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LimeLight

Video tracking for Open Field – Plus Maze – Radial Arm Maze – Zero Maze – Novel Object Recognition – Conditioned Place Preference

LimeLight is a video tracking system for behavioral experiments. It is extremely flexible and powerful, with a complete range of features, and yet it is easy to set up and operate, and it is affordable. Developed in close collaboration with behavioral neuroscientists, LimeLight gets high praise from its users.

Tracking. LimeLight can track up to 4 arenas at once. Use 1 camera for all 4 arenas (one in each quadrant of the image), or for maximum flexibility, 1 camera for each arena. No special lighting is necessary, and black or white animals are tracked automatically on almost any background. If you can see it, so can LimeLight. Any number of stationary items can be placed in the arena without affecting tracking. Collect images at up to 30 frames per second for one animal or up to 8 frames per second when tracking 4 arenas at once. Tracking is robust even at extremely low light levels so that the environment can be tailored to the animal’s needs, not the camera’s. Images are compressed on-the-fly and stored on the hard drive for later review and for off-line scoring of complex behaviors. All or part of the trial video can easily be exported in AVI or QuickTime format for presentation purposes.

Arena Setup. LimeLight simplifies the process of setting up grids and zones, but allows complete flexibility. Dedicated interfaces for Open Field testing (circular or square) and Elevated Plus Maze require only a few mouse clicks to configure. The Custom zone setup allows almost infinite flexibility for more specialized designs, such a novel object recognition or zero-maze, again with a simple click-and-drag interface. Zone configurations can be changed in the analysis phase, after the trials are recorded.

Behavior Scoring. User-defined behaviors can be scored either during a trial, or while viewing the trial from stored video. Store multiple sets of behaviors for rapid switching between experiments. Each behavior can be assigned a key stroke, or use the mouse to click buttons for scoring observed behaviors. For complex experiments, stored video can be reviewed repeatedly and at the users pace, each time adding in the scores for a different behavior or behavior set. Statistics on the scored behaviors can then be viewed and exported to Excel.

Stimulus Control. LimeLight has 8 output signals (TTL format) for controlling the experimental apparatus. The user can specify that a line be turned on when the animal enters a particular zone in the arena, with numerous options for sequencing. Initial grace period, stimulus duration, delay and lock-out are adjustable. The 8 output lines are provided on a plug-and-play USB interface.

PcolorMapHigh throughput. LimeLight was specifically designed for high throughput experiments. Some of our users record trials from hundreds of animal per day. All setup parameters are stored for rapid switching between experiments. Analysis windows and batch export allow rapid review of large numbers of trials.

Analysis. LimeLight excels at analysis. The main analysis window gives an informative overview of most aspects of a trial in graphical form. The display at the right, for example, shows a colormap of the time spent in each grid-square, and is instantly available for any animal in a data file. Video is also instantly available for playback for any animal.

Export zone-by-zone values for many different measures, or export summary data for all available measures and all recorded animals. Exported measurements are automatically displayed in a new window in Excel, as well as being saved to a text-based file. Collect data from any number of animals into a single data file and afterward sort animals into user-specified groups: When an Analysis is performed, data are reported for animals in each group, together with mean and standard deviation for the group as a whole. Analyses can also be prepared for user-defined portions of each trial. For example, one can split each trial into 2 equal intervals and analyze each interval separately. Analyses are designed in collaboration with our users and are tailored to the specific needs of each type of experiment. For all experiments, each analysis parameter can be exported by grid square or user-defined groups of grid squares, maze arm, or custom zone. Parameters include:

  • Distance travelled in each region (cm or %)
  • Time spent in each region (sec or %)
  • Crossings into each region
  • Body length from nose to base of tail (mm), together with stretch-attend bouts
  • Pointing Direction (orientation)
  • Mean distance from center
  • Latency to Zone
  • Distance and Velocity vs. time
  • Statistics for scored behaviors (1st and 2nd order)
  • Statistics on occurrence of stimuli (output control)
  • Zone sequence
  • Circling
  • Rotation

Ease of use. LimeLight’s interface makes it a pleasure to use (and we are not the only ones to say so). Dozens of shortcuts make traversing the many features of the program nearly instantaneous. Most displays, for example, can be double-clicked to display more details of an analysis or to change the displayed parameters, without searching through the menus for the relevant item.

Scoring Behaviors#

The window below shows the offline scoring of complex behaviors. The user is rerunning the video from a previously recorded trial and scoring a set of 4 behaviors.

  • The video can be played frame-by-frame, backwards and forwards, at real-time speed or fast forward speed, with key commands for each mode. Or scroll quickly through the video by dragging the green slider beneath the image.
  • Behaviors can be selected by clicking on the green buttons, or by the key commands selected of each behavior. Here, the behaviors are set to be mutually exclusive (selecting one turns of all others). Or, behaviors can be set up so that multiple ones can be selected simultaneously.
  • The user can choose to preserve previous scores for some behaviors while concentrating on scoring others. This way, different behaviors can be scored on different reviews of the video.
  • Up to 20 different sets of behaviors, with 16 behaviors each, can be stored and selected for use on any trial.

Exporting Movies#

During the recording of a trial, LimeLight allows you to save the images collected by the program. In the LimeLight Analysis program, the movies can be easily viewed. In addition, an option is provided for converting any portion of the image file to a QuickTime movie format that can be imported into PowerPoint and other presentation software.

Behavior Analysis#

A more detailed view of scored behaviors. Totals can be exported to Excel, along with zone-wise measures of behaviors and 2nd order measures (how often one behavior follows another).

