Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

This method will allow you to identify the precise location of ice nucleation and propagation in three dimensions, which can be difficult with some infrared cameras. The advantage of this technique is that it provides depth information, which isn't possible with normal two-dimensional video. Because humans can't visualize infrared radiation, a camera which detects infrared and converts it into the visible spectrum is the only way to visualize heat generating events, such as freezing.

Even though we use this system to illustrate freezing in a strawberry plant, it could be used in any project where depth is important in a visual image. The protocol might be difficult for some because it involves using a software with a somewhat difficult learning curve. To begin, with fastening straps and a small block of wood, set up two infrared, or IR, cameras to produce the correct convergence angle of the lenses.

The two cameras must be trained on the same object, but at slightly different angles, corresponding to the distance between the eyes. Mount both cameras to a 10-by-10 centimeter laboratory jack. After programming a freezer according to the text protocol, place one six-week old strawberry plant with two to five flowers in a one-liter container inside.

Use the jack to position both cameras vertically so that the two images contain the whole plant and a portion of the soil. Then, with the USB connector, connect the two cameras to the computer, and plug the two AC outlets into both cameras to allow a continuous monitoring of the plants. To set up the computer and software for recording, open two windows of the software by double-clicking the icon for the IR camera software two times.

Then, follow the instructions in the help menu to connect the left camera to the left window and the right camera to the right window. Open the screen capture software, and adjust the capture frame by clicking and dragging the frame so that it includes both cameras to allow simultaneous screen capture of both cameras. Video streams from the two cameras must be captured simultaneously so that the right and left images are precisely synchronized.

The screen capture software will allow you to do this very easily. Next, in the controller menu, start the freezer program by selecting Run, and begin the screen capture. Then, click on the Recording button on the window, and ensure that the outline showing the region of the screen being captured turns red.

Record the strawberry plant freezing down to minus eight degrees Celsius, and hold the temperature of the freezer for one hour. To process the videos, first convert the MP4 file from the screen capture software to a mov file, using commercially available file conversion software. Then, double-click anywhere inside the project panel to import the mov file of interest into the imaging software, and drag the file to the Composition icon at the bottom of the project panel.

Then, save the project to the same folder containing the original videos. Along the bottom of the preview window, click on the Region of Interest icon, and outline only the recording from the left camera. Drag the same mov video to the Composition icon to create a second composition of the same video.

Then, repeat this step with the right camera. Now, select Composition, Crop Comp to Region of Interest for the left view. Then, repeat this step for the right view.

Rename each composition to indicate which is left and right. Highlight the left composition by clicking on it, and, in the main menu at the top, select Composition, Add to Render Queue. In the render queue, click on Output Module, and make sure the video will be rendered as a video.

Click on Output To, name the video Strawberry Left, and save it to the same folder as the original recording and the project. Click Save, and then click the Render button at the top right side of the render panel. Repeat the rendering for the Strawberry Right composition.

Next, double-click the project panel, and import the rendered Strawberry Left and Strawberry Right videos. Highlight both videos, and drag them to the Composition icon at the bottom of the project panel. In the pop-up screen asking for the Still Duration, enter three with five zeros for a three-hour duration.

Highlight the top layer of the composition panel, and, from the menu at the top, select Effect, Perspective, 3D Glasses. In the control panel, click the box to the right of left view. List the two videos in the composition panel in a drop-down menu, and highlight the video in the list for the left view.

Then, repeat this step for the right view. In the box to the right of 3D View, select Red Blue LR.Using red-blue glasses, inspect the view in the project panel. If the 3D view seems to be incorrect, click Swap Left-Right.

Adjust Scene Convergence and Vertical Alignment to eliminate any ghosting and eye strain. When the 3D aspect of the video is acceptable, highlight the composition by clicking on it, and select Composition, Add to Render Queue, as demonstrated earlier in this video. Finally, render the video.

This IR video shows that leaves froze at a higher temperature than flowers, although the freezing did not necessarily begin at the same position on each leaf. Once the leaves froze, freezing progressed down the petiole to the crown of the plant. When the temperature dropped one to two degrees, the flowers froze, beginning at the calyx, before spreading to the petals and receptacle.

When comparing the 2D infrared image with the 3D wearing glasses, the 3D image made it easier to precisely determine the order in which the leaves and flowers froze. These wheat roots were submerged in growing medium composed of peat, and ice shavings were added prior to the freezing to ensure the roots would freeze. Freeze nucleation occurred at about minus 0.5 degrees Celsius, midway along a root on the right side.

The freezing then progressed upwards to the crown of the plant, causing the base of the outer leaves to freeze, and then downwards into the roots, at the back of the plant. Note that, without the 3D perspective, it is nearly impossible to determine the order in which the specific roots froze. It's important to position the camera so each one is aimed at the same location on the object being filmed.

Using the cameras we used in this demonstration will ensure that the angle is correct for producing a 3D anaglyph video. This procedure can be used to generate video, as well as 3D still images of any object by taking only two pictures from different angles.