Name: a rapid laser probing method facilitates the non-invasive and contact-free determination of leaf thermal properties
Uploaded: 2017-01-07T17:00:00
Description: Watch this Scientific Journal Video about A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties at JoVE.com

The authors are grateful to Dr. Thomas Rademacher and Ibrahim Al Amedi for cultivating the plants used in this study. We would like to thank Dr. Richard M. Twyman for his assistance with editing the manuscript. This work was in part funded by the European Research Council Advanced Grant "Future-Pharma", proposal number 269110, the Fraunhofer Zukunftsstiftung (Future Foundation), the Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.

1. Plant Cultivation and Sample Preparation
<ol>
	<li>Flush each mineral wool block with 1-2 L of deionized water and subsequently with 1 L of 0.1% [m/v] fertilizer solution. Place one tobacco (Nicotiana tabacum or N. benthamiana) seed in each block and gently flush with 0.25 L of fertilizer solution without washing away the seed.</li>
	<li>Cultivate the plants for 7 weeks in a greenhouse or phytotron with 70% relative humidity, a 16-h photoperiod (180 &#181;mol s&#8722;1 m&...

<ol>
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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=Effectiveness+of+cuticular+transpiration+barriers+in+a+desert+plant+at+controlling+water+loss+at+high+temperatures.">Effectiveness of cuticular transpiration barriers in a desert plant at controlling water loss at high temperatures.</a> AoB PLANTS. 8, (2016).</li><li>Parker, W. J., Jenkins, R. J., Abbott, G. L., Butler, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+Method+of+Determining+Thermal+Diffusivity,+Heat+Capacity,+and+Thermal+Conductivity.">Flash Method of Determining Thermal Diffusivity, Heat Capacity, and Thermal Conductivity.</a> J Appl Phys. 32, 1679-1684 (1961).</li><li>Hays, R. 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The contact-free, non-destructive measurement method described above can be used to determine cp,s and &#654; in a simultaneous and reproducible manner. The calculation of &#654; in particular depends on several parameters that are sensitive to errors. Nevertheless, the impact of these errors was either linear or sub-proportional, and the coefficient of variation for all parameters was found to be less than 10%. Even though the method can thus be regarded as robust, some technical improvements can be ...

