This research was funded by the Austrian Science Fund (FWF): P23681-B16.

1. Preparation of Plant Materials
<ol>
	<li>Use either leaves or whole plants of subject plant material (Hosta spp. or Phaseolus vulgaris).</li>
</ol>
2. Preparation of Water Solutions Containing Ice Nucleation Active (INA) Bacteria
<ol>
	<li>Culture the INA bacterium, Pseudomonas syringae (Strain Cit-7) in petri dishes at 25 &#176;C on Pseudomonas Agar F prepared with 10 g/L of 100% glycerol per the manufacturer&#8217;s direction.</li>
	<li>After cultures have grown sufficiently, place at 4 &#176;C until needed but keep at 4 &#176;C for two days prior to ensure a high level of ice nucleation activity.</li>
	<li>Scrape bacteria from a single plate from the surface of the agar with a plastic, disposable or re-usable metal spatula at the time of use and place in 10-15 ml of deionized water in a 25 ml disposable cuvette. The concentration should be in the range of 1 x 107 to 1 x 109 &#183; ml -1. The solution will appear cloudy. There is no need to confirm the concentration using a hemocytometer or spectrophotometer, as concentration need only be approximate.</li>
	<li>Vortex the cuvette for a minimum of 10 sec to distribute the bacteria. 
	Note: The specific concentration of the resulting INA mixture is not important and the protocol described will provide more than an adequate level of ice nucleation activity. This mixture of INA bacteria and water will be used later in the nucleation experiments.</li>
</ol>
3. Setting Up a Freezing Experiment
<ol>
	<li>Place the high resolution infrared camera (SC-660) inside the protective acrylic box so that the lens projects through the opening in the front of the box and the wires connecting the camera to a laptop or recording device exit through the rear opening of the box. Secure the lid of the box and place the box inside the environmental chamber or freezer in a location that will allow the subject plant material to be seen.
	<ol>
		<li>Provide a dark background around the plant material by lining the walls of the chamber with black construction paper to prevent interference from reflected infrared energy.</li>
		<li>Fit the chamber with LED lighting to minimize heating from the light source when recording images in visible wavelengths is required. Only a minimum of lighting, such as a battery-operated closet light or other small LED device, is required for the plants to be visible by the camera.
		<ol>
			<li>Once visible images of the subject plant material are taken, turn off the LED lighting. Distribute all external wired connections (firewire connection to computer, power cord, etc.) to the camera via a port or other opening in the chamber.</li>
		</ol>
		</li>
		<li>Fill any extra space in the port or opening with insulating foam material to avoid or reduce temperature gradients within the chamber. Set the initial temperature of the chamber at 1 &#176;C.</li>
	</ol>
	</li>
	<li>Align plants or plant parts so that the plant material is in the field-of-view of the camera and the plant material is visible on the remote viewing screen or within the chosen software.</li>
	<li>Allow plants to equilibrate at 1 &#176;C for 30 min to 1 hr, depending on the size of the plant material, prior to initiating a controlled freezing experiment. This ensures that the temperature of the plant will not lag behind air temperature by many degrees once the freezing experiment is initiated. Equilibration is achieved when the temperature of the plant material is within 0.5 &#176;C of air temperature.
	<ol>
		<li>Place a layer of Styrofoam insulation on top of the soil of potted plants if potted plants are used. Once the plants have equilibrated, commence cooling of the chamber. 
		Note: The layer of insulation on the soil surface of the pot reduces the amount of continued heat loss from the pot to the air surrounding the plant, and prevents the roots from freezing, as this would not typically occur during a frost event in nature due to the massive reservoir of residual heat present in the soil.</li>
	</ol>
	</li>
	<li>Set the desired camera parameters (color palette, temperature range, specific areas of interest, etc.), as discussed in 3.4.1-3.4.4.
	<ol>
		<li>Select the rainbow palette to display the temperature variations while viewing the live image.</li>
		<li>Set the temperature span to 5 &#176;C by adjusting the temperature bar located just below the image in the software.</li>
		<li>Choose the linear scale (algorithm) for converting the infrared data into the false color image as defined by the selected palette (rainbow) and set the range of temperature to 5 &#176;C and to track automatically based on the image. Alternatively, adjust the set range manually while conducting the experiment.
