Name: protocol for producing three-dimensional infrared video of freezing in plants
Uploaded: 2018-09-12T14:30:00
Description: Watch this Scientific Journal Video about Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants at JoVE.com

This work was supported by USDA in-house funding.

1. Preparation
<ol>
	<li>Gather equipment, materials, and software to record and process the video of plant freezing.
	<ol>
		<li>Start a programmable freezer by setting the power switch to On, and set the temperature to 0 &#176;C. Program the freezer to reach -8 &#176;C at 1 &#176;C/h.</li>
	</ol>
	</li>
	<li>Place one 6-weeks-old strawberry plant with 2 - 5 flowers that was grown in a 1 L container into the freezer.</li>
	<li>Set up 2 IR cameras (e.g., FLIR T620 cameras) using fastening stra...

<ol>
	<li>Geng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+display+technologies.">Three-dimensional display technologies.</a> Advances in Optics and Photonics. 5, 456-535 (2013).</li><li>Ceccardi, T. L., Heath, R. L., Ting, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-temperature+exotherm+measurement+using+infrared+thermography.">Low-temperature exotherm measurement using infrared thermography.</a> HortScience. 30, 140-142 (1995).</li><li>Wimmer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> History of thermal imaging, Security Sales and Integration. , (2011).</li><li>Wisniewski, M., Lindow, S. E., Ashworth, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+of+ice+nucleation+and+propagation+in+plants+using+infrared+video+thermography.">Observations of ice nucleation and propagation in plants using infrared video thermography.</a> Plant Physiology. 113, 327-334 (1997).</li><li>Kuprian, E., Tuong, T., Pfaller, K., Livingston, D. P., Neuner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+supercooling+of+reproductive+shoots+is+enabled+by+structural+ice+barriers+being+active+despite+an+intact+xylem+connection.">Persistent supercooling of reproductive shoots is enabled by structural ice barriers being active despite an intact xylem connection.</a> Public Library of Science ONE. 11, e0163160 (2016).</li><li>Livingston, D. P., Tuong, T. D., Murphy, J. P., Gusta, L., Wisniewski, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-definition+infrared+thermography+of+ice+nucleation+and+propagation+in+wheat+under+natural+frost+conditions+and+controlled+freezing.">High-definition infrared thermography of ice nucleation and propagation in wheat under natural frost conditions and controlled freezing.</a> Planta. 247, 791-806 (2017).</li><li>Boyles, R. P., Raman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+climate+patterns+and+trends+in+North+Carolina+(1949-1998).">Analysis of climate patterns and trends in North Carolina (1949-1998).</a> Environment International. 29 (2-3), 263-275 (2003).</li><li>Poling, E. B., Poling, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+Cold+Events.">Managing Cold Events.</a> A Growers' Guide to Production, Economics and Marketing. , 75-97 (2015).</li><li>Hacker, J., Neuner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+porpagaion+in+plants+visualized+at+the+tissue+level+by+infrared+differential+thermal+analysis+(IDTA).">Ice porpagaion in plants visualized at the tissue level by infrared differential thermal analysis (IDTA).</a> Tree Physiology. 27, 1661-1670 (2007).</li><li>He, J. Q., Harrison, R. J., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+imaging+system+for+strawberry+phenotyping.">A novel 3D imaging system for strawberry phenotyping.</a> Plant Methods. 13, 93-101 (2017).</li></ol>

Two IR cameras are necessary for this protocol, and they must be aimed at the subject from slightly different angles1. This will require the lenses to be from 5 - 8 cm apart, but both must be aimed at the same place on the subject to be filmed. Think of the 2 camera lenses as a kind of surrogate for the viewer&#39;s eyes. The left camera is analogous to the left eye and the right camera to the right eye. The post-processing software will tint the left image to a red color and the right image to a ...

