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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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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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，因为扁平叶子表面是优选的测量，但样品自然具有波浪形表面。这个问题可以通过设计具有精确地调节到叶样品， 例如 ，叶片的厚度和宽度的几何形状的专用样本保持器，在喜欢夹持样品来克服能方向。这种方法将使得测量更重现，但由于样品和保持器之间的牢固的接触，将需要拉叶面平会妨碍测量的非接触式的性质。因此使用这种夹持器的好处将取决于测量， 即的上下文中，测量的精度或无接触性质是否是最重要的。与此相反，这样的考虑可能并不在所有的叶必要具有固有平坦表面， 例如，水稻及相关物种。 在样品的环境由于对流热传递至空气运动应该在测量过程中被保持为最小，因为这会严重影响使用C P，S的计算和ʎ18。该装置因此应该由空调系统，散热器或其他设备，如计算机产生的空气流位于酒店整体的散热风扇。这也是重要的，因为在之前或测量由于蒸发过程中可能发生的叶子19，其可以增加空气流动20的相对水分含量的变化，并没有占在模型中。因此，测量结果，尤其是已分离的叶子，应迅速在协议部分中描述，以避免在数据采集期间的错误进行。在未来，蒸发的对测量的影响可以减少，或者如果测量是在一个至少部分封闭的测量室与实施的湿度控制进行了避免。 中 c p，S和ʎ值的准确度，可以通过测量在相应的方程中使用更精确的参数来增加。在C p的情况下，S这些参数是激光功率，最大和环境温度和样品的体积， 即 ，拉斯的产物呃点面积和厚度，和样品密度（式8）。后两个参数必须伴随实际测量实验确定，如果几个代表性的生物学重复进行测试的可靠性可以提高。然而，即使当使用一个简单的拨号表压测量，相比于显微分析在叶片厚度的差异只有11％，这影响由同一程度对C P，S和ʎ算出的值。相反，温度和激光功率可以在整个测量进行监测。 中 c p的准确性，s时，可以如这些在线数据被用于固定值，而不是对激光功率和环境温度被提高，并且使用公校准传感器收集的数据。这些考虑也适用于ʎ，但环境和样品的温度是最重要的参数，因为这两个影响由四个功率的计算值。 ʎ的当前计算基于关于对流热传导和热辐射的几个假设。例如，发射率（ε）和对流热传递系数（H）未测量或在上面提出的方法明确地计算，但是从先前的出版物18,21而得。 ʎ的精度可因此通过实际测量条件下确定这两个参数加以改进。然而，使用用于计算所述文献数据尽管如此，由于其系统发育到烟草物种和它们的生理学， 即，草本植物3产生了分别用于其相似的性质，可以预期其它植物物种实验确定的范围内ʎ值。即使对于ε和h的值超过为ε先前用于在植物中这些值报告的整个范围内， 例如 ，0.93-0.98变化 21，其上ʎ的最终值效果&lt;10％，因此内这里观察到的自然变异。 以上提出的方法不仅能够确定完整无恙叶和离体叶片的热性能，但它也正确地识别不同类型的测量前有意引入的更严重的伤害。因此，不同类型的叶片样品中可以很容易区分，提供了一个工具来删除，分析之前，任何可怜样，将产生低质量的数据。这一功能可以监测生物材料， 例如 ，当样品不能满足规格的中 c p，的条款及ʎ可以从进一步的处理中排除被用于质量控制。这将是在一个高度调节过程的上下文中的资产，如分子农业4。 相比更为在这个新方法的优点文献包括快速样品处理，最少的准备，C P，S和ʎ的和非破坏性无接触同时测量，并使用共同的设备，可以在许多光学实验室发现。这将有利于相比，这些需要专门而昂贵的设备，例如差示扫描量热仪的方法的更广泛的应用。此外，量热法需要与样品22直接接触，从而有损坏的危险，并且该方法通常限于特定的热容量22测量。与此相反，而热成像可检测坏死或在无接触方式23叶或整个植物的物理变化，它也需要这可能在未来被更便宜和更强大的红外线摄像机被克服复杂的图像分析和专用专门装置24和伴随外围设备。谱分析是另一个接触-fr的的水含量和叶绿素水平25分析ee值的方法，但它尚未用于确定比热容量和/或导热性。 本文所报告的测量方法是一种可靠的方法来确定的热特性的植物叶具有低投资成本和短测量时间。它被成功地用于测定在普通烟草和本氏烟 ，两个物种是在分子农业4的面积相关中 c p，S和ʎ。基于叶片温度分布这两个参数的计算值均与之前的其他植物3报道的一致。该方法是非破坏性的，接触的，并且不需要复杂的样品制备，提供超过对热性能的分析当前的所有可供选择的方法的优点。简单的设计也可以促进手HEL发展D器件以增加灵活性。

