Knowledge Base

Set Up EthoVision XT

Prerequisites

Connect the DanioVision Observation Chamber to the EthoVision XT computer.

Optionally, set up the water-flow system.

Turn on the DanioVision Observation Chamber with the switch on the front panel. Wait at least 15 minutes for the DanioVision chamber to start up.

Make sure to remove the lens cap from the camera.

Assign Treatments to Wells

To prevent systematic errors as a result of potential variation in temperature, light intensity or other unknown variables across a well-plate, it is recommended to either assign larvae from different treatments randomly to the wells or make sure that wells containing different treatments (that is, different solutions or different concentrations of the same solution) are evenly distributed across the well-plate. For example, one type of solution should not be placed only in the center wells.

Create a DanioVision Experiment

Preferred Method

With this method, all arenas are created automatically but need to be manually adjusted using the Arrange Arenas option. Unnecessary functionality like manual scoring is removed from the interface and detection settings are optimized for DanioVision.

1. In the EthoVision XT Startup window, under New Experiment, click New from Template, or choose File > New from Template.
2. In the Select a Template option window, click Apply a Pre-Defined Template. Next, follow the instructions described below.
3. Which video source will you use? Select the option Live Tracking (and Saving Video Files), click the Sources button and select the Basler camera. Click Next.
4. Which subjects will you track? Select Fish and then Zebrafish Larvae from the list. Click Next.
5. How is the arena configured? Select an Arena template from the list, then click Next.
   • Select one of the Well Plate or Open Field templates if you have a number of wells other than those listed in the other templates.
   • Arena templates are also present for older DanioVision DVOC versions: DVOC 001x to DVOC 004x.
   • To know which DanioVision version you have, see the code DVOC-00xx on the label located on the back panel of the DanioVision chamber.
6. Initialize the template experiment. Check that all settings are correct and, if so, click Finish.
7. Enter the name of the experiment and click OK. The EthoVision XT Overview window opens.
8. Follow all the next steps:
   • Experiment Settings
   • Arena Settings
   • Trial Control Settings
   • Detection Settings

Notes:

• If you select a template which contains more arenas than needed, you can exclude the unused arenas with Data Selection.
• If you select a template for a DanioVision 1–3 version, it is possible that the DanioVision detection method does not give optimal detection of the larvae with images from your camera. If that is the case, try changing the Sensitivity or use another detection method in the Advanced section of the Detection Settings pane. See also Configure Detection Settings in the EthoVision XT Help.
• You can also create an experiment based on a previous experiment — for example, when you want to replicate an experiment with the same number of arenas and the same trial control procedures. Choose File > New from Template > Apply a Custom Template. For details, see Create a New Experiment Based on an Existing Experiment in the EthoVision XT Help.
• You can also create a DanioVision experiment with no predefined settings. With this option you must draw the arenas and arrange them manually. Choose File > New and in the window that appears select the option DanioVision Experiment. In the Experiment Settings, next to Number of Arenas, enter the number of wells. Creating an experiment with a predefined template is advised because the arenas are defined automatically. For more information on creating an experiment manually, see Set Up an Experiment in the EthoVision XT Help.
• No matter which method you choose, when you create a DanioVision experiment, unnecessary functionality like Manual Scoring Settings is removed from the interface and detection settings are optimized for DanioVision.

Experiment Settings

Choose Setup > Experiment Settings.

Video Source

1. Under Video Source, click the Video icon on the right side of the row of your camera.
2. In the Video Settings window that appears, click the Formats tab and select the Resolution and Frame Rate. The default resolution is 1280 x 960 at 30 frames per second. If you create an experiment based on a template with the Tapping Device, set the frame rate to 60 fps and reduce the resolution according to your setup. From the Color Space list, choose Y800. This is the color space for a monochrome video image. The other options, suitable for color images, do not apply to DanioVision.

Tracked Features

Specify whether you want to track the center point of the fish (default) or the center point, the nose point, and the tail base. Choose the latter option only if you are tracking fish in a close-up image.

Body Point Detection Technique

The Contour-based technique is selected. The Deep Learning option is not available in DanioVision experiments.

Analysis Options

If you are interested in how long and how frequently your subjects have been active versus inactive, select Activity Analysis under Analysis Options. Make sure you also adjust the Activity settings in the Detection Settings. For more information about setting up an experiment, see Set Up an Experiment in the EthoVision XT Help.

