One of the most commonly studied behavior in zebrafish (Danio rerio) is the startle response. It is triggered through various stimuli either mechanical, acoustic or visual. This study consists in administering two stimuli with a predefined interval and score if the fish reacts to both stimuli or not, or to characterize the reaction to the stimuli such as the latency to the response or distance traveled. Those studies are often performed in IR lighting so as to not perturb the fish and to control the origin of the stimulation.
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To perform mechanical stimulation, 2 general approaches have emerged: the plate can be vibrated at different frequencies and amplitudes or a focal impact on the plate (tap) can be applied. The MCAMTM provides both means of stimulation. In the following Application Note, our engineers characterized important intrinsic temporal properties of the system of the plate tapper Module.

If equipped with the plate tapper option, the MCAM provides a method to use a set of solenoids to provide a momentary tap to a well plate during a video recording. The MCAM provides options to vary the relative strength of the provided tap and the time when the solenoids are energized.

It may be useful to better understand the precise time when the tap is delivered to the well plate. Due to the operating principles of the solenoids used in the plate tapper, there will always be a relative delay from the time when the solenoid is energized, to the time when it strikes the well plate holder. Here we provide a sample experiment that can be used to help calibrate this delay.

To quantify the solenoid delay, we placed the solenoid driver under the MCAM in such a way that both an LED, which is programmed to illuminate at the same time the solenoid is energized, and a solenoid are visible under a single micro camera. The LED, when turned on, will appear as a bright point under the MCAM. When the solenoid is extended, it will appear as a dark shadow. To analyze the delay from the solenoid tapper, we divide the video into two regions of interest, that of the solenoid, and that of the LED, and plot the relative brightness of the video as a function of time.

Figure 1. The solenoid plate tapper is placed under the MCAM’s field of view. The solenoid plunger, circled in purple, is carefully placed under a mount point so as to be imaged in transmission. The LED on the microcontroller is circled in orange.
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To measure the delay of the solenoid tapper, the solenoid is placed directly under a microcamera to simultaneously record the movement of the solenoid and the LED at a frame rate of 500 frames per second using the MCAM (field of view set to 512 x 3072 with a binning of 4). The recording was repeated 60 times to take an average of the intensity.

Figure 2. a) The solenoid on the left, and the LED on the right, in their resting position. b) The solenoid on the left, and the LED on the right in their fully activated position.
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The normalized brightness of the LED and opacity of the solenoid are plotted in the figure below. A noticeable delay between the LED and the solenoid are visible in Figure 3.
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Figure 3. Intensity plots for the solenoid and the LED for the full duration and the rising and falling phases.

While the turn on time delay of the LED is on the order of 1 to 2 ms, the plot shows that the time for the solenoid to be fully extended is on the order of 13–15 milliseconds. Users should take this delay into account when quantifying the delay in the response time of animals to a given stimulus.

To conclude, the MCAM user interface provides tools to control the control signals that trigger the start of a stimulus. However, delays in the physical apparatus can impact when the stimulus is delivered to a well plate. These delays can be on the order of 10 to 100 milliseconds which can skew the measurement of the response time of animals to a given stimulus especially when observing them at high speeds. Users of the MCAM are encouraged to conduct calibration experiments with their full apparatus that would reveal the time when a stimulus is received by animals.

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