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="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54835/54835fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</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

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米的范围内。确定与拨号规厚度得到的〜0.28×10值- 3米（N = 10），这是从微观测量结果的一个标准差范围内。因此，拨号计测量可以优于微观方法在日常应用的厚度决定，因为它是更容易申请和对C P，S和ʎ结果从劳动密集型技术偏离小于10％。 普通烟草和烟草本塞姆氏叶子的密度为750±10公斤米- 3（N = 20），其中631-918公斤米比赛- 3系列以前在其他3种报告叶子。 比热容的计算 收集烟草物种的温度分布显示出超过该激光脉冲直到最大温度（T 最大 ）时的快速增加是在不到1秒达到。脉冲后，将温度呈指数下降，直到达到环境温度（T 0）（ 图3A - C）。的比热容（C P，S </子&gt;）根据公式±323公斤Ĵ-1 K -1为普通烟草和2252±285公斤Ĵ-1 K -1的烟草本塞姆氏 3661 8屈服值计算。两个栽培设定和持续时间用于每个 种（参见1.2节），但是这丝毫没有影响ÇP，S（ 图4）。然而，C P，S的值从旧的（底部）线性减少到年轻（顶部）的叶（R 2 = 0.85）在普通烟草 （ 图4A），其相关的水含量[GG的情况-1生物质]已被确定为湿生物质的在收获时间的差异，并在60℃下11日之后72小时培养的质量。水含量和比热容量之间的这种相关性是在其他作者13与以前的观察一致。观察到N. benthamia呈负相关娜（R 2 = 0.79），其中不同程度的成熟的叶子的比热容量之间的差;相比于普通烟草 21％（底=旧顶端=年轻）分别只有13％。这种差异可能的事实，在烟草本塞姆氏叶的水含量是在不同程度的叶熟化11的几乎恒定起源。敏感性分析发现，在中 c p差异，S分别正比于测量参数波动方程8所述的效果的反射和透射激光功率为子比例，因为这些参数没有在方程式7个体因素。因此，在这两个参数中的差错的影响比那些引起的激光功率或环境温度的波动较小。在一般情况下，测量被认为是坚固的，因为所有的参数涉及在C p的计算，S有一个系数小于10％的变异（ 图4C和D）。 <img alt="图4" src="/files/ftp_upload/54835/54835fig4.jpg" /> 图4:比热容量和热导率值确定为普通烟草和本塞姆氏 。 一 。比热容和树叶根据对植物的叶位普通烟草的热导率（=底部老叶;中间=成熟的叶片;顶=幼叶）。明星和三角形表明，分别为49和56天，分别植物。 B点 。比热容量与烟草本塞姆氏的热导叶根据对植物的叶的位置。星和三角形表示被培养在人工气候温室或分别植物。 ℃。比热容量的值的敏感性，以对输入参数的变化。 TRiangles显示从10％增长产生（红，向上）或比热容值单一模型参数减小（蓝色，向下）。 ð。热导率值的灵敏度于输入参数的变化。三角形标记从10％增长产生（红，向上），或在单模型参数鞋导热性值降低（蓝色，向下）。在A和B误差棒表示标准差（N≥3），而在C和D所代表在10％的变化的灵敏度分析所获得的值的完整范围。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54835/54835fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 热导率的计算 热导率（ʎ）从通过的指数的温度分布计算接头（ 图3）与方程传导和对流热传递以及热辐射相结合。 15式产生了0.49±0.13缩略词平均值- 1秒- 1 的 K - 1（N = 19） 普通烟草和0.41±0.20缩略词- 1秒- 1 的 K - 1（N = 25）为N.本生 。有ʎ和植物年龄或栽培环境之间没有相关性，虽然观察到烟草本塞姆氏 （ 图4B）的叶龄和ʎ之间的相关性，与其他植物物种14以前报道年龄依赖性差异同意。如上所讨论的，水含量是因为它被认为是在整个对烟草本塞姆氏变成熟叶均质这种差别不太可能原因。相反，我们推测的L的更改电炉组织， 例如 ，细胞壁组成，负责此观察通过改变叶片的热传递性能，从而影响ʎ的值。 ʎ的确定是在环境温度变化敏感。敏感性分析发现，±2.3的K波动由64-125％改变ʎ的值。根据公式15，环境温度具有由四个电源上的热辐射的效果，因此直接影响ʎ的值。 测量仪器的评估 这是可能的设置测量组件3小时内。一旦完成后，该系统的启动时间是每测量系列大约15分钟。单次测量了不到3分钟，包括样品制备和整个测量周期。的激光的曝光时间分析揭示，一个的0.5秒的加热时间导致了19.9±4.3℃的温度增加（N = 55）是一个良好的信噪比（SNR）和所需的高ΔT之间的最佳折衷（通过长的激光脉冲实现）低ΔT（由短激光脉冲实现）需要以避免组织损伤。脉冲持续时间大于0.5秒长导致了质量的来自样品的损失，可能反映的水和/或损坏的蒸发到叶组织为最多时达到70℃的样品温度，而只有42.9±4.2℃，（正观察了0.5秒的激光脉冲= 55）。对于小于0.5秒，±0.31的K温度噪声（标准偏差，N = 25）的持续时间占ΔT的5％以上并且因而ΔT的显著一部分。与此相反，在0.5秒的噪声仅占据了信号的2.5％，由此认为是微不足道的。此外，该样品不加热到超过〜45℃，这是烟草植物还可以在天然热带暴露到亚热带栖息地和其中温度仅有损于苔原生境15中发现的植物品种。激光器的功率密度为170千瓦米-2，而天然的太阳辐射通常在1.0-1.4千瓦米-2 16,17的范围内。然而，由于脉冲的时间很短，这种较高的能量剂量并可能不是由一个最近出版显微分析11所指示损坏的叶组织。因为在此期间仅用于计算ʎ温度数据被限制在激光脉冲之后的最初20秒那样的噪声（±0.31 K）占样品的温度信号的不到5％，并因此被视为无关紧要。当从超过20秒的时间内温度数据使用，ʎ计算值下降（ 图3F）。一个可能的解释是，一些假设的为ʎ的计算ΔT的低值不适用的。尤其，描述在等式15的热辐射的术语可能已经因为它是由温度的第四功率的影响的影响。此外，周边暴露于激光试样斑叶面积可能略微加热，因此可能不是理想的散热器在模型减小有效ΔTx和最终所计算的ʎ假定。

