Name: a rapid laser probing method facilitates the non-invasive and contact-free determination of leaf thermal properties
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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.植物栽培和样品制备<ol><li>冲洗每个矿棉块用1-2升去离子水，随后用1升0.1％[M / V]肥料溶液。放置一个烟草（ 烟草或烟草本塞姆氏 ）种子在每块0.25升肥料溶液冲洗轻轻没有洗去种子。 </li><li>培育7周的植物在温室或人工气候室用70％相对湿度，16小时光周期（180微摩尔的 S - 1米- 2;λ= 400-700纳米）和25/22℃的光照/黑暗温度政权。 </li><li>移动至植物的测定装置。如果植物是不动的，收获单叶热性能的测量。 </li></ol> 2.确定叶片厚度和密度<ol><li>确定叶片厚度<ol><li>在磷酸盐缓冲盐水制备2％的[M / V]的琼脂糖溶液（PBS）中，并高压灭菌它。让溶液冷却到40℃并嵌入放置在培养皿叶样品。通过在4℃下放置在培养皿在冰箱中30分钟固化琼脂糖。 </li><li>切使用振动为15°的刀片切削角琼脂糖块成200微米的切片。使用为1.0mm秒的切削速度-1和0.5mm的振幅。 </li><li>安装使用氰基丙烯酸酯为固定液载玻片5个横向叶的部分。确定叶片厚度用20×物镜的显微镜，并用10×放大率目镜下，使用根据制造商的说明内置于显微镜软件的测量工具。 </li><li>在确定样本地区叶片厚度无脉。 </li><li>可替代地，确定与在所述叶片的一个自由静脉区域拨号轨距的叶片厚度。确保拨号隔距保持垂直于叶片的平面。 CAUTION:氰基丙烯酸酯是一种皮肤的刺激性，如果不小心处理也可能粘上手指并拢。 </li></ol></li><li>确定叶面密度<ol><li>确定干燥比重瓶的空质量（M 0），然后加满水，并再次确定质量（M 1）。完全干燥的比重瓶，里面放上一张叶片和确定质量（m 2）一次。随着里面的叶子，认真填写了水的比重瓶并确定质量（m 3）。 </li><li>用式1计算的叶密度（PS）。 方程1: <img alt="方程" src="/files/ftp_upload/54835/54835eq1.jpg" /></li></ol></li></ol> 3.确定叶的光谱透射率与思考<ol><li>由采样保持夹具之间固定它放置在一个UV / VIS分光光度计的样品室中的叶。对于传输测量，将叶在DET前面<html>埃克特。对于反射测量放置叶在检测室的后部。 </li><li>启动分光光度计控制软件。选择从900nm的光谱1600纳米。开始新的扫描，并记录值传输（μT）和反射（μr）通过UV / VIS分光光度计软件显示，基于光谱曲线上。 </li><li>具有至少三个生物学重复执行所有测量。增加生物学重复的数量为五个或更多，如果一个异质样品质量可以预期， 即，在叶片的表面形态和厚度变化。 </li><li>通过根据等式2和3由所测量的激光功率P 的激光测量μT或μ 的R值乘以计算传输（P T）和反射性（P R）的电力。 公式二:ftp_upload / 54835 / 54835eq2.jpg"/&gt; 公式三: <img alt="方程" src="/files/ftp_upload/54835/54835eq3.jpg" /> 注:该变速器可以在测量过程中的光电二极管传感器（见6.3）来也就确定了。 </li></ol> 4.设置测量仪<ol><li>装入一个光纤耦合单条近红外二极管激光（波长= 1,550nm处）插入一个不锈钢支架25.4毫米直径的锥。连接控制器到近红外激光的输出功率（P 激光 ）设定为4-6 W. </li><li>放置具有25.4毫米的焦距的双凸透镜在所述锥体的端部的光束宽度调整到13毫米。 </li><li>放置一个光电二极管功率传感器的透镜的底部之下354毫米。然后通过将一个中性滤光片为1.0的光密度和传感器上方的22毫米的陶瓷层衰减的光电二极管。 </li><li>光电二极管功率传感器连接到使用同轴电缆的示波器。 </li><li>接一个10×10厘米框架，其具有与测量设置在308毫米镜头下方（ 图1）的高度在支架6×6厘米样品曝光区域。通过将其安装到10×10厘米框架固定在空间上叶位。 </li><li>连接使用通用串行总线（USB）电缆一个近红外检测器的个人计算机，为检测器安装接口软件。 </li><li>将检测器以45°角的激光束在陶瓷层135毫米的位置。由直到最大温度信号观察改变传感器的位置和角度对准检测器样品上的激光光点的测定区域。 </li><li>使用激光控制接口软件，以调节输出激光功率5瓦和激光脉冲的到0.5秒的持续时间。在激光功率的图示下方的控制选项窗口中选择"电流控制"命令，输入"5"调整激光功率成＆＃34;功率[W] 0.5"变成了"时间[s]"场"。现场通过键入调整激光脉冲持续时间"。 </li><li>以确定每个组实验中的绝对的激光功率，替换用热表面吸收功率传感器的光电二极管功率传感器在每一组实验中的端部，并测量20秒的激光输出功率，而不会用样品。 </li></ol> 5.准备叶片样品<ol><li>为测量使用完好无损的叶子。 </li><li>如果相关的调查，通过用手术刀刺入叶，揉乳胶手套之间的叶，叶暴露于明火或2-3个激光束，或使用其他技术来模拟其他模仿典型叶片损害类型类型的损害。 </li><li>小心，但很快安装采样保持夹具之间的叶子样品。 </li></ol> 6.取温度测量<ol><li>避免叶片和陶瓷之间的直接接触衰减器放置在光电传感器上面，以防止与C P，S，λ的计算人工干扰传热（见第9节）。 </li><li>使用温度测量软件收集叶样品的温度曲线用于经由近红外检测器共60秒。首先，记录温度基准10秒钟，然后启动激光为0.5秒，并继续收集数据为49.5秒。 <ol><li>通过点击"测试"，然后"新测量"开始测量。之后单击热分布的图形表示上方的绿色箭头。减通过点击"保存"图标的轮廓的图形表示上方的温度分布（一个程式化的盘）。 </li></ol></li><li>通过使用和不使用连接示波器叶子样本计算测量信号的差确认使用光电二极管功率传感器所发射的激光功率经由同轴电缆在光电二极管功率传感器（ 图2）。 <ol><li>确定两个侧面的高度（F 1，S和f 2，S）在与示波器获得的电压曲线。 </li><li>重复测量未经叶样品作为基准（ 六1,0和f 2,0）。根据公式4（也参见图2）计算这些测量值的比变速器μ 吨 。 公式4: <img alt="方程" src="/files/ftp_upload/54835/54835eq4.jpg" /></li></ol></li></ol><img alt="图2" src="/files/ftp_upload/54835/54835fig2.jpg" /> 图2:测量使用光电二极管功率探头叶传输。 一 。对于没有叶子样品参照实验典型电压档用示波器观察。 B点 。电压曲线与安装在装置中的叶样品。在这两种情况下，所发射的激光功率正比于每两个侧翼的。 <a href="http://ecsource.jove.com/files/ftp_upload/54835/54835fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 7.计算样品叶的比热容<ol><li>通过从最大叶温度T 毫安 X [K]（式5）中减去室温T 0的 [K]计算在激光脉冲期间的最大温度差ΔT[K]。 公式5: <img alt="方程" src="/files/ftp_upload/54835/54835eq5.jpg" /></li><li>计算基于有效激光功率和激光脉冲持续时间（等式6），其中P - [R [W]是反射的激光功率和（E S [J]）由叶子吸收的能量PT [W]是发射激光功率。 EQUA化6: <img alt="方程" src="/files/ftp_upload/54835/54835eq6.jpg" /></li><li>计算加热叶面积的质量用（M S [千克]）等式7中，其中，d S [米]是根据2.1）的叶厚度，R 激光 [米]是激光光斑的半径，V S [米3]是加热的叶体积，ρS [公斤米-3]是根据2.2叶密度）。 公式7: <img alt="方程" src="/files/ftp_upload/54835/54835eq7.jpg" /></li><li>计算中 c p，S [J千克-1 K -1]。根据由加热的叶面积质量m S和最大温度差ΔT的乘积除以所吸收的能量（E S）等式8。 公式8: <img alt="方程" src="/files/ftp_upload/54835/54835eq8.jpg" /></li></ol> 8.准备千卡的温度分布数据人电导率计算<ol><li>使用近红外传感器控制软件的"导出"命令，将时间和温度的原始数据导出为* .dat文件并打开该文件在电子表格处理器。 </li><li>应用1:100的数据缩减， 例如，使用一个"IF（MOD（值100）= 0;"×"，"0"）"命令，导致每0.1秒的一个数据点的数据密度。 </li><li>计算平均基线温度T B [℃]为每个温度分布在最初的10秒的测量，在此期间，激光器仍然关闭的。然后，计算T B和实际环境温度T 0 [℃]之间的差异。 </li><li>使用这种差别在使用，如果T B移它向T 0（Y-标准化）， 例如，单独正常化每个配置文件; - T 0 = 2.0 K，然后从各个温度值与温度减去2.0ķ公关OFILE（ 图3A）。 </li><li>各归温度曲线（X-正常化）的时间坐标删去最高样品温度之前，每一个数据点（T max）和分配开始T = 0 T 最大 （ 图3B）新的时间值。 </li><li>屏幕的每个信息对突然的温度变化， 即，温度差是超过三倍基线噪声水平，这是典型的3×0.31ķ≈1.0，因为它们对应于测量工件K.取出从数据集中这些区域（ 图3C ）。 </li><li>使用电子表格处理器，其中T T [K]是在时刻t [秒]的拟合叶样品的温度适合的指数衰减函数（等式9）的数据，T 0是环境温度，A [K]时的振幅和T 1 [S]衰变常数（ 图3D）。 公式9: <img alt="方程" src="/files/ftp_upload/54835/54835eq9.jpg" /></li><li>使用嵌合功能的激光脉冲之后，计算从0-80 S中的叶片样品中的温度下降。 </li><li>通过加入273.15的值各温度数据点（ 图3E）变换在[℃]测得的温度数据给[K]的规模。 </li></ol><img alt="图3" src="/files/ftp_upload/54835/54835fig3.jpg" /> 图3:为λ的计算数据处理方案。 一 。削减数据后，将温度分布是归一化至环境温度。 B点 。接着，将最大样本温度（T 最大 ）前的所有数据点都被删除。 ℃。测量工件（在"不一致"数据集示出）根据温度鉴定转移比第大稀土元素倍基线噪音和从之前拟合指数函数数据集移除。 ð。摄氏温标被转换为开氏温标。 E.对于每个时间间隔，λ是基于温度分布进行计算。 F。第20号窗口是指在相关的温度变化可以观察。 -G。基于所选择的时间窗上的平均和标准偏差的计算为λ。 小时 。代表性的结果为两个不同的普通烟草叶样品。橙色箭头和线表示上所呈现的数据相应的处理步骤的效果。 <a href="http://ecsource.jove.com/files/ftp_upload/54835/54835fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 9.计算叶样品的热导率<ol><li>计算温度各色叶样品，并根据公式10，其中ΔT×〔K〕是温差为每个0.1秒间隔环境之间NCE，T（T）[℃]是拟合叶样品温度和T 0 [℃]的环境温度（ 图3E）。 公式10: <img alt="方程" src="/files/ftp_upload/54835/54835eq10.jpg" /></li><li>假设在温度的下降是由于对流热传递，热辐射和热传导的综合效果。使用相应的能量平衡（等式11），作为λ的计算，其中，ΔE 温度 [D]是在两个连续的时间点，在样品的热能量的差的基础上，ΔE 拉德 [D]是由于能量差热辐射，ΔECONV [J]就是能量差，由于对流换热，和Δ，E COND [J]就是能量差，由于热传导。 公式11: <img alt="方程" src="/files/ftp_upload/54835/54835eq11.jpg" /></li><li>替代在能量平衡的一般术语的实际的物理性质，得到公式12，其中ΔTT [K]是在嵌合叶样品的温度差，ε无单位的发射率，σ[千克小号-3ķ-4]史蒂芬-Boltzmann不变，A 拉德 [M 2]热辐射的面积，H [J -1米-2 K -1]的对流换热系数，A CONV [M 2]对流换热面积，A COND [米2]的热传导和L [m]表示特征长度的面积。 公式12: <img alt="方程" src="/files/ftp_upload/54835/54835eq12.jpg" /></li><li>计算恰拉基于相关cteristic长度1:1 = V / A。 </li><li>使用加热的样品体积V S和叶样品的截面积来计算[米2]。截叶面积，根据式13，其中A cond是哪里传导发生河激光是激光光斑的半径以及d s是叶片厚度的区域对应于COND。 公式13: <img alt="方程" src="/files/ftp_upload/54835/54835eq13.jpg" /></li><li>计算根据公式14，其中A 激光是激光光斑面积A 拉德和A CONV。 公式14: <img alt="方程" src="/files/ftp_upload/54835/54835eq14.jpg" /></li><li>替代方程9，12和13入公式11并解决λ后，产生式15其中t 激光为t他的激光脉冲持续时间[S]。 公式15: <img alt="方程" src="/files/ftp_upload/54835/54835eq15.jpg" /></li><li>假定0.94为ε的值，并计算每0.1 - S的时间间隔λ在第一20秒的温度分布的。平均的200个值以这种方式得到的λ和计算标准偏差（ 图3F - H）。 </li></ol>