Despite living in a world of three physical dimensions, researchers are often limited to reporting visual observations in 2D. Although 2D images are generally sufficient to convey important information, this lack of information about depth restricts our ability to perceive and understand the complexity of real-world objects.1
This deficiency in information about the depth provided an incentive to produce 3D videos mainly in the commercial film industry since the early 1900s1. However, generating clear 3D information in still images and video is hindered by the complexities involved in producing those images. The simplest approach to generating 3D film is based on principles used in stereoscopic photography. Stereoscopic photography utilizes two images of the same object from slightly different angles that conveys a 3D image in the brain.To make this possible, each eye must look only at its respective image (i.e., the left eye at the left image and the right eye at the right image). Since the eyes will not naturally do this, stereoscopic headgear was designed to make this possible1. Several stereoscopic viewing techniques, as well as polarization-interlaced, time-multiplexed, and head-mount display techniques, have been used during the development of 3D films, but the color-interlacing or anaglyph method using red and green (or cyan) glasses is one of the simplest and least expensive techniques. For a comprehensive review of 3D imaging and the various techniques involved, see the review by Geng1.
Monitoring freezing in plants using IR thermography is based on the principle that when water freezes, it must give up internal energy2. This energy is in the form of heat, which is detectable in the IR region of the electromagnetic spectrum. Cameras able to record the IR energy have been in use since 19293. The first published report using IR technology to film freezing in plants is from Cecardi et al.2, but the resolution of the camera used makes it difficult to accurately determine the tissue where the freezing is initiated. Wisniewski et al.4 determined more precise sites of ice nucleation in several plant species using a higher resolution camera. As the technology used in IR thermography improved, higher resolution images led to discoveries such as barriers to freezing5 and the precise cellular localization of ice formation6.
One difficulty in filming subjects in IR is caused by small differences in temperatures. This will cause most objects in the field of view to be a similar color, making it difficult to determine precisely which object(s) is/are freezing. This can be important when determining the order of freezing in specific tissues, such as leaves or roots in wheat6. If the IR video of plants freezing could be imaged in 3D, the accuracy of determining which part of the plant is freezing at a certain point in time could be improved.
Strawberry is a crop in certain areas of the United States in which freezing temperatures are of considerable concern for the growers. Under some growing conditions, it is common for strawberry flowers to appear 2 - 3 weeks before the average last spring freeze. A freeze event can occur as late as June in some areas of the Appalachian Mountains7 and usually results in the death of the flower. Frost protection is, therefore, critical for strawberry growers in areas subject to these freeze events. Strawberry growers in North Carolina, for example, must frost-protect, on average, between 4 - 6 frost events before bloom and 1 - 2 hard freezes during the early bloom period8. To help develop strawberry genotypes that are more freezing tolerant, it is important to understand various aspects of the freezing, such as the sites of ice nucleation and propagation into other parts of the plant. IR thermography provides an effective means to address these issues.
Here, we use strawberry to illustrate a technique for recording freezing events in 3D using the anaglyph method. Strawberry is well suited for this example because the leaves and flowers are widely distributed in the 3D space and can be difficult to differentiate when viewed in 2D infrared videos.

<table border="0" cellpadding="0" cellspacing="0" width="811">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">T620 Infrared Camera and software</td>
			<td style="width:71px;">FLIR</td>
			<td style="width:119px;">55903-5122</td>
			<td style="width:323px;">2 cameras are needed. Software works only on a Windows-based computer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:299px;">After Effects</td>
			<td style="width:71px;">Adobe</td>
			<td style="width:119px;">15.0.1.73</td>
			<td style="width:323px;">Post-Production Video Editing Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Bandicam</td>
			<td style="width:71px;">Bandisoft</td>
			<td style="width:119px;">4.1.2.1385</td>
			<td>Screen Capture Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Laboratory Scissor Jack&#160;&#160;</td>
			<td style="width:71px;">Eisco</td>
			<td style="width:119px;">CH0642A</td>
			<td>Steel Platform 13X15 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fastening Strap</td>
			<td style="width:71px;">Velcro</td>
			<td style="width:119px;">90441</td>
			<td>To hold camera on jack.&#160; Should be at least 60cm long by 2cm wide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Media Converter</td>
			<td style="width:71px;">iSkysoft</td>
			<td style="width:119px;">10.0.6</td>
			<td>Software to convert mp4 files to .mov&#160;</td>
		</tr>
	</tbody>
</table>

protocol for producing three-dimensional infrared video of freezing in plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Surprisingly, the IR video of the strawberry plant freezing (Supplemental Video 1) indicated that not all leaves/flowers froze at the same time. The leaves and flowers both froze individually at different temperatures, but the leaves froze earlier than the flowers and at a higher temperature. In addition, the freezing began in the leaves but not necessarily at the same position on each leaf. While these results have not been described previously in strawberry, similar res...

Watch this Scientific Journal Video about Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants at JoVE.com

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

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

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