		<ol>
			<li>Use the temperature of a specific point or an average temperature within the defined area of interest provided by the software. Retrieve the temperature data of all pixels from the recorded video sequence or from the information embedded in the image file. Figure 3 shows a typical screenshot from within ResearchIR software.</li>
		</ol>
		</li>
		<li>Place a cursor on a location on the plant tissue that represents a specific point of interest. Define the area of interest as points (1 -3 pixels in size), boxes, lines, ellipses, or circles. Multiple combinations of points or shapes can be located over the image.</li>
	</ol>
	</li>
	<li>Recording a video sequence
	<ol>
		<li>Set the camera to record at 60 Hz and for the recording to be stopped manually.</li>
		<li>Indicate the location on the computer or external drive where the recorded video file will be placed.</li>
		<li>Commence recording. 
		Note: Recording to an external hard drive is highly recommended since large video files will be generated. Recorded video files can be later edited to contain only the portion containing the necessary information. This will greatly reduce the file size.</li>
		<li>Lower the temperature of the chamber incrementally by 0.5 -1.0 &#176;C. Wait until the plant temperature equilibrates with air temperature and then lower the temperature again by 0.5-1.0 &#176;C. Depending on the mass of the plant tissue being observed and its morphology, equilibration can take 10 to 15 min. Thus, giving a cooling rate of about 4 &#176;C/hr.</li>
		<li>Continue in this manner until the plant freezes and observations are completed. End the recording when the freezing process has been completed. 
		Note: The plant tissue has equilibrated with air temperature when the plant material and background are the same color since they are at the same temperature. Since the background temperature and the temperature of the plant tissue are the same, it may be difficult to visualize the plant material until you again lower the temperature and there is temperature differential between the plant tissue and air temperature.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Taschler, D., Beikircher, B., 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=Frost+resistance+and+ice+nucleation+in+leaves+of+five+woody+timberline+species+measured+in+situ+during+shoot+expansion.">Frost resistance and ice nucleation in leaves of five woody timberline species measured in situ during shoot expansion.</a> Tree Physiol. 24, 331-337 (2004).</li><li>Neuner, G., Hacker, J., L&#252;tz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+formation+and+propagation+in+alpine+plants.">Ice formation and propagation in alpine plants.</a> Plants in alpine regions: Cell Physiology of adaptation and survival strategies. , 163-174 (2012).</li><li>Ladinig, U., Hacker, J., Neuner, G., Wagner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+endangered+is+sexual+reproduction+of+high-mountain+plants+by+summer+frosts?+-+Frost+resistance,+frequency+of+frost+events+and+risk+assessment.">How endangered is sexual reproduction of high-mountain plants by summer frosts? - Frost resistance, frequency of frost events and risk assessment.</a> Oecologia. 171, 743-760 (2013).</li><li>Wisniewski, M. E., Gusta, L. V., Fuller, M. P., Karlson, D., Gusta, L. V., Wisniewski, M. E., Tanino, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+nucleation,+propagation+and+deep+supercooling:+the+lost+tribes+of+freezing+studies.">Ice nucleation, propagation and deep supercooling: the lost tribes of freezing studies.</a> Plant Cold Hardiness: from the laboratory to the field. , 1-11 (2009).</li><li>Bokhurst, S., Bjerke, J. W., Davey, M. P., Taulavuori, K., Taulavuori, E., Laine, K., Callaghan, T. V., Phoenix, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impacts+of+extreme+winter+warming+events+on+plant+physiology+in+a+sub-Arctic+heath+community.">Impacts of extreme winter warming events on plant physiology in a sub-Arctic heath community.</a> Physiol. Plant. 140, 128-140 (2010).</li><li>Taulavuori, K., Laine, K., Taulavuori, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+on+Vaccinium+myrtillus.and+Vaccinium+vits-idea.in+relation+to+air+pollution+and+global+change+at+northern+high+latitudes:+A+review.">Experimental studies on Vaccinium myrtillus.and Vaccinium vits-idea.in relation to air pollution and global change at northern high latitudes: A review.</a> Env. Exp. Bot. 87, 191-196 (2013).