The large-scale processing of biological materials often requires heat-treatment steps such as pasteurization. The equipment for such processes can be designed more precisely if the thermal properties of the biological materials are well characterized, including the specific heat capacity (cp,s) and thermal conductivity (&#955;). These parameters can be determined easily for liquids, suspensions and homogenates by calorimetry 1. However, measuring such parameters in solid samples can be labor intensive, and often requires direct contact with the sample or even its destruction 2. For example, photothermal techniques require direct contact between the sample and detector 3. Such limitations are acceptable during food processing, but are incompatible with highly regulated processes such as the production of biopharmaceutical proteins in plants in the context of good manufacturing practice 4. In such a context, repeated (e.g., weekly) monitoring of thermal properties may be required during a seven-week growth period for individual plants as a quality control tool. If such a monitoring would require and consume a leaf for each measurement, there would be no biomass left to process at the time of harvest.
Additionally, using only leaf parts instead would cause wounding to the plant and increase the risk of necrosis or pathogen infection, again diminishing the process yield. The likelihood of pathogen infection may also increase if a method with direct contact to the sample would be used, inducing the risk that an entire batch of plants can be infected through contact with a contaminated sensor device. Similar aspects have to be considered for the monitoring of plant stresses like drought, e.g., in an ecophysiological context. For example, water loss is often monitored by a change in the fresh biomass, which requires an invasive treatment of the plants under investigation 5, e.g., dissecting a leaf. Instead, determining the specific heat capacity, which depends on the water content of a sample, in a non-invasive manner as describe here, can be used as a surrogate parameter for the hydration status of plants. In both scenarios (pharmaceutical production and ecophysiology), artificial stresses induced by destructive or invasive measurement techniques would be deleterious as they can distort the experimental data. Therefore, previously reported flash methods 6 or the placement of samples between silver plates 7 are unsuitable for such processes and experiments because they either require direct contact to the sample or are destructive. The parameters cp,s and &#955; must be determined in order to design the process equipment for a blanching step that can simplify product purification and thus reduce manufacturing costs 8-10. Both cp,s and &#955; can now be rapidly determined by contact-free non-destructive near infrared (NIR) laser probing in a consistent and reproducible manner 11 and this new method will be explained in detail below. The results obtained with this method were successfully used to simulate heat transfer in tobacco leaves 12, allowing the design of appropriate processing equipment and the selection of corresponding parameters such as the blanching temperature.
The method is easy to set up (Figure 1) and has two phases, measurement and analysis, each of which comprises two major steps. In the measurement phase, a leaf sample is first locally heated by a short laser pulse and the maximum sample temperature is recorded. The temperature profile of the sample is then recorded for a duration of 50 s. In the analysis phase, leaf properties such as density (easily and accurately determined by pycnometric measurement) are combined with the maximum sample temperature to calculate cp,s. In the second step, the leaf temperature profile is used as the input for an energy balance equation, taking conduction, convection and radiation into account, to calculate &#955;.
Detailed step-by-step instructions are provided in the protocol section, expanding on the contents of the accompanying video. Typical measurements are then shown in the results section. Finally, the benefits and limitations of the method are highlighted in the discussion section along with potential improvements and further applications.
<img alt="Figure 1" src="/files/ftp_upload/54835/54835fig1.jpg" /> 
Figure 1: Apparatus used to determine leaf thermal properties. A. Photograph of the measurement apparatus used to determine the specific heat capacity and thermal conductivity of leaves. The peripheral devices (computers, oscilloscope) are not shown. B. Schematic representation of the measurement apparatus. The laser and connected equipment are highlighted in red, the NIR detector for temperature measurement is shown in purple, the leaf sample is green and the photodiode power sensor is blue. C. Drawing of the elements of the measurement setup with the same color code as in B. The size bar indicates 0.1 m. D. Screenshot illustrating the typical elements of the laser control software. <a href="http://cloudflare.jove.com/files/ftp_upload/54835/54835fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1207">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">1&#34; tube</td>
			<td style="width:259px;">Thorlabs</td>
			<td style="width:136px;">SM1L10E</td>
			<td style="width:403px;">Tube for fiber holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Agarose</td>
			<td style="width:259px;">Sigma Aldrich</td>
			<td>A0701</td>
			<td>Agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bi-Convex lense f=25.4</td>
			<td style="width:259px;">Thorlabs</td>
			<td>LB1761</td>
			<td>Lense</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:411px;">Digital Handheld Optical&#160;Power and Energy Meter Console</td>
