Knowledge Base

System Configurations

EthoVision® XT Base allows you to perform complete tests with one animal in one arena. You can extend the functionality with one or more of the eight available modules:

• External Data Module – Integrate EthoVision XT data with co-acquired external data.
• Multiple Arenas Module – Track animals in up to a hundred arenas simultaneously.
• Social Interaction Module – Track multiple animals within each arena.
• Trial & Hardware Control Module – Create complete test protocols to initiate and control interaction with external equipment, based on real-time analysis of the animal's behavior.
• Quality Assurance Module – Assign different rights to different users and comply with Good Laboratory Practice guidelines.
• Track3D – Record movement of an animal in a test chamber (such as a wind tunnel or swim tank), visualize the trajectory in a three-dimensional image, and calculate a number of movement parameters.

Data Management

1. Setup

Template Experiments

EthoVision XT contains a user-guided set-up with templates for several test paradigms. At the start of the program you can either start a brand-new experiment, re-use the settings from a previous experiment, or use the user-guided set-up to open an experiment template suited to a specific test (water maze, plus maze, 96-well plate, etc.). With these templates, many basic settings are pre-set, such as arena definition, detection settings, and parameters for analysis. You can fine-tune these settings to fit your specific needs.

DanioVision Experiments

If you use DanioVision™, a system specifically designed for observing and analyzing zebrafish larvae behavior, you can select the DanioVision experiment option when creating a new experiment. Furthermore, the automated arena detection detects individual wells in standardized well-plates automatically and the automated detection settings detect the subjects — for example, zebrafish larvae — and optimize detection with a few mouse clicks.

Good Laboratory Practice

Additionally, for those conducting Quality Assurance experiments, particularly in environments adhering to Good Laboratory Practice (GLP) standards, EthoVision XT offers specialized options to meet these rigorous requirements. This ensures that your experiments not only yield reliable data but also comply with industry standards for quality and reproducibility.

Flexibility

For researchers seeking maximum flexibility in their experimental design, the "New from Scratch" option allows for complete customization of the experiment setup. This feature is ideal for innovative research that goes beyond standard protocols, offering the freedom to tailor every aspect of the experiment to unique research needs.

Experiment Options

• Tracking can be done live or from a video file.
• You can use digital cameras or analog cameras (but not a combination of the two).
• You can connect external data acquisition systems to EthoVision XT.¹
• Track in one or multiple arenas, with a maximum of 99 arenas.²
• Track one or multiple subjects per arena, with a maximum of 16 subject roles.²
• For mice, rats, and zebrafish you can use center point tracking, or center of gravity, nose, and tail base. For mice and rats you have the option between a contour-based technique or a deep learning based technique. You can also track based on color or color marker.²
• Activity analysis – a non-tracking option analyzing overall activity per arena based on pixel changes. This is very useful for freezing detection.
• Distance can be measured in millimeters, centimeters, meters, or inches.
• Time can be measured in milliseconds, seconds, minutes, or hours.
• Rotation can be measured in degrees, radians, gradians, or rotations.

Manual Scoring

The Manual Event Recorder is a built-in coding system which allows you to define a list of behaviors that you can score manually during and after data acquisition (live or offline).

• Score live.
• Score (also) offline, to score at a lower or faster playback speed. You can also edit scored events.

Different types of behaviors:

• Start-Stop Behaviors – Behaviors that are independent from each other.
• Point Events – Behaviors without a duration.
• Mutually Exclusive Behaviors – Behaviors grouped in such a way that activating one behavior automatically stops the other behavior.

You can define different behaviors using the keys from the computer keyboard. Without key codes defined, you can manually score events using the mouse.

Arena Settings

The arena is the region in the video image where the animal is tracked. You can define:

• The arena(s).
• Subdivisions of the arena (zones).
• Cumulative zones (define individual smaller zones to be taken into account for cumulative analysis).
• Hidden zones (used for shelters) and entry zones to analyze entries and exits.
• Zones organized in layers, so you can define and analyze zones that overlap.
• Points of interest.

You can define an unlimited number of zones in an arena, and create zones of any shape. Calibration of your arena will allow EthoVision XT to calculate parameters such as velocity and distances to certain points. If you are tracking in multiple arenas, you can copy and paste the settings from one arena to the other.

Multiple Arena Definition

When your experiment contains multiple arenas — for instance using a multi-well plate or a group of open fields or home cages — you can use the multiple arena set-up functionality. This means that you do not need to draw each arena separately. EthoVision XT allows you to easily rotate, scale, transpose, resize, and reposition the series of arenas simultaneously. In DanioVision experiments, automated arena detection detects the individual wells in standardized well-plates automatically.

Arena/Zone Auto-Subdivision

This option allows the automated subdivision of circular and rectangular arenas into multiple equal zones. It includes auto-labeling of the subdivisions.

Multi-Camera Set-Up

In EthoVision XT you can combine the input of multiple cameras. This way you can track from multiple arenas (in combination with the Multiple Arenas Module) without the need to place them all under one camera.

Trial Control Options

You can program EthoVision XT to automatically start and stop tracking, based on any of the parameters offered by EthoVision XT. This feature also includes an external command option, which allows you to start another computer program when a condition becomes true. For example, present a visual stimulus on a computer screen or play a sound file when the animal enters a particular zone.

Detection Settings

Besides the options mentioned above to optimize the detection and identification of the subject(s), contrast settings can be fine-tuned, subject size can be defined, and video smoothing can be applied. There are also some additional detection settings specific to when you are using the Rat or Mouse Behavior Recognition Module.

EthoVision XT offers four different detection methods:

• DanioVision – For DanioVision, the detection method is chosen automatically. It provides great ease to set as a method because it is tailored specifically to larvae.
• Gray Scaling – Defines all connecting pixels with a gray value between two threshold values as a possible object.
• Static Subtraction – Looks at differences between pixels in a reference image (without the animal) and the current video image (with the animal in the arena).
• Dynamic Subtraction – Like the previous method, looks at differences between a reference image and the current image, while updating the reference image at each sample.
• Differencing – Makes a statistical comparison between pixels in a reference image and the current image. The statistical comparison uses the variance in contrast between the current and reference image and determines whether each pixel has changed enough to be considered part of the subject.

Tracking Methods (Body Point Estimate)

The tracking method specifies how the body points are estimated once the subject is detected. Depending on how many subjects and body points you specify in the Experiment Settings, one or more methods for tracking of body points are available in the Method section of the Advanced Detection Settings.

Contour-Based Body Point Detection Technique:

• Any Species – Default – This detection method analyzes the contour of the area detected as subject at each sample to assign the nose-point and tail-base. Make sure in the detection settings that the tail is fully detected. With this method it may be possible to track non-rodent shapes, but the method is not designed for it. This method is automatically activated when you track the center-point in one animal per arena and you specified Other Species in the Automated Setup.
• Adult Fish – For Occlusions – Use this method to track zebrafish and other fish of similar shape, when viewed from above. Make sure that the whole body of the fish is detected, including the tail.
• All Species – For Occlusions – This method does not make any assumption about the shape of the subject. Choose this method to track, for example, insects, crustaceans, and large animals. Also use this method if you track fish from the side view of the water tank and the Adult Fish method does not give good results. However, with this method you only track the center point.

Contour adjustments can be made in one of two ways or by combining both methods:

• Erosion – Decreases the animal's surface area by 1–10 pixels.
• Dilation – Increases the animal's surface area by 1–10 pixels.

Trial List

The trial list consists of trials that are planned, carried out, or skipped, including system variables and user-defined independent variables. All are organized in a cross table and data can be copied to and from Excel sheets. In EthoVision XT you can schedule a list of trials based on pre-recorded video files. This list can be automatically acquired as a batch in data acquisition. The trial list also allows you to import trials from previously acquired experiments, with the aim of analyzing data collected in two or more identical experiments as one data set.

2. Data Acquisition

EthoVision XT offers the following acquisition methods:

1. Acquire Data Live – Live tracking requires no disk space for video storage, but creates no video backup either. The maximum trial duration without video recording (as tested) is 72 hours.
2. Acquire Data Live and Record Video – Live tracking while EthoVision XT records a backup video to an MPEG4 video file. The maximum trial duration with video recording (as tested) is 72 hours depending on the hardware used.
3. Longer Trials – To acquire trials longer than mentioned above, you can split your multi-day testing into multiple trials.
4. Record Video and Acquire Data – EthoVision XT records a video to an MPEG4 file which you can use for data acquisition later. This method is recommended if the computer is not fast enough for live data acquisition.

With the additional module for Trial & Hardware Control, you can define complete test protocols to initiate and control interactions between your animals and external sensor and stimuli devices. Real-time analysis of any of the parameters of EthoVision XT allows you to control complete test protocols based on your animal's behavior. You can use it for complex learning tasks such as the radial arm maze with automatically controlled doors and food delivery using pellet dispensers. Attach and control all standard (TTL-based) hardware.

If you are using multiple arenas, conditions are applied to each arena individually.

¹ Only available with the Physiology Integration Module.
² Depending on whether you are using one of the modules.

Source: Technical Specs - EVXT 18 - Zebrafish, Noldus Information Technology