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G., 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=Thermal+infrared+imaging+of+crop+canopies+for+the+remote+diagnosis+and+quantification+of+plant+responses+to+water+stress+in+the+field.">Thermal infrared imaging of crop canopies for the remote diagnosis and quantification of plant responses to water stress in the field.</a> Funct. Plant Biol. 36, 978-989 (2009).</li><li>Defraeye, T., Verboven, P., Ho, Q. T., Nicolai, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convective+heat+and+mass+exchange+predictions+at+leaf+surfaces:+Applications,+methods+and+perspectives.">Convective heat and mass exchange predictions at leaf surfaces: Applications, methods and perspectives.</a> Comput. Electron. Agric. 96, 180-201 (2013).</li><li>Arndt, S. K., Irawan, A., Sanders, G. 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The authors have no conflicts of interest to disclose.

上述的非接触式的，非破坏性测量方法可用于确定以同时和再现的方式中 c p，S和ʎ。特别ʎ的计算取决于几个参数是错误敏感。尽管如此，这些错误的影响是线性或分比例，并为所有参数变化系数被认为是少于10％。即使该方法可因此被认为是坚固的，一些技术改进可制成以减少错误的剩余源。 安装样品放入组件在技术上是挑战性的，因为扁平叶子表?...

生物材料的大规模加工通常需要的热处理步骤，例如巴氏杀菌。对于这样的方法的设备可以更精确地设计，如果生物材料的热性能是公特征在于，包括特定的热容量（C P，S）和热导率（λ）。这些参数可以容易地为液体，悬浮液和匀浆由量热法1来确定。然而，固体样品中测量这种参数可以是劳动密集型的，并且经常需要与样品或者甚至它的破坏2直接接触。例如，光热技术需要在样品和检测器3之间的直接接触。这种限制是食品加工过程中可以接受的，但是是高度调节的过程不相容如在良好生产规范4的上下文中在植物中生产的生物制药蛋白质。一世n个这样的背景下，可在七周的生长期为植物个体作为一种质量控制工具所需热性能的重复（ 例如，每周）的监测。如果这样的监测需要和消耗对于每个测量一个叶，就没有生物质留在收获时间来处理。 此外，仅使用叶份代替会导致伤害的植物和增加坏死或病原体感染的风险，又减少了工艺产率。病原体感染的可能性也会增加，如果将用于直接接触到样品的方法，诱导该植物的整个批次可以通过与污染的传感器装置接触被感染的危险。类似方面必须考虑对植物监测强调像干旱， 例如 ，在一个生理生态环境。例如，失水往往是由在鲜生物量的改变，这需要一种侵入特雷监视正在调查5， 如植物，解剖叶的atment。相反，在确定比热容量，这取决于样品的水含量，以非侵入性方式在这里描述，可以用作植物的水合状态的替代参数。在这两种情况下（药品生产和生态生理学），通过破坏或侵入性的测量技术人工诱导应力将是有害的，因为他们可以扭曲的实验数据。因此，先前报道闪光方法6或样品的银板7之间的位置不适合于这样的工艺和实验，因为它们要么需要直接接触到样品或者是破坏性的。所述参数c P，S和λ必须以设计的工艺设备用于预煮步骤，可以简化产物纯化，从而降低制造成本8-10来确定。使用CP，S和λ现在可以通过无接触的非破坏性近红外（NIR）激光以一致和可再现的方式11探测被快速确定，并且这种新的方法将在下面进行详细说明。用这种方法得到的结果进行了成功地用于模拟传热在烟叶12，允许适当的处理设备的设计和相应的参数，如热烫温度的选择。 该方法是容易设置（ 图1），并具有两个阶段，测量和分析，其每一个包括两个主要步骤。在测量阶段，叶子样品首先在本地通过短激光脉冲加热和最大采样温度被记录下来。然后将样品的温度分布被记录为50秒的持续时间。在分析阶段，叶属性，如密度（轻松，准确地通过比重瓶measurem确定耳鼻喉科）与最大样本温度相结合来计算中 c p，第在第二步骤中，叶的温度分布被用作输入一个能量平衡方程，以传导，对流和辐射进去，来计算λ。 在协议部分提供了详细的一步一步的指导，扩大对影随行的内容。典型的测量，然后在结果部分中。最后，该方法的优点和限制在讨论部分与潜在的改进和进一步的应用沿着突出。 <img alt="图1" src="/files/ftp_upload/54835/54835fig1.jpg" /> 图1:用于确定叶热性能的装置。 一 。所述测量装置的照片来确定比热容量和乐的热导率鸟类。外围设备（计算机，示波器）未示出。 B点 。的测定装置的示意图。激光器和连接的设备以​​红色突出显示，用于温度测量的近红外检测器示于紫，叶样品是绿色和光电二极管功率传感器是蓝色的。 ℃。绘制测量设置具有相同的颜色代码作为B的元件的尺寸条表示0.1米。 ð。屏幕截图示出激光控制软件的典型元素。 <a href="http://ecsource.jove.com/files/ftp_upload/54835/54835fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>1&quot; tube</td><td>Thorlabs</td><td>SM1L10E</td><td>Tube for fiber holder</td></tr><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A0701</td><td>Agarose</td></tr><tr><td>Bi-Convex lense f=25.4</td><td>Thorlabs</td><td>LB1761</td><td>Lense</td></tr><tr><td>Digital Handheld Optical&amp;nbsp;Power and Energy Meter Console</td><td>Thorlabs</td><td>PM100D</td><td>Console for thermal surface absorber sensor</td></tr><tr><td>Digital Phosphor Oscilloscope&amp;nbsp;</td><td>Tektronix</td><td>DPO7104</td><td>Oscilloscope</td></tr><tr><td>DMR light microscope</td><td>Leica</td><td>n.a.</td><td>Light microscope</td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-432-2</td><td>Pycnometer</td></tr><tr><td>Ferty 2 Mega</td><td>Kammlott</td><td>5.220072</td><td>Fertilizer</td></tr><tr><td>Fiber holder</td><td>Thorlabs</td><td></td><td>Fiber holder</td></tr><tr><td>Forma -86 &amp;#176;C ULT freezer</td><td>ThermoFisher</td><td>88400</td><td>Freezer</td></tr><tr><td>Greenhouse</td><td>n.a.</td><td>n.a.</td><td>For plant cultivation</td></tr><tr><td>Grodan Rockwool Cubes 10 x 10 cm</td><td>Grodan</td><td>102446</td><td>Rockwool block</td></tr><tr><td>Infrared Detector Optris CT</td><td>Optris</td><td>OPTCTLT15</td><td>Infrared detector</td></tr><tr><td>Infrared Detector Software Compact Connect</td><td>Optris</td><td>n.a.</td><td>Control software for infrared detector</td></tr><tr><td>Lambda 1050 UV/Vis spectrophotometer</td><td>PerkinElmer</td><td>L1050</td><td>UV/VIS Spectrophotometer</td></tr><tr><td>Laser 400 &amp;mu;m, 1,550&amp;nbsp;nm Conduction Cooled Single Bar Fiber Coupled Module</td><td>DILAS</td><td>M1F-SS2.1</td><td>Laser</td></tr><tr><td>Laser cover</td><td>Amtron</td><td>LM200</td><td>Laser Cover</td></tr><tr><td>Laser Driver&amp;nbsp;</td><td>Amtron</td><td>CS 408</td><td>Laser Driver</td></tr><tr><td>Osram cool white 36 W</td><td>Osram</td><td>4930440</td><td>Light source</td></tr><tr><td>Photodiode sensor&amp;nbsp;</td><td>Thorlabs</td><td>PDA20H-EC</td><td>Power sensor for transmission measurements</td></tr><tr><td>Precision weight Ohaus Analytical Plus</td><td>Ohaus</td><td>80251552</td><td>Precision weight</td></tr><tr><td>Sample frame</td><td>Fraunhofer ILT</td><td>n.a.</td><td>Fixation of the leaf sample</td></tr><tr><td>Software Pyro Control</td><td>Amtron</td><td>n.a.</td><td>Laser Power Control Software</td></tr><tr><td>Stainless-steel-holder</td><td>n.a.</td><td>n.a.</td><td>Holder for measurement set-up</td></tr><tr><td>Teflon plates 2 cm</td><td>Fraunhofer ILT</td><td>n.a.</td><td>Teflon attenuation</td></tr><tr><td>Thermal surface absorber Power sensor</td><td>Thorlabs</td><td>S314C</td><td>Sensor for laser power measurements</td></tr><tr><td>Vibratome</td><td>Leica</td><td>1491200S001</td><td>Vibratome</td></tr><tr><td>Zoc/Pro 6.51&amp;nbsp;</td><td>EmTec Innovative Software</td><td>n.a.</td><td>Laser Control Software&amp;nbsp;</td></tr></tbody></table>

