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:

• Mouse Behavior Recognition Module - Automatically detect ten mouse behaviors such as grooming, rearing, and digging.
• Rat Behavior Recognition Module - Automatically detect ten rat behaviors such as grooming, rearing, and sniffing.
• 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 and 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.

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, 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 the DanioVision system for multi-well plates you can select the DanioVision experiment option which optimizes the arena settings and detection settings for zebrafish larvae in well plates.

Live Mouse Tracker Experiments

If you use the Live Mouse Tracker acquisition system (https://livemousetracker.org) and want to analyze these data in EthoVision XT you can select the Live Mouse Tracker experiment option when creating a new experiment. This creates a modified version of a standard, multi-subject, multi-body point experiment, with predefined, permanent settings for Live Mouse Tracker.

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 (only available with the External Data Module).
• Track in one or multiple arenas, with a max of 100 arenas (only available with the Social Interaction Module).
• Track one or multiple subjects per arena, with a maximum of 16 subject roles (only available with the Social Interaction Module).
• 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 (only available with the Social Interaction Module).
• 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.

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 and score those categories with keyboard keys or with the mouse. 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/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 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 in multiple equal zones, including auto-labeling of these subdivisions. For example, it can be used to divide a water maze into quadrants, simplifying the process of analyzing behavior across different sections of the arena.

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.

With the additional module for Trial and 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.

Tracking Methods - Body Point Estimate for 1 Animal per Arena

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.

Deep Learning Based Body Point Detection Technique

When using this technique a new algorithm based on a convolutional neural network finds the nose- and tail-base points of rodents with high accuracy and few or no nose-tail swaps. The network was trained to extract features from video images of rodents of various colors and in various backgrounds, with videos in which the nose and the tail-base were annotated. During tracking, the network analyzes a portion of the image that includes the detected subject. It creates a map of probability of occurrence for both the nose- and the tail-base points, and finally makes an estimate of the position of the nose point and the tail-base point, based on the highest probability.

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.
• Rodents - Default - This detection method analyzes the varying shape of the contour of the area detected as subject and builds up a rodent model. It is more robust than the Shape-based method because it does not require the nose and tail to be visible: it can predict the position of the nose and the tail based on previous samples. Make sure in the detection settings that the tail is removed from the body contour with Erode and Dilate. Choose this method when you want to track a single rodent without occlusions or without difficult tracking conditions.
• Rodents - For occlusions - This method can handle severe shape distortions, for example when the animal's body is occluded or when multiple animals touch. However, it requires a lot of computer performance. Choose this method for rodents in a social interaction context, or when you track rodents that can be occluded, for example, by bars or other objects in the cage. Make sure in the detection settings that the tail is removed from the body contour with Erode and Dilate.
• Other 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 with 1-10 pixels.
• Dilation - increases the animal's surface area with 1-10 pixels.

Besides the above-mentioned options 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 for when you are using the Rat or Mouse Behavior Recognition Module.

Tracking Methods - Multiple Subjects per Arena

Color Marker tracking: The color marker itself is tracked and used for the subject identification.

Contour based body point detection offers 2 options:

1. Center of gravity tracking only.
2. Center of Gravity and Nose point and Tail-base tracking.

In this case 2 subject identification methods are available:

• Marker-Assisted identification - Identities are distinguished based on a color-marker.
• Unmarked subjects - identities may switch when paths cross.

Deep Learning Based Body Point Detection - Multiple Subjects

This method requires the animals have uniform color but appear somewhat different in the camera image. When 2 identical mice or rats are used, it is recommended to use one of the following methods to make them appear different for the deep learning network:

• Use a tail marker.
• Bleach the fur on the back of one animal.
• Shave part of the fur on the back of one animal.

Hooded rats (2-tone rodents) are not supported.

Detection Settings

EthoVision XT offers four different detection methods:

• Gray scaling - Defines all connecting pixels with a gray value between two threshold values as a possible subject.
• Static subtraction - Looks at differences between a reference image (without the animal) and the current video image (with the animal in the arena).
• Dynamic subtraction - Like the previous, looks at differences between a reference image and the current image, while updating the reference image on 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.

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 to analyze 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 and video recording simultaneously.)
5. Acquire data from existing video files - Track data from video recorded with programs other than EthoVision XT.
6. Acquire data from a batch of existing video files at once - Track data from a series of pre-recorded video files.

In options 2 through 5, the video file is available for post-acquisition visualization in combination with the track file and analysis results. If you choose option 3, 4, or 5, it is possible to analyze the samples at a rate faster than the actual sample rate, depending on the processor speed.

EthoVision XT supports a number of cameras. While tracking is done live it is possible to create video files as a backup and for integrated visualization (acquisition method 2). EthoVision XT is designed to work with MPEG2, MPEG4 and H.264 AVC video formats.

Series of Trials from Live Video Feed

You can use a live video feed to acquire a series of trials from. You can predefine start and stop conditions and an inter-trial interval.

Acquire Additional Behavioral Data

You can score behaviors manually, you can add scores live (while the test is running) or offline (by reviewing the video file). For this you need to predefine a list of behaviors. This list of behaviors is visible in the scoring panel of the acquisition screen. You can score behaviors by directly clicking them in the panel or by pressing the predefined keys. In addition, EthoVision XT is able to automatically recognize ten rat or mouse behaviors (in combination with the Rat Behavior Recognition Module or the Mouse Behavior Recognition Module respectively).

Acquire Data with DAQ System

With the External Data Module, external data acquired with a separate DAQ system can be synchronized with the tracking data acquired with EthoVision XT. When you simultaneously acquire tracking data and physiological data, EthoVision XT sends out a synchronization signal to the external DAQ system. After data acquisition, you can import the physiological data into EthoVision XT and the two data streams are synchronized. You can only co-acquire external data when you track data live or use EthoVision XT to record the video (options 1 through 3). EthoVision XT offers import profiles for a number of DAQ systems, such as DSI's Ponemah.

You can import:

• Signals sampled at a constant rate (with equidistant time stamps).
• Signals sampled at a non-constant rate (with non equidistant time stamps).
• Physiological data stored in ASCII or EDF format.
• Ultrasonic vocalization data recorded with UltraVox XT.

Analyzing data - You can:

• Analyze external data after resampling to the EthoVision XT sample rate. For example, you can resample EEG from 250 Hz to 25 Hz, and then calculate the mean of the signal and other statistics.
• Convert a signal, for example cage temperature, to a state variable with possible values 1 and 0, based on the value of the signal. You can use this state variable to analyze specific time intervals. For example, analyze all time intervals when cage temperature was above 25 degrees C.
• Synchronize the external data with the video and the track data after import.

Exporting data - You can export the original external data as well as the resampled data.

3. Edit Data

In the Track Editor the stored video file can be used to evaluate the tracking result. This allows you to quickly and easily find wrong data points and correct them. Furthermore it is possible to select samples automatically. In the latter case EthoVision will find samples that match a specific criterion, for instance to find missing points or samples that are separated by a distance greater or smaller than a specific value. With the Track Editor, points can be deleted, moved to another position or interpolated. Furthermore it can be used to swap subjects in case these were assigned incorrectly, or correct the nose-point and the tail-base in the case they were swapped.

You can save a copy of the track with the edits when you are satisfied, so you always go back to that last copy when you make a mistake in the edits. Furthermore the original track data also stays stored.

4. Smooth Tracks

Small (erratic) movements, such as body wobbles or outliers, may lead to an overestimation of certain parameters.

• The Lowess filtering method, which essentially smooths the acquired tracks of the body center point.
• As an alternative you can apply Minimal distance moved smoothing. This allows you to specify the minimal distance moved between samples that is taken into account as actual movement. This allows you to exclude micro movements.
• You can also remove outliers using the Maximal distance moved.

5. Select Data

EthoVision XT offers four methods for data selection or separation:

1. Filtering - Picking out entire tracks, based on the value of system variables, user-defined variables, or the presence of co-acquired external data.
2. Nesting - Picking out parts of tracks, according to the zone the animal (or one of its body points) was in, the behavioral state the animal was in, or during a certain time interval.
3. Time bins - Time intervals of constant duration that you can define to analyze data as if the tracks were split into segments. (Length can vary between 1 second and 24 hours.)
4. Results per zone - An easy way to calculate the results of individual analysis parameters per zone.

6. Visualize Data

You can plot all the selected tracks, or partial tracks, in one matrix. In this view you can sort rows and columns according to one or more independent variables. You can also visualize integrated data: the video, the animal's track, and the values of the selected parameters in one dynamic, synchronized view. With the built-in screen recording tool you can make a screen recording of either full screen or the module specific area (tracks and plots) of the integrated visualization.

All output variables can be presented in tables and graphs. You can customize the layout of the statistics results table, making data export to other applications straightforward.

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Download the original PDF: EthoVision XT 18 - Technical Specifications (Rodents) (PDF)