</li><li>Wisniewski, M., Glenn, D. M., Gusta, L., Fuller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+infrared+thermography+to+study+freezing+in+plants.">Using infrared thermography to study freezing in plants.</a> HortScience. 43, 1648-1651 (2008).</li><li>Gu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2007+eastern+US+spring+freeze:+increased+cold+damage+in+a+warming+world.">The 2007 eastern US spring freeze: increased cold damage in a warming world.</a> BioScience. 58, 253-262 (2008).</li><li>Augspurger, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spring+warmth+and+frost:+phenology,+damage,+and+refoliation+in+a+temperate+deciduous+forest.">Spring warmth and frost: phenology, damage, and refoliation in a temperate deciduous forest.</a> Func. Ecol. 23, 1031-1039 (2007).</li><li>Neuner, G., Erler, A., Hacker, J., Ladinig, U., Wagner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frost+resistance+of+reproductive+tissues+in+various+reproductive+stages+of+high+alpine+plant+species.">Frost resistance of reproductive tissues in various reproductive stages of high alpine plant species.</a> Physiol. Plant. 147, 88-100 (2013).</li><li>Skre, O., Taulavuori, K., Taulavuori, E., Nilsne, J., Igeland, B., Laine, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+hardening+and+winter+temperature+for+growth+in+mountain+birch+populations.">The importance of hardening and winter temperature for growth in mountain birch populations.</a> Env. Exp. Bot. 62, 254-266 (2008).</li><li>H&#228;nninen, H., Tanino, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tree+seasonality+in+a+warming+climate.">Tree seasonality in a warming climate.</a> Trends in Plant Science. 16, 412-416 (2011).</li><li>Katz, R. W., Brown, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+events+in+a+changing+climate:+variability+is+more+important+than+averages.">Extreme events in a changing climate: variability is more important than averages.</a> Climate Change. 21, 289-302 (1992).</li><li>Wisniewski, M., Gusta, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+plant+cold+hardiness:+an+opinion.">Understanding plant cold hardiness: an opinion.</a> Physiol. Plant. 147, 4-14 (2013).</li><li>Wisniewski, M., Gusta, L., 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=Adaptive+mechanisms+of+freeze+avoidance+in+plants.+A+brief+update.">Adaptive mechanisms of freeze avoidance in plants. A brief update.</a> Env. Exp. Bot. 99, 133-140 (2014).</li><li>Burke, M. J., Gusta, L. V., Quamme, H. A., Weiser, C. J., Li, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freezing+and+injury+in+plants.">Freezing and injury in plants.</a> Annu. Rev. Plant Physiol. 27, 507-528 (1976).</li><li>Ishikawa, M., Price, W. S., Ide, H., Arata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+freezing+behaviors+in+leaf+and+flower+buds+of+full-moon+maple+by+nuclear+magnetic+resonance+microscopy.">Visualization of freezing behaviors in leaf and flower buds of full-moon maple by nuclear magnetic resonance microscopy.</a> Plant Physiol. 115 (4), 1515-1524 (1997).</li><li>Ishikawa, M., Sakai, A., Li, P. H., Sakai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+freezing+avoidance+in+comparison+with+freezing+tolerance:+a+demonstration+of+extra-organ+freezing.">Characteristics of freezing avoidance in comparison with freezing tolerance: a demonstration of extra-organ freezing.</a> Plant cold hardiness and freezing stress. , 325-340 (1982).</li><li>Buchner, O., 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=Freezing+cytorrhysis+and+critical+temperature+thresholds+for+photosystem+II+in+the+peat+moss+Sphagnum+capillifolium.">Freezing cytorrhysis and critical temperature thresholds for photosystem II in the peat moss Sphagnum capillifolium.</a> Protoplasma. 243 (1), 63-71 (2010).</li><li>Pearce, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ice+and+cell+shape+in+frost-stressed+cereal+leaves:+A+low+temperature+scanning+electron+microscopy+study.">Extracellular ice and cell shape in frost-stressed cereal leaves: A low temperature scanning electron microscopy study.</a> Planta. 175, 313-324 (1988).</li><li>Ashworth, E. N., Anderson, J. A., Davis, G. A., Lightner, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+formation+in+Prunus+persica.+under+field+conditions.">Ice formation in Prunus persica. under field conditions.</a> J. Am. Soc. Hort. Sci. 110 (3), 322-324 (1985).</li><li>Ashworth, E. N., Davis, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+formation+in+woody+plants+under+field+conditions.">Ice formation in woody plants under field conditions.</a> HortSci. 21, 1233-1234 (1986).</li><li>Pramsohler, M., 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=Freezing+pattern+and+frost+killing+temperature+of+apple+(Malus+domestica.)