a rapid laser probing method facilitates the non-invasive and contact-free determination of leaf thermal properties

植物可以产生有价值的物质如次级代谢产物和重组蛋白。从植物生物质后者的纯化可以通过热处理（热烫）精简。一个热烫装置可以更精确地设计，如果叶子的热性质详细地说， 即 ，比热容和热导率是已知的。这些特性的测量是耗时且劳动密集型的，并且通常需要将样品直接接触侵入性的方法。这样可以减少产品的产量和可能是遏制需求， 如不兼容，在良好生产规范的范围内。为了解决这些问题，一种非侵入性，无接触方法的开发，用于确定在一分钟左右的比热容和完整植物叶的热导率。该方法涉及定义的长度和强度的短激光脉冲的到的一个小区域的应用程序叶样品，引起正在使用近红外传感器测量的温度上升。的温度上升与已知的叶性质（厚度和密度）相结合，以确定比热容量。然后热导率是根据随后的温度下降的信息计算出的，以热辐射和对流热传递考虑。相关的计算和样品处理关键方面进行了讨论。

叶性质的测量 使用上述显微镜方法中，0.22-0.29×10叶片厚度- 3米确定为普通烟草 （0.25±0.04×10 - 3米时 ，N = 33）和本氏烟 （0.26±0.02×10 - 3 M，N = 24），这是很好的0.20-0.33×10内-先前关于各种植物品种3的叶子报道3米的范?...

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.

一种快速探测激光方法有利于叶热性能的非侵入性和无接触测定

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

a-rapid-laser-probing-method-facilitates-non-invasive-contact-free

Research

JoVE Journal

Biochemistry

7.2K 次观看.  Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.. 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.

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

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.

RGB 和光谱根成像植物表型和生理研究: 实验装置和成像协议

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

科研

环境科学

High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient optical micro-imaging technique which can be used to map and quantify such behavior. Europium thenoyltrifluoroacetonate (EuTFC) has a 612 nm luminescence line whose activation efficiency drops strongly with increasing temperature, due to T-dependent interactions between the Eu3+ ion and the organic chelating compound. This material may be readily coated on to a sample surface by thermal sublimation in vacuum. When the coating is excited with ultraviolet light (337 nm) an optical micro-image of the 612 nm luminescent response can be converted directly into a map of the sample surface temperature. This technique offers spatial resolution limited only by the microscope optics (about 1 micron) and time resolution limited by the speed of the camera employed. It offers the additional advantages of only requiring comparatively simple and non-specialized equipment, and giving a quantitative probe of sample temperature.

Europium thenoyltrifluoroacetonate (EuTFC) has an optical luminescence line at 612 nm, whose activation efficiency decreases strongly with temperature. If a sample coated with a thin film of this material is micro-imaged, the 612 nm luminescent response intensity may be converted into a direct map of sample surface temperature.

高分辨率热显微成像采用铕螯合物的荧光涂料

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient ...