Use of Trial Control Hardware

Follow this section if you use stimulus devices like the white/color lights and the tapping stimulus device.

1. In the Experiment Settings, under Trial Control Hardware, make sure that Use of Trial Control Hardware is selected and click the Settings button.
2. In the Hardware Interface window that opens, choose the type of system you have. If you have DanioVision DVOC-0050, choose DanioVision DVOC-005x/TCP. The other options (DanioVision DVOC-001x – 004x), if present, are meant for older versions of the DanioVision Observation Chamber.
3. In the Device Configuration window, under Device Features, the hardware devices activated for this experiment are listed.
4. Optionally, the default status of the DanioVision White Bottom Light is Light Off. This means that when you start a trial, the White Bottom Light is switched off. To use the White Bottom Light as a stimulus, program its activation with Trial Control. If, however, you want to use the White Bottom Light as a default lighting condition and use darkness as a stimulus, do the following:
   • Click the White Light Default button.
   • Under Default Device State, select Light On.
   • Choose a percentage of intensity (0–100) and click OK. Then in the Trial Control procedure you can program when to switch the light off.
5. Optionally, if you connect additional hardware, under Plug-in Devices, select the Device Type in the corresponding rows depending on which ports on the DanioVision back panel you use to connect those devices.
   • TTL Port A / TTL Port B: For example, if the DanioVision Temperature Control Unit is connected to the DanioVision chamber through TTL Port A, choose DanioVision TC (DVTCU-001x) from the list next to TTL Port A. If you connect other devices, choose Custom Hardware.
   • Trigger Out: If you connected the Optogenetics triggering devices or LED controllers to the DanioVision chamber through the Trigger Out BNC connectors, choose Optogenetics from the list next to Trigger Out. If you connected other devices through the BNC connectors, choose Custom Hardware.
6. Optionally, edit the name in the Device ID field.
7. Click OK.
8. Optionally, if you set the default state of the White Bottom Light to Light On, open the Trial Control Settings. This will confirm your selection. From that moment on, the White Bottom Light switches on at the start of the trial.

Notes:

• If you upgraded an experiment from an older EthoVision XT version, do not change the Trial Control Hardware Settings in the Experiment Settings. For example, if you see Noldus Mini USB-IO Box selected in the Hardware Interface window, leave it as is. Otherwise the Trial Control rules — for example, to control the white light — may not work.
• When you change the default state of the White Light Stimulus to Light On, the white light is only switched on in one of the following cases:
  • When you open the Trial Control screen.
  • When you open the Arena – Hardware Mapping window.
  • When you start a trial.
• For more information on Trial and Hardware Control, see the EthoVision XT – Trial and Hardware Control – Reference Manual.

IP Settings

To ensure communication between DanioVision DVOC-0050 and EthoVision XT, the IP address of the Ethernet port on your computer connected to DanioVision must be configured correctly.

DanioVision DVOC-0050 is connected to your computer through the Ethernet port at the back of the chamber labeled Ethernet. This port has a specific, unique IP address: 192.168.20.50. If you purchased the computer from Noldus, the IP address is already set and you can skip this section. Otherwise, follow the instructions below.

1. In the Experiment Settings, under Trial Control Hardware, make sure that Use of Trial Control Hardware is selected and click the Settings button.
2. In the Hardware Interface window that opens, choose DanioVision DVOC-005x/TCP.
3. In the DanioVision Configuration window, click Settings and verify that the IP address is 192.168.20.50.
4. Open the Network and Sharing Center in Windows. In the Windows Search field, type Control. Under Best Match, click Control Panel, then Network and Sharing Center, then click Change Adapter Settings at the top-left corner of the window.
5. Open the adapter settings and locate the item that corresponds to the port on your PC connected to the Ethernet connector on the back panel of DanioVision. To identify which port each item refers to, disconnect the cable from that port and check where the Disconnected symbol appears.
6. Set the IP address, for example 192.168.20.1, and assign the IP address to the Ethernet card.

Notes:

• If the DanioVision Configuration window shows the message Not Connected to DanioVision, do the following:
  • Make sure that the DanioVision chamber is switched on. Check the green LED light on the front panel.
  • Check the cables between the computer and DanioVision.
  • Make sure that the IP addresses of the Ethernet ports on the computer match those in DanioVision.
  • Click Refresh.

Source: DanioVision DVOC-0050 Reference Manual - Set Up EthoVision XT, Noldus Information Technology