+wood+under+controlled+conditions+and+in+nature.">Freezing pattern and frost killing temperature of apple (Malus domestica.) wood under controlled conditions and in nature.</a> Tree Physiol. 32 (7), 819-828 (2012).</li><li>Wisniewski, M., Lindow, S. E., Ashworth, E. N. <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 Physiol. 113 (2), 327-334 (1997).</li><li>Lutze, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+atmospheric+[CO2]+promotes+frost+damage+in+evergreen+tree+seedlings.">Elevated atmospheric [CO2] promotes frost damage in evergreen tree seedlings.</a> Plant Cell Environ. (6), 631-635 (1998).</li><li>Ball, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Space+and+time+dependence+of+temperature+and+freezing+in+evergreen+leaves).">Space and time dependence of temperature and freezing in evergreen leaves).</a> Func. Plant Biol. 29 (11), 1259-1272 (2002).</li><li>Sekozawa, Y., Sugaya, S., Gemma, H. <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+flowers+of+Japanese+Pear+(Pyrus+Pyrifolia).">Observations of ice nucleation and propagation in flowers of Japanese Pear (Pyrus Pyrifolia).</a> Nakai) using infrared video. 73 (1), 1-6 (2004).</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+propagation+in+plants+visualized+at+the+tissue+level+by+IDTA+(infrared+differential+thermal+analysis).">Ice propagation in plants visualized at the tissue level by IDTA (infrared differential thermal analysis).</a> Tree Physiol. 27, 1661-1670 (2007).</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+propagation+in+dehardened+alpine+plant+species+studied+by+infrared+differential+thermal+analysis+(IDTA).">Ice propagation in dehardened alpine plant species studied by infrared differential thermal analysis (IDTA).</a> Arc. Antarc. Alp. Res. 40 (4), 660-670 (2008).</li><li>Hacker, J., Spindelb&#246;ck, 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=Mesophyll+freezing+and+effects+of+freeze+dehydration+visualized+by+simultaneous+measurement+of+IDTA+and+differential+imaging+chlorophyll+fluorescence.">Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence.</a> Plant Cell Environ. 31, 1725-1733 (2008).</li><li>Neuner, G., XU, B., Hacker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Velocity+and+pattern+of+ice+propagation+and+deep+supercooling+in+woody+stems+of+Castanea+sativa.,+Morus+nigra.+and+Quercus+robur.+measured+by+IDTA.">Velocity and pattern of ice propagation and deep supercooling in woody stems of Castanea sativa., Morus nigra. and Quercus robur. measured by IDTA.</a> Tree Physiol. 30, 1037-1045 (2010).</li><li>Hacker, J., Ladinig, U., Wagner, 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=Inflorescences+of+alpine+cushion+plants+freeze+autonomously+and+may+survive+subzero+temperatures+by+supercooling.">Inflorescences of alpine cushion plants freeze autonomously and may survive subzero temperatures by supercooling.</a> Plant Sci. 180, 149-156 (2011).</li><li>Kuprian, E., Briceno, V., Wagner, 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+barriers+promote+supercooling+and+prevent+frost+injury+in+reproductive+buds,+flowers+and+fruits+of+alpine+dwarf+shrubs+throughout+the+summer.">Ice barriers promote supercooling and prevent frost injury in reproductive buds, flowers and fruits of alpine dwarf shrubs throughout the summer.</a> Env. Exp. Bot. 106, 4-12 (2014).</li><li>Wisniewski, M., Glenn, D. M., Fuller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+hydrophobic+particle+film+as+a+barrier+to+extrinsic+ice+nucleation+in+tomato+plants.">Use of a hydrophobic particle film as a barrier to extrinsic ice nucleation in tomato plants.</a> HortScience. 127, 358-364 (2002).</li><li>Aryal, B., &amp;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=Leaf+wettability+decreases+along+an+extreme+altitudinal+gradient.">Leaf wettability decreases along an extreme altitudinal gradient.</a> Oecologia. 162, 1-9 (2010).</li><li>Wisniewski, M., Fuller, M. P., Margesin, R., Schinner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ice nucleation and deep supercooling in plants: new insights using infrared thermography. In: Cold adapted organisms. Ecology, physiology, enzymologyandmolecularbiology. , 105-118 (1999).</li><li>Franks, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biophysics and biochemistry at low temperatures. , (1985).</li><li>Neuner, G., Kuprian, E., Hincha, D., Zuther, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+thermal+analysis+of+plant+freezing+processes.">Infrared thermal analysis of plant freezing processes.</a> Methods in Molecular Biology: Plant Cold Acclimation. , 91-98 (2014).</li></ol>

