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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S., Philip, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermophysical+Properties+of+Plant+Leaves+and+Their+Influence+on+the+Environment+Temperature.">Thermophysical Properties of Plant Leaves and Their Influence on the Environment Temperature.</a> International Journal of Thermophysics. 31, 2295-2304 (2010).</li><li>Buyel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+development+strategies+in+plant+molecular+farming.">Process development strategies in plant molecular farming.</a> Curr. Pharm. Biotechnol. 16, 966-982 (2015).</li><li>Schuster, A. 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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 ...

The overall goal of this procedure is to determine the thermal properties of leaves in a contact-free manner. This method can help answer key questions in the field of molecular farming about in-process control of plant growth and health. The main advantages of this technique are that it is contact-free, fast, and does not damage the sample.The implications of this technique extend toward automated inspection of plantations with a mobile detector to define feeding and nutrition on a plant-by-plant basis. To begin plant cultivation, flush mineral wool blocks with one to two liters of deionized water each, then flush each block with one liter of a 0.1%fertilizer solution. Place one tobacco seed in each block and flush with 250 milliliters of fertilizer solution, ensuring that the seed is not washed away.Cultivate the plants for seven weeks in a greenhouse or phytotron. Once the plants have grown, either harvest intact, undamaged single leaves for plant characterization or use intact plants. To begin leaf thickness determination, prepare in autoclave 50 milliliters of a 2%mass by volume agarose solution and phosphate-buffered saline.Allow the solution to cool to 40 degrees celsius and then add the solution to a Petri dish containing a single leaf sample. Cool the sample at four degrees celsius for 30 minutes to solidify the agarose and embed the leaf. Then use a vibratome to cut the agarose block into 200 micrometer slices.Mount five transversal leaf sections on a glass slide with cyanoacrylate. Use a microscope or dial gauge to determine the thickness of vein-free areas of the leaf sample. Use another single leaf sample in water to determine leaf density with a pycnometer.Next, clamp another single leaf in front of the detector in a UV vis spectrophotometer sample chamber. In the spectrophotometer software, select a spectrum of 900 to 1, 600 nanometers. Perform the scan and record the transmission value based on the spectral curve.Then, clamp the leaf behind the detector and repeat the scan. Determine the reflection value from the spectral curve. Obtain transmission and reflection measurements from at least three leaf samples.To begin, mount a 1, 550 nanometer fiber coupled single bar near-IR diode laser in the cone. Then secure a biconvex lens with a 25.4 millimeter focal length at the end of the cone. Clamp a 25.4 millimeter diameter cone with a stainless steel holder to a laboratory stand.Place a neutral density filter with an optical density of 1.0 in a 22 millimeter thick ceramic plate above the censor to attenuate the laser beam. Mount a photo diode power sensor 354 millimeters below the bottom of the lens. Connect the photo diode power sensor to an oscilloscope.Affix a 10 centimeter by 10 centimeter frame with a six centimeter by six centimeter sample exposure area 308 millimeters below the lens. Mount the detector 135 millimeters above the ceramic attenuator. Connect a near-IR detector to a computer and prepare the software.Put an absorber plate on the leaf and turn on the laser. Adjust the detector angle to 45 degrees relative to the laser beam. Continue adjusting the detector angle and height until the maximum temperature reading is observed.In the laser control software, select power control. Set the output laser power to five watts and the pulse duration to 0.5 seconds. To begin the measurements, either bring a whole plant to the measurement apparatus or harvest whole, undamaged single leaves.Immediately mount the leaf sample in the frame, taking care not to damage the leaf. Do not allow the leaf to contact the ceramic attenuator. In the near-IR software, start a new 60 second measurement.Record the baseline temperature profile for 10 seconds before activating a single 0.5 second laser pulse. Continue recording data for the remaining 49.5 seconds. When the measurement is complete, click on the stop button above the thermal profile and then save the profile.Export the raw time and temperature data as a dot dat file for further analysis. Then in the oscilloscope software, determine the flank heights in the voltage profile, which were automatically collected during the laser pulse. Repeat the temperature profile measurement with a single 0.5 second laser pulse for each sample.Then repeat the laser signal measurement without a leaf sample as a reference. Determine the flank heights from this voltage profile and calculate the transmission values for each sample. Use spreadsheet software to calculate and graph the specific heat capacity and thermal conductivity of the samples.Single leaf samples of the plant species Nicotiana tabacum, known as tobacco, and Nicotiana benthamiana were analyzed with this method. Both species showed a rapid increase to maximum temperature in less than one second in response to the laser pulse, followed by an exponential decrease to ambient temperature. The specific heat capacity and thermal conductivity values were calculated for both species from leaves collected at the bottom, middle, and top of the plant.An increase in thermal conductivity for leaves further up the plant was observed for both species, however Nicotiana tabacum showed an inverse trend in specific heat capacity. No correlation between cultivation duration and thermal conductivity was observed in Nicotiana tabacum. Further, no correlation between cultivation conditions and thermal properties were observed in Nicotiana benthamiana.The effect of individual variables on specific heat capacity and thermal conductivity was examined. The thermal conductivity of Nicotiana benthamiana was highly sensitive to ambient temperature. Specific heat capacity was not significantly sensitive to any of the parameters involved in its calculation.Once mastered, this technique can be done in a few minutes per sample if it is performed properly. While attempting this procedure, it is important to remember to correctly mount the leaf at the given precision to ensure high quality measurements and reproducible results. Following this procedure, other methods like fluorescence measurements can be performed in order to answer additional questions like recombinant protein expression.After its development, this technique paved the way for researchers in the field of plant biotechnology to explore plant growth in Nicotiana species. After watching this video, you should have a good understanding of how to determine the specific heat capacity and thermal conductivity of leaves in a contact-free and non-destructive manner. Don't forget that working with lasers can be hazardous to your eyes and precautions such as wearing the appropriate safety glasses should always be taken while performing this procedure.

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Research

JoVE Journal

Biochemistry

7.2K visualizações.  Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.. The overall goal of this procedure is to determine the thermal properties of leaves in a contact-free manner. This method can help answer key questions in the field of molecular farming about in-process control of plant growth and health. The main advantages of this technique are that it is contact-free, fast, and does not damage the sample.The implications of this technique extend toward automated inspection of plantations with a mobile detector to define feeding and nutrition on a pl...