FreezeFrame for Optogenetics

Screening out motion of the fiber optic cable

FreezeFrame has become the standard for automated scoring of fear conditioning experiments. Optogenetics, however, has added a new level of complexity to video-based scoring systems for fear conditioning: The fiber optic used for delivering light stimuli can vibrate and sway, even when the animal is perfectly still. The camera is sensitive enough to detect this motion, which can then artificially reduce the measured freezing time of the animal. FreezeFrame 5 has a new freezing detection algorithm that screens out the motion of the cable and reports motions of only the animal.FreezeFrame Optogenetics Algorithms

This new algorithm has been tested against human scoring in a lab with considerable experience in both fear conditioning and optogenetics. For the graph below, hand scores were performed independently, before the trials were analyzed in FreezeFrame, and the FreezeFrame scores were performed without reference to the human scores.

Pulsing stimuli

For some protocols, it is useful to pulse the light source in order to best activate cells optogenetically. The new digital interface will generate pulsing stimuli with frequencies of up to 500 Hz, and pulses as short as 1 ms duration. All of the stimulus parameters frequency, duration, pulse width, and on and off times during the trial, can be easily specified in the FreezeFrame program.

Screening out the stimulus light

For pulsing stimuli, light can sometimes leak from the cable and head fixture. To prevent pulsing stimulus light from creating motion artifacts, the chamber can be illuminated with far-red or infrared light. An infrared filter is then placed on the camera. Stimulus light is then filtered out and freezing is calculated from the infrared image. No change in camera is needed.