The authors have no competing financial interests or conflicts of interest.

Water has the ability to supercool to temperatures well below 0 &#176;C and the temperature at which water will freeze can be quite variable.36 The temperature limit for supercooling of pure water is about -40 &#176;C and is defined as the homogeneous nucleation point. When water freezes at temperatures warmer than -40 &#176;C it is brought about by the presence of heterogenous nucleators that enable small ice embryos to form which then serve as a catalyst for ice formation and growth.37 There are a...

Freezing temperatures that occur when plants are actively growing can be lethal, particularly if the plant has little or no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic and arctic ecosystems 1-6. Episodes of severe spring frosts have had major impacts on fruit production in the USA and South America in recent years 7-9 and have been exacerbated by the early onset of warm weather followed by more typical mean low temperatures. The early warm weather induces buds to break, activating the growth of new shoots, leaves, and flowers all of which have very little to no frost tolerance 1,3,10-12. Such erratic weather patterns have been reported to be a direct reflection of ongoing climate change and are expected to be a common weather pattern for the foreseeable future 13. Efforts to provide economical, effective, and environmentally-friendly management techniques or agrochemicals that can provide increased frost tolerance have had limited success for a host of reasons but this can be partly attributed to the complex nature of freezing tolerance and freezing avoidance mechanisms in plants. 14
The adaptive mechanisms associated with frost survival in plants have traditionally been divided into two categories, freezing tolerance and freezing avoidance. The former category is associated with biochemical mechanisms regulated by a specific set of genes that allow plants to tolerate the stresses associated with the presence and the dehydrative effect of ice in its tissues. While the latter category is typically, but not solely, associated with structural aspects of a plant that determine if, when, and where ice forms in a plant 14. Despite the prevalence of freeze avoidance as an adaptive mechanism, little research has been devoted in recent times to understanding the underlying mechanisms and regulation of freeze avoidance. The reader is referred to a recent review 15 for greater details on this subject.
While the formation of ice at low temperatures may seem like a simple process, many factors contribute to determining the temperature at which ice nucleates in plant tissues and how it spreads within the plant. Parameters such as the presence of extrinsic and intrinsic ice nucleators, heterogeneous vs. homogeneous nucleation events, thermal-hysteresis (antifreeze) proteins, the presence of specific sugars and other osmolytes, and a host of structural aspects of the plant can all play a significant role in the freezing process in plants. Collectively, these parameters influence the temperature at which a plant freezes, where ice is initiated and how it grows. They can also affect the morphology of the resulting ice crystals. Various methods have been used to study the freezing process in plants under laboratory conditions, including nuclear magnetic resonance spectroscopy (NMR) 16, magnetic resonance imaging (MRI) 17, cryo-microscopy 18-19, and low-temperature scanning electron microscopy (LTSEM). 20 Freezing of whole plants in laboratory and field settings, however, has mainly been monitored with thermocouples. The use of thermocouples to study freezing is based on the liberation of heat (enthalpy of fusion) when water undergoes a phase transition from a liquid to a solid. Freezing is then recorded as an exothermic event. 21-23 Even though thermocouples are the typical method of choice in studying freezing in plants, their use has many limitations that limit the amount of information obtained during a freezing event. For instance, with thermocouples it is difficult to nearly impossible to determine where ice is initiated in plants, how it propagates, if it propagates at an even rate, and if some tissues remain free of ice.
Advances in high-resolution infrared thermography (HRIT) 24-27, however, have significantly increased the ability to obtain information about the freezing process in whole plants, especially when used in a differential imaging mode. 28-33 In the present report, we describe the use of this technology to study various aspects of the freezing process and various parameters that affect where and and at what temperature ice is initiated in plants. A protocol will be presented that will demonstrate the ability of the ice-nucleation-active (INA) bacterium, Pseudomonas syringae (Cit-7) to act as an extrinsic nucleator initiating freezing in a herbaceous plant at a high, subzero temperature.
High-resolution Infrared Camera
The protocol and examples documented in this report utilize a high resolution infrared video radiometer. The radiometer (Figure 1) supplies a combination of infrared and visible spectrum images and temperature data. The spectral response of the camera is in the range of 7.5 to 13.5 &#181;m and provides 640 x 480 pixel resolution. Visible spectrum images generated by the built-in camera can be fused with IR-images in real time, which facilitates the interpretation of complex, thermal images. A range of lenses for the camera can be used to make close-up and microscopic observations. The camera can be used in a stand-alone mode, or interfaced and controlled with a laptop using propietary software. The software can be used to obtain a variety of thermal data embedded in the recorded videos. It is important to note that a wide variety of infrared radiometers are commercially available. Therefore, it is essential that the researcher discuss their intended application with a knowledgeable product engineer and that the researcher test the ability of any specific radiometer to provide the information needed. The imaging radiometer used in the described protocol is placed in an acrylic box (Figure 2) insulated with Styrofoam in order to deter exposure to condensation during the warming and cooling protocols. This protection is not needed for all cameras or applications.