LimeLight Analysis#

The heart of the LimeLight program is the analysis window, shown here at not quite full size. The complete list of animals contained in a data file is shown to the left. Clicking on an animal instantly brings up its motion path. If the images from the trial were stored, a movie of the trial is available for viewing using the video buttons (and attached key commands) below the image.

  • The analysis of the trial is shown to the right. Here the number of crossings per grid square are shown as an intensity graph. A checkbox to the right changes the display to table format with numbers for each grid square. The pull down menu currently set to “Crossings” is used to select from the many different analysis parameters, such as distance travelled, time in each grid square, mean length in each grid square, latency to each square and so on. Double-clicking the colormap exports the data to a text file and opens the file into Excel.
  • A portion of each trial can be analyzed (instead of the whole trial) by toggling the All/Part checkbox and setting the starting and ending time of the part of the trial one wants to analyze. Or, cut into multiple intervals.
  • Scored behaviors are shown to the lower right. Double clicking the graph opens a more detailed analysis.
  • Adjust the grid or create custom zones from arbitrary groups of grid squares by double-clicking the image.
  • Separate windows are accessible for viewing distance and speed against time, mean distance to center, animal length and circling. Any selected parameter can be exported directly to an open Excel spreadsheet for instant viewing and statistical analysis.
  • The current path shows the track of the animal’s body (center of mass). The pull-down menu to the right allows display of the track taken by the animal’s nose or tail.
  • Menu items open windows for the display and export of more complex analyses, such as animal length, speed and distance against time, mean distance to center, etc.

User-Specified Groups#

Using the Groups Window below, the user can divide all the trials stored in a data file into any number of groups. Here, there are 4 groups specified (unimaginatively named “Group 1”, “Group 2” etc). The number of groups and group names are specified in the controls to the left. Once the groups are named, then each group is selected in turn using the green buttons, and trials are assigned to that group by clicking on the list to the right. Once groups are specified, batch exporting of analyses can then be performed by group. That is, within the spreadsheet generated by LimeLight, animals are ordered according to group, and the mean, std and sem of the analyzed parameters are calculated and reported separately for each group. In this example, Distance travelled has been exported by Zone, and two groups of 8 animals each have been analyzed separately.

https://actimetrics.com/products/limelight/batch-analysis/

Body Length Calculation#

As a trial proceeds, in addition to tracking the animal’s position LimeLight also calculates the length of the animal from nose to the tail base (blue and red cursors in the image to the right). In this case, finding the nose and tail is pretty straightforward, but LimeLight is also extremely accurate even when the animal has its tail curled up around its body, or when the animal is huddled in the corner of the arena.

In the window below is shown a histogram of the animal’s length during the trial. Using the two green cursors, the user then defines 3 different length ranges. Length as a function of time is shown at the bottom of the window, and length as a function of position is shown to the upper right. Each point is color coded according to the defined length ranges. It is also possible to make measures of stretch-attend behavior.

With Length ranges defined in this way, it is then possible to perform a Batch Analysis of length data by Zone or Grid. Possible length parameters are % short, %medium, %long, and mean length for each zone or grid square.

The shortest length is often useful to define rearing, since when the animal rears up toward the camera its profile becomes extremely short.

Circling Analysis#

Circling Analysis In collaboration with one of our users, we have developed a novel measure of circling behavior. The analysis is based on spectral methods of analysis — similar to a sound spectrogram — applied to the animal’s path. In the image below, a drug has been administered at the 1800-second mark and animal begins circling. The animal’s locomotion energy starts to concentrate into a small band of frequencies, as shown in the spectrogram (colormap). As a result, the circling index, which integrates over all frequencies, rises. LimeLight allows the user to zoom in on any portion of the graphs below, select a point in the graph and display the animal’s behavior at that point, and export the circling index for multiple trials.

Rotation#

LimeLight keeps track of the relative angle of the body — the line between the nose and tail. With this information, it is possible to count the number of times an animal rotates on its axis during the course of a trial. In the image below, the animal is rotating continuously in the clockwise direction. For display purposes, the trace is reset to 0 degrees every time the animal completes a 180-degree turn (red dots). When two consecutive 180-degree rotations in the same direction are detected within a set time period (in this case, 10 seconds), a rotation is counted. A total of 52 complete clockwise rotations occurred in this particular trial (lower image). The number of rotations in each direction for a complete set of trials can be calculated automatically and batch exported to a spreadsheet file.

Ordering LimeLight#

LimeLight has 2 different hardware configurations. The 1-camera system can be used to record 1 to 4 animals at once. For multiple animals, each one is placed in a separate arena in one quadrant of the image. The 4-camera system is also used to record up to 4 animals, but each arena is viewed by a separate camera. The additional hardware adds to the system cost, but the 4-camera setup reduces image distortion since each arena is viewed from its center and not from a corner. In addition, it increases flexibility in the placement of arenas. With each system, trials in each arena can be started and stopped together or independently. Trials can be started from the keyboard or from remotes switches (provided at no extra charge on request).

Order WaterMaze, LimeLight and/or FreezeFrame together as a single, cost-effective system that will perform almost any behavioral task.

System Components

  • 1 to 4 USB cameras with zoom lenses
  • Cables and connectors
  • Interface box for shocker control outputs
  • Data acquisition and analysis software

System requirements

  • Laptop or desktop computer
  • Windows 7/8/10
  • May be run on Macintosh Computers using Parallels Desktop for Mac in OS 10.6.8 or later