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

Nanyang Technological University

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

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 detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

激光显微切割RNA测序实验设计:从玉米叶片发展的经验分析

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

科研

生物化学

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 target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

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

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

生产，结晶和结构测定<em&gt;℃。难辨</em&gt;通过Microseeding和锌SAD PPEP-1

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 (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

差示扫描量热 - 用于评估蛋白质抗原的热稳定性和构象的方法

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 highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

测定蓝细菌中的糖原含量

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 phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

HEK 细胞 Mitoribosome 的快速分离

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 plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

叶喷雾质谱: 一种快速的环境电离技术直接评价植物组织中的代谢产物

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 to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

原子力显微镜成像在核细胞结构和动力学研究中的应用

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 step to conservation of flowers that are threatened or endangered. Because floral fragrance is critical for attracting pollinators, this method could be used to better understand or even enhance pollination. We present a protocol using a portable charcoal air filter and vacuum to collect floral fragrance volatiles, which are then analyzed by a GC-MS. By using this method, fragrance volatiles can be sampled using a non-destructive method with a machine that is easily transported. This methodology uses a rapid sampling procedure, cutting sampling time down from 2-3 hours to approximately 10 minutes. Using GC-MS, the fragrance compounds can be identified individually, based on authentic standards. The steps used for collecting fragrance and control data are presented, from material setup to collecting the data output.

Here, we present a protocol for collecting the floral fragrance volatiles from blooming flowers, using a non-destructive sampling procedure.

使用头空间挥发性收集技术快速收集花香挥发物，用于 GC-MS 热解吸采样

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

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

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 withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

测量天然产品生物调节特性的平面运动分析

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