Knowledge Base

4 - Prepare Data Acquisition 4.1 Train the animals................................................................................. Before you start acquisition, it is recommended to train the animals to cross the glass plate. This results in better data. With the Detection Settings the CatWalk system distinguishes the paw prints from the background. 4.3 Specify the acquisition settings .......................................................... In the Acquisition Settings, you can view the snapped Background Image, specify how CatWalk should treat acquired runs and set the Sound feedback. 4 - Prepare Data Acquisition

4.1 Train The Animals

Training Protocol

Before you start acquiring runs, animals need to learn to cross the walkway without interruptions or hitches so all animals start from a similar baseline. This is important in order to be able, during an experiment, to compare different runs and pick up gait abnormalities that are the result of effects of treatment rather than differences in quality of runs. Below you find a description of a training procedure that you can adapt to suit your experimental setup: 1. Habituate the animal to the CatWalk system by placing it in the corridor for 1 minute on a number of consecutive days until it freely explores the walkway. Make sure the corridor walls are together as close as possible so the animal is still able to comfortably walk in between the walls but is not able to meander too much from side to side. 2. One week prior to treatment, train the animals to run across the glass plate with a minimum of three runs per day. You can condition the animals to run by signaling the availability of a food pellet at the other end of the walkway using a conspicuous auditory or visual cue placing food pellets. If necessary, subject the animals to mild food restriction or deprivation. You can use all sorts of incentives to facilitate runs across the walkway. You can use normal food pellets but also special rodent sweets or candy. Using the special goal box animal crossing the walkway. 3. The training criterion could then be that the animal crosses the walkway without any interruptions or hitches, in three consecutive runs. The time it takes for the average animal to cross the glass plate can be used as a basis for the run criteria (see page 39).

Tips From Catwalk Xt Users

Training animals in a different environment

Some users find it more convenient to train an animal to cross a certain distance, similar to the length of the walkway, in another apparatus than the CatWalk system. For example, you can start training an animal to run from one side of an open field to the other. Once it has learnt to run this distance, you can let it run on the walkway. 4 - Prepare Data Acquisition

Using home cage mates

If you use the goal box, let all animals from a home cage habituate together in the goal box. For example, the first time for five minutes, the next time for two-three minutes. In that case you must make sure that only one rat crosses the walkway and the others stay in the goal box, for example using a sliding door between the walkway and the goal box entrance. When the animal approaches the goal box, open the sliding door and close it carefully to avoid pinching the animals' tail after it has entered that cage.

How long should you wait?

Let the animal find its own way to the other end of the walkway, without trying to force it in any way. Wait up to for example 15-20 minutes. Experience shows that patience produces good results.

4.2 Configure The Detection Settings

Introduction To Detection Settings

Detection settings and profiles

The aim of the Detection Settings is to achieve an optimal intensity range for the paw prints and to filter out background noise. This is done by putting an animal of average weight on the walkway and then adjusting the Camera Gain and Green Intensity Threshold. Optionally, you can adjust the voltage (intensity) of the green and red lights in, respectively, the walkway and the illuminated ceiling. CatWalk provides quantitative feedback in order to define an optimal range between the minimum and maximum print intensity. Make sure to turn the lights off before configuring the Detection Settings. Alternatively, you can work under red light conditions. Make sure you avoid the red ambient light from shining directly into the camera. Make sure you completely open the camera's aperture and you focus the camera. Detection Settings are stored in a Detection Settings profile. You can create multiple Detection Settings profiles of which one is active during acquisition (see also Profiles on page 150). When you start up CatWalk XT for the first time, two Detection Settings profiles are present: Rats and Mice. 4 - Prepare Data Acquisition You can configure Detection Settings both automatically and manually. We recommend to first carry out auto detection and then manually fine-tune the settings (see below).

Basic procedure

1. Carry out auto detection (see page 49) to get optimal values for Camera Gain and Green Intensity Threshold.
2. Adjust the Camera Gain and Green Intensity Threshold to further optimize detection (see manual detection on page 51).
3. If necessary, adjust the Green Walkway Light and/or Red Ceiling Light to further increase the quality of the paw prints (see Adjust the intensity of the Green Walkway Light and/or the Red Ceiling Light on page 52).