<table><tbody><tr><td>Infrared Camera</td><td>FLIR</td><td>SC-660</td><td>Many models available depending on application</td></tr><tr><td>Infrared Analytical Software</td><td>FLIR</td><td>ResearchIR 4.10.2.5</td><td>$3,500</td></tr><tr><td>Pseudomonas syringae (strain Cit-7)</td><td></td><td></td><td>&lt;a href=&quot;mailto:icelab@berkeley.edu&quot;&gt;Kindly provided by Dr. Steven Lindow, University of California&amp;nbsp; Berkeley icelab@berkeley.edu&lt;/a&gt;</td></tr><tr><td>Pseudomonas Agar F</td><td>Fisher Scientific</td><td>DF0448-17-1</td><td></td></tr></tbody></table>

the use of high-resolution infrared thermography (hrit) for the study of ice nucleation and ice propagation in plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

Ice-nucleating activity of the Ice+ bacterium, Pseudomonas syringae (strain Cit-7)
A 10 &#181;l drop of water and 10 &#181;l of water containing P. syringae (Cit-7) were placed on the abaxial surface of a Hosta leaf (Hosta spp.) (Figure 4). As illustrated, the drop of water containing the INA bacteria froze first and was responsible for inducing the leaf to freeze while the drop of water on the leaf surface remained unfrozen.
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Watch this Scientific Journal Video about The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants at JoVE.com