			<td style="width:259px;">Thorlabs</td>
			<td style="width:136px;">PM100D</td>
			<td>Console for thermal surface absorber sensor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Digital Phosphor Oscilloscope&#160;</td>
			<td style="width:259px;">Tektronix</td>
			<td style="width:136px;">DPO7104</td>
			<td>Oscilloscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DMR light microscope</td>
			<td style="width:259px;">Leica</td>
			<td style="width:136px;">n.a.</td>
			<td>Light microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Falcon 50mL Conical Centrifuge Tubes</td>
			<td style="width:259px;">Fisher Scientific</td>
			<td style="width:136px;">14-432-2</td>
			<td>Pycnometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Ferty 2 Mega</td>
			<td style="width:259px;">Kammlott</td>
			<td style="width:136px;">5.220072</td>
			<td>Fertilizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fiber holder</td>
			<td style="width:259px;">Thorlabs</td>
			<td style="width:136px;"></td>
			<td>Fiber holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Forma -86C ULT freezer</td>
			<td style="width:259px;">ThermoFisher</td>
			<td style="width:136px;">88400</td>
			<td>Freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Greenhouse</td>
			<td style="width:259px;">n.a.</td>
			<td style="width:136px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Grodan Rockwool Cubes 10x10cm</td>
			<td style="width:259px;">Grodan</td>
			<td style="width:136px;">102446</td>
			<td>Rockwool block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Infrared Detector Optris CT</td>
			<td style="width:259px;">Optris</td>
			<td style="width:136px;">OPTCTLT15</td>
			<td>Infrared detector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Infrared Detector Software Compact Connect</td>
			<td style="width:259px;">Optris</td>
			<td style="width:136px;">n.a.</td>
			<td>Control software for infrared detector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Lambda 1050 UV/Vis spectrophotometer</td>
			<td style="width:259px;">PerkinElmer</td>
			<td>L1050</td>
			<td>UV/VIS Spectrophotometer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:411px;">Laser 400&#956;m, 1550nm Conduction Cooled Single Bar Fiber Coupled Module</td>
			<td style="width:259px;">DILAS</td>
			<td style="width:136px;">M1F-SS2.1</td>
			<td>Laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Laser cover&#160;</td>
			<td style="width:259px;">Amtron</td>
			<td style="width:136px;">LM200</td>
			<td>Laser Cover</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Laser Driver&#160;</td>
			<td style="width:259px;">Amtron</td>
			<td style="width:136px;">CS 408</td>
			<td>Laser Driver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Osram cool white 36 W</td>
			<td style="width:259px;">Osram</td>
			<td style="width:136px;">4930440</td>
			<td>Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Photodiode sensor&#160;</td>
			<td style="width:259px;">Thorlabs</td>
			<td style="width:136px;">PDA20H-EC</td>
			<td>Power sensor for transmission measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Precision weight Ohaus Analytical Plus</td>
			<td style="width:259px;">Ohaus</td>
			<td style="width:136px;">80251552</td>
			<td>Precision weight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sample frame</td>
			<td style="width:259px;">Fraunhofer ILT</td>
			<td style="width:136px;">n.a.</td>
			<td>Fixation of the leaf sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Software Pyro Control</td>
			<td style="width:259px;">Amtron</td>
			<td style="width:136px;">n.a.</td>
			<td>Laser Power Control Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Stainless-steel-holder</td>
			<td style="width:259px;">n.a.</td>
			<td style="width:136px;">n.a.</td>
			<td>Holder for measurement set-up</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Teflon plates 2cm</td>
			<td style="width:259px;">Fraunhofer ILT</td>
			<td style="width:136px;">n.a.</td>
			<td>Teflon attenuation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Thermal surface absorber Power sensor</td>
			<td style="width:259px;">Thorlabs</td>
			<td style="width:136px;">S314C</td>
			<td>Sensor for laser power measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Vibratome</td>
			<td style="width:259px;">Leica</td>
			<td style="width:136px;">1491200S001</td>
			<td>Vibratome</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Zoc/Pro 6.51&#160;</td>
			<td style="width:259px;">EmTec Innovative Software</td>
			<td style="width:136px;">n.a.</td>
			<td>Laser Control Software&#160;</td>
		</tr>
	</tbody>
</table>

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.

Measurement of Leaf Properties
Using the above microscopic method, a leaf thickness of 0.22-0.29 &#215; 10&#8722;3 m was determined for both N. tabacum (0.25&#177;0.04 &#215; 10&#8722;3 m, n=33) and N. benthamiana (0.26&#177;0.02 &#215; 10&#8722;3 m, n=24), which is well within the 0.20-0.33 &#215; 10&#8722;3</sup...

Watch this Scientific Journal Video about A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties at JoVE.com

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

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 ...

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Biochemistry

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential Scanning Calorimetry ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling

Fragrances of many flower families have been sampled and the volatiles analyzed. Knowing the compounds that make up the fragrances can be an important ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...