Auto Detection

1. First, put an animal on the walkway. Make sure the illuminated ceiling is lowered.
2. In the Profiles Explorer, double-click the Detection Settings profile you want to use: Mice or Rats. The difference between the Mice and Rats Detection Settings profile is the default intensity of the Green Walkway Light (Mice: 16.5 Volts, Rats: 16.0 Volts). For both Mice and Rats, the default value for Camera Gain is 20, for Green Intensity Threshold is 0.10 and for Red Ceiling Light is 17.7 Volts. Figure 4.1 shows the initial values for Camera Gain and Green Intensity Threshold under Detection Settings, and Maximum and Minimum Green Intensity under Results, before the start of Auto Detection. 4 - Prepare Data Acquisition
3. Click the Reset Prints button and then the Start Auto Detect button. The program now automatically adjusts both Camera Gain and Green Intensity Threshold in consecutive steps to achieve the optimal range. Adjustment of the Camera Gain results in a value for Maximum Green Intensity between 197 and 203 based on the prints in the Print view. Adjustment of the Green Intensity Threshold ensures that prints that do not contain pixels with an intensity above the Minimum Green Intensity are removed from the Print view. It is important in this step that the animal walks through the camera view a number of times for proper determining of the detection settings. If the animal just sits still, the detection settings might not work for a moving animal.
4. When Auto Detection is finished successfully, a message appears.
5. You can now do the following: You can accept the settings and proceed to Acquisition Settings (see page 53). You can manually adjust the Camera Gain and Green Intensity Threshold to tweak the quality of the paw prints (see manual detection on page 51). Furthermore, you can adjust the intensity of the Green Walkway Light and/or Red Ceiling Light (see Figure 4.1 Part of the Detection Settings window. This example shows the default values under Detection Settings and Auto Detection Settings before auto detection. 4 - Prepare Data Acquisition page 52). This might be useful if you work with either very heavy or very light animals and the default settings in the Detection Settings profiles Rats and Mice do not result in optimal detection of the paws. Also, decreasing the intensity of the Red Ceiling Lights might result in better detection of toes for use with the Interactive Footprint Measurements.

Auto Detection Settings

Under Auto Detection Settings you can set the following:

Maximum range from - The Camera Gain is automatically adjusted until the Maximum Green Intensity (displayed under Results) reaches a value within this range. The default range is between 197 and 203. Frames before delta - This parameter determines how many video frames with prints should be detected before the Camera Gain is auto adjusted. A print should contain at least one pixel with an intensity above the Intensity Minimum value. The default number of frames is 5. Note: the frame rate of the CatWalk camera is 100 frames per second. Intensity Minimum - This is the minimum value for the intensity. Each print should contain at least one pixel with an intensity above the Intensity Minimum. The default value is 85. Prints caused by background noise (such as, other light sources than the green LEDS, dirt) that do not contain a single pixel with an intensity above 85 are automatically removed by an increase of the Green Intensity Threshold.

Manual Detection

1. First, put an animal on the walkway. Make sure the illuminated ceiling is lowered.
2. Choose Setup > Detection Settings. As soon as the Detection Settings window opens, the Print view shows the animal's cumulative prints. Under Results, the Maximum and Minimum Green Intensity of all prints shown in the Print view are displayed, based on the current settings of the Camera Gain and Green Intensity Threshold shown under Detection Settings. The Camera Gain (dB) determines the Maximum Green Intensity, the Green Intensity Threshold determines the Minimum Green Intensity.
3. Under Detection Settings, adjust the Camera Gain until the Maximum Green Intensity value for these prints is approximately 203. The absolute maximum intensity for prints is 255. 4 - Prepare Data Acquisition As a result, under Results, the Maximum and Minimum Green Intensity, and Range are changed, based on the current values for Camera Gain and Green Intensity Threshold. Each time you adjust Camera Gain, the prints in the Prints view are reset.
4. If necessary, increase the Green Intensity Threshold to remove noise, that is, prints in the Print View that are not paws. As a result, in the Print view, the noise is filtered out and under Results, the Minimum Green Intensity and Range both change.
5. Click OK when you are done.

Adjust the intensity of the Green Walkway Light and/or the Red Ceiling Light

Adjust the intensity of the Green Walkway Light when the Camera Gain reaches its maximum or minimum value; this can occur if, respectively, the animal's paws exert too little pressure on the glass plate or if there is too much background noise. You get an error message if this happens. For example, with a large, heavy rat, the Camera Gain can drop below the minimum value of 6. In that case, decrease the Green Walkway Light intensity from the default 16.0 to 15.5. Next, re-do auto detection (see page 49). If detection of the toes is important (for example, if you want classify toes in the Interactive Footprint Measurements to calculate the Sciatic Functional Index), make sure to check whether manually adjusting the intensity of the Green Walkway Light (in steps of 0.25) might result in even better detection of the toes, especially for light-weighted animals. Also, reducing the intensity of the Red Ceiling Light might increase the detection of the toes (see Figure 4.2 for an example of what the optimal intensity of the Red Ceiling Light should look like). Figure 4.2 Two examples of Red Ceiling Light intensities. In the top picture, the intensity is too low. As a result, the animal is only detected when it already has entered the walkway. Furthermore, the body contour is not fully visible which results in poor performance of the automatic classification. The bottom picture shows an example of optimal intensity of the Red Ceiling Light. 4 - Prepare Data Acquisition We recommend to adjust the intensity of the Green Walkway Light and/or the Red Ceiling Light after determining the optimal values for Camera Gain and Green Intensity Threshold. As a rule of thumb, the optimal value for Camera Gain ranges between 10 and 20 dB. If the value of Camera Gain is far outside this range (for example, 6 or 35), adjust the intensity of the Green Walkway Light and re-do auto-detection until the Camera Gain is within the optimal range (10 - 20 dB).

4.3 Specify The Acquisition Settings

Choose Setup > Acquisition Settings.

Background Image

When you have snapped a Background Image before acquisition (indicated by a check mark icon; see page 58), you can view it by clicking the View Background Image button. When the Background Image does not cover the area of interest of the walkway, you should (re-)define the walkway.

Run Settings

You can specify the following settings: Auto abort runs when the duration exceeds ... seconds - When selected, this option automatically stops acquisition when the set time is exceeded. The Auto abort run... option is different from the Maximum run duration in the run criteria (see page 39). When the animal exceeds the Maximum run duration, the run is marked 'non-compliant' but acquisition is not aborted. CatWalk XT stops recording automatically when the run duration reaches 60 seconds. Runs longer than 60 seconds generate so much data that it can impede the computer's performance. Discard auto aborted runs - When selected, CatWalk automatically deletes runs that exceed the time set in the Auto abort runs... option. Discard user aborted runs - When selected, runs that are stopped by the user are deleted. 4 - Prepare Data Acquisition When this option is not selected, when you manually stop acquisition during a run, the run is saved. Discard runs not compliant with the Run Criteria - When selected, runs that do not meet the run criteria (see page 39) are automatically deleted. Discard incomplete prints at the start and end of a run - When selected, paw prints or part of paw prints on the border of the walkway are not included in the classification (that is, CatWalk draws no rectangle around the print). Including incomplete prints in the analysis affects parameters, such as, Print Area or Max Contact Area. Please note that if a paw print consists of sub-prints (for example, toes), the sub-prints that are not on the border of the walkway are not discarded. As a result, sub-prints that are not on the border are included in the classification, sub-prints on the border are not (see Figure 4.4 for an example). To discard the complete print, select all labeled sub- prints and label them as Junk.

Sound Feedback

It might be useful to get auditory feedback (system beep) on the status of acquisition, especially when you are not able to view the runs on the monitor. You can select the following: Run start (1 beep) - When selected, you get one system beep when acquisition starts. Run stop (2 beeps) - When selected, you get two system beeps when acquisition stops.

Figure 4.3 Example of the result of selecting the option "Discard

incomplete prints at the start and end of a run" in Classification. A paw print directly on the border of the walkway is not included for classification (CatWalk draws no rectangle around the print) and therefore available for labeling.

Figure 4.4 Example of a paw print of which a sub-print (on the

border of the defined walkway) is not included in the classification and another one (labeled RH) is included in the classification. To exclude the labeled sub-print, reset the label and label the print as Junk. 4 - Prepare Data Acquisition Error (e.g. camera connection lost, 3 beeps) - When selected, you get 3 system beeps if an error occurs. Turn Sound Feedback off when you suspect it might disturb the animals.

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Source: CatWalk XT 10.7 - Reference Manual