工程学

Subsurface Defect Localization by Structured Heating Using Laser Projected Photothermal Thermography

The presented method is used to locate subsurface defects oriented perpendicularly to the surface. To achieve this, we create destructively interfering thermal wave fields that are disturbed by the defect. This effect is measured and used to locate the defect. We form the destructively interfering wave fields by using a modified projector. The original light engine of the projector is replaced with a fiber-coupled high-power diode laser. Its beam is shaped and aligned to the projector&#39;s spatial light modulator and optimized for optimal optical throughput and homogeneous projection by first characterizing the beam profile, and, second, correcting it mechanically and numerically. A high-performance infrared (IR) camera is set up according to the tight geometrical situation (including corrections of the geometrical image distortions) and the requirement to detect weak temperature oscillations at the sample surface. Data acquisition can be performed once a synchronization between the individual thermal wave field sources, the scanning stage, and the IR camera is established by using a dedicated experimental setup which needs to be tuned to the specific material being investigated. During data post-processing, the relevant information on the presence of a defect below the surface of the sample is extracted. It is retrieved from the oscillating part of the acquired thermal radiation coming from the so-called depletion line of the sample surface. The exact location of the defect is deduced from the analysis of the spatial-temporal shape of these oscillations in a final step. The method is reference-free and very sensitive to changes within the thermal wave field. So far, the method has been tested with steel samples but is applicable to different materials as well, in particular to temperature sensitive materials.

This method aims at locating vertical subsurface defects. Here, we couple a laser with a spatial light modulator and trigger its video input to heat a sample surface deterministically with two anti-phased modulated lines while acquiring highly resolved thermal images. The defect position is retrieved from evaluating thermal wave interference minima.

使用激光投射光热法构造加热的地下缺陷定位

The presented method is used to locate subsurface defects oriented perpendicularly to the surface. To achieve this, we create destructively interfering ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

Field Measurement of Effective Leaf Area Index using Optical Device in Vegetation Canopy

Leaf area index (LAI) is an essential canopy variable describing the amount of foliage in an ecosystem. The parameter serves as the interface between green components of plants and the atmosphere, and many physiological processes occur there, primarily photosynthetic uptake, respiration, and transpiration. LAI is also an input parameter for many models involving carbon, water, and the energy cycle. Moreover, ground-based in situ measurements serve as the calibration method for LAI obtained from remote sensing products. Therefore, straightforward indirect optical methods are necessary for making precise and rapid LAI estimates. The methodological approach, advantages, controversies, and future perspectives of the newly developed LP 110 optical device based on the relation between radiation transmitted through the vegetation canopy and canopy gaps were discussed in the protocol. Furthermore, the instrument was compared to the world standard LAI-2200 Plant Canopy Analyzer. The LP 110 enables more rapid and more straightforward processing of data acquired in the field, and it is more affordable than the Plant Canopy Analyzer. The new instrument is characterized by its ease of use for both above- and below-canopy readings due to its greater sensor sensitivity, in-built digital inclinometer, and automatic logging of readings at the correct position. Therefore, the hand-held LP 110 device is a suitable gadget for performing LAI estimation in forestry, ecology, horticulture, and agriculture based on the representative results. Moreover, the same device also enables the user to take accurate measurements of incident photosynthetically active radiation (PAR) intensity.

Fast and precise leaf area index (LAI) estimation in terrestrial ecosystems is crucial for a wide range of ecological studies and calibrating remote sensing products. Presented here is the protocol for using the new LP 110 optical device for taking ground-based in situ LAI measurements.

利用光学装置对植被冠层有效叶面积指数进行野外测量

Leaf area index (LAI) is an essential canopy variable describing the amount of foliage in an ecosystem. The parameter serves as the interface between ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

通过叶反射和叶绿素荧光分析同步测量的光合行为评价

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

生物工程

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

测量叶片导水率和气孔导度和他们的反应，辐射和脱水蒸发通量法（EFM）

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

生物学

Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 &#181;m is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase.
To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time.
The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.

An original experimental setup for heating cells in a culture dish using 1.94 &#181;m continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

连续波ium激光加热培养细胞以研究细胞热效应

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser ...

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.

赫连图斯安努斯大型热成像筛选装置新稳压器的鉴定

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 ...

气候相关胁迫下的马铃薯恢复力分析

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar (Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to reveal&#160;the dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

作者聚焦:使用 PlantScreen 机器人平台揭示植物对非生物胁迫的响应

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

基于图像的高通量表型分析用于确定马铃薯对单一胁迫和联合胁迫的形态和生理响应

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions ...