of Zebrafish (Danio rerio) larvae at a user selectable frame rate for a controlled duration of time.

One key stimuli that can be delivered to the zebrafish is a controlled flash of light. The zebrafish assay panel as of owl version 0.19.23 provides the ability to customize the color of the flash delivered to the zebrafish.

The discussion below provides a brief overview of how one can tune the parameters to obtain a repeatable, calibrated and reproducible assay.

To complete the calibration process, you must have access to a lux meter. Inexepensive digital lux meters are available on Amazon and other online retailers and can be obtained for less than $50.

Calibration procedure

To start the calibration process, set the illumination mode to be “Transmission IR850” at 50% brightness. This ensures that the flash stimuli’s brightness can be maximized. To calibrate the system, we will use the Background Illumination controls to dynamically adjust adjust the settings before recording them as stimuli.

  1. Place the detector of the lux meter face down on the surface of the diffuser in the center.

  2. With only the IR lights on, the lux meter should read a lux value less than 5 lux.

  3. Set the Lux meter’s range to be able to display values in the thousands. Typically this is denoted by a little 100x multiplier on the diplay of the lux meter.

  4. Select the color of the flash you desire. Otions include White, Red, Green, Blue and Custom. As a starting point, set the lux to 1000.

  5. Note that the hue of White flash may appear different than what you expect. The Custom option allows you control the hue by adjusting the fraction of the red green and blue LED values. Adjust the values of the red, green, and blue intensity as desired.

  6. Adjust the LUX until the desired reading on the LUX meter is achieved. Note that the measurement on the lux meter is dependent on the distance between the lux meter and the diffuser. This is true for any diffuse light source.

  7. Repeat steps 5 and 6 until the desired reading is achieved.

The figure below was generated by measuring a few different values of with the setup described above on a typical MCAM at the Ramona Optics facilities.

Calibration plot of measured lux versus the lux setpoint entered in the MCAM interface, showing a near 1:1 relationship.

As shown in the figure above, the lux setpoint in the entry box was calibrated to close to a 1:1 relationship for the measured lux in the White color dropdown. However users may change to adjust the hue of the flash by selecting the Custom option (the default values are those of the white setpoint) and adjusting them ever so slightly.

Once you record your settings, they may be used in the flash stimuli toolbox to programatically change the values of the illumination throughout the acquisition.