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

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9.5K Views.  Agricultural Research Service (USDA-ARS), Kearneysville, WV. Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Video: The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be streamlined by heat treatment (blanching). A blanching apparatus can be designed more precisely if the thermal properties of the leaves are known in detail, i.e., the specific heat capacity and thermal conductivity. The measurement of these properties is time consuming and labor intensive, and usually requires invasive methods that contact the sample directly. This can reduce the product yield and may be incompatible with containment requirements, e.g., in the context of good manufacturing practice. To address these issues, a non-invasive, contact-free method was developed that determines the specific heat capacity and thermal conductivity of an intact plant leaf in about one minute. The method involves the application of a short laser pulse of defined length and intensity to a small area of the leaf sample, causing a temperature increase that is measured using a near infrared sensor. The temperature increase is combined with known leaf properties (thickness and density) to determine the specific heat capacity. The thermal conductivity is then calculated based on the profile of the subsequent temperature decline, taking thermal radiation and convective heat transfer into account. The associated calculations and critical aspects of sample handling are discussed.

A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample.

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be ...

Biochemistry

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

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.

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 ...

Simulating Impacts of Ice Storms on Forest Ecosystems

Ice storms can have profound and lasting effects on the structure and function of forest ecosystems in regions that experience freezing conditions. Current models suggest that the frequency and intensity of ice storms could increase over the coming decades in response to changes in climate, heightening interest in understanding their impacts. Because of the stochastic nature of ice storms and difficulties in predicting when and where they will occur, most past investigations of the ecological effects of ice storms have been based on case studies following major storms. Since intense ice storms are exceedingly rare events it is impractical to study them by waiting for their natural occurrence. Here we present a novel alternative experimental approach, involving the simulation of glaze ice events on forest plots under field conditions. With this method, water is pumped from a stream or lake and sprayed above the forest canopy when air temperatures are below freezing. The water rains down and freezes upon contact with cold surfaces. As the ice accumulates on trees, the boles and branches bend and break; damage that can be quantified through comparisons with untreated reference stands. The experimental approach described is advantageous because it enables control over the timing and amount of ice applied. Creating ice storms of different frequency and intensity makes it possible to identify critical ecological thresholds necessary for predicting and preparing for ice storm impacts.

Ice storms are important weather events that are challenging to study because of difficulties in predicting their occurrence. Here, we describe a novel method for simulating ice storms that involves spraying water over a forest canopy during sub-freezing conditions.

Ice storms can have profound and lasting effects on the structure and function of forest ecosystems in regions that experience freezing conditions. ...

Near-Infrared Temperature Measurement Technique for Water Surrounding an Induction-heated Small Magnetic Sphere

A technique to measure the temperature of water and non-turbid aqueous media surrounding an induction-heated small magnetic sphere is presented. This technique utilizes wavelengths of 1150 and 1412 nm, at which the absorption coefficient of water is dependent on temperature. Water or a non-turbid aqueous gel containing a 2.0-mm- or 0.5-mm-diameter magnetic sphere is irradiated with 1150 nm or 1412 nm incident light, as selected using a narrow bandpass filter; additionally, two-dimensional absorbance images, which are the transverse projections of the absorption coefficient, are acquired via a near-infrared camera. When the three-dimensional distributions of temperature can be assumed to be spherically symmetric, they are estimated by applying inverse Abel transforms to the absorbance profiles. The temperatures were observed to consistently change according to time and the induction heating power.

A technique utilizing wavelengths of 1150 and 1412 nm to measure the temperature of water surrounding an induction-heated small magnetic sphere is presented.

A technique to measure the temperature of water and non-turbid aqueous media surrounding an induction-heated small magnetic sphere is presented. This ...

Engineering

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Biology

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

High-Resolution Imaging of Chlorophyll Biosynthesis in Arabidopsis Seedling

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Author Spotlight: Non-Invasive High-Resolution Measurement of Chlorophyll Synthesis During De-Etiolation

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Summary

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Chapters in this video

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

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

Simulating Impacts of Ice Storms on Forest Ecosystems

Near-Infrared Temperature Measurement Technique for Water Surrounding an Induction-heated Small Magnetic Sphere

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation