它承认艺术和科学和教务长的Concordia大学办公室的学院院长办公室支持这项研究的乐趣。 作者要感谢GA克罗斯比他对本次调查的许多贡献。

1.样品制备和装载的低温光谱<ol><li>制备〜3毫升发光发色团的〜10 -3 -10 -4 M溶液在适当的溶剂。 注意:虽然许多溶剂，可以使用水和各种醇溶剂（ 例如 ，乙醇，乙醇/甲醇的混合物，乙二醇，和甘油）提供的溶解性和表面张力特性的用于低温工作的良好组合。 </li><li>通过加捻裸铜线的长度~0.4毫米直径围绕钉制备样品环或螺旋，得到单个环路〜3毫米直径后跟〜30毫米编织铜线直线长度。 </li><li>冲洗用95％的乙醇样品环以除去手指油和其它污染物并晾干。以确保环的清洁度，第一冲洗用硝酸，第二用蒸馏水，最后用乙醇。 </li><li>通过浸渍第加载样品溶液到样品环路Ë循环到样品溶液（步骤1.1）的溶液中，让表面张力保持在循环中的溶液的薄膜。 </li><li>马上浸装载样品环到液氮冷冻和稳定的循环，注意用眼保护（护目镜）和手部防护（手套）薄膜样品溶液。 </li></ol> 2.热电偶准备，调整和设置<ol><li>通过在两个铜 - 康铜结从非绝缘0.4毫米直径的铜导线的两个长度和0.4毫米直径的非绝缘康铜（铜镍合金）线之一长度制备的铜 - 康铜（T型）热电偶:将试样结和一个标准参比端（0 ℃的冰/水混合物）。 注意:虽然结可银焊接在一起，它是完全令人满意的扭转铜和康铜导线在一起以形成结。 <ol><li>夹紧铜和康铜导线磕磕碰碰呃与一〜90°角，尖嘴钳。而在两根电线拉牢，紧贴编织在一起了五六个圈。 </li></ol></li><li>安排所有热电偶导线所以他们不会比在两路口以外的任何点触摸。如果热电偶导线覆盖有薄绝缘涂层，它们可能接触并且比结其他点。 <ol><li>当使用绝缘热电偶电线，刮掉在形成所述结和所述电触点与所述电压表端子的导线的端部绝缘。小心检查导线上的绝缘材料不发光。通过放置在样品环一小片绝缘线，激发样品与所选择的波长的光，并寻找一个发射信号时，没有样品存在测试热电偶导线绝缘作为杂散发光的潜在来源。 </li></ol></li><li>从SAM连接两根铜热电偶导线PLE和参考路口高阻51/2位数字电压表的输入端。 </li><li>放置无论是在0℃的水/冰浴的样品和参考结和零电压表。 </li><li>对准装载样品环，并在液氮充满光学杜瓦热电偶的样本的结到适当的高度和激发光光束的方向一致。保持热电偶结和样品环路并拢尽可能和正好在杜瓦同一高度。 </li></ol> 3.一般机械支撑和样品环的对齐和热电偶结点在光学杜瓦<ol><li>要对齐并保持在光学杜瓦所需的高度样品环，通过张贴小电器鳄鱼夹到5毫米直径×30毫米长木榫准备一个可调节高度的固定夹。 <ol><li>在适合snugl软木塞钻孔稍微较小直径大于5毫米ý到光杜瓦的顶部。夹住鳄鱼夹样品环的编织线部分，然后在软木塞向上或向下滑动销实现杜瓦所需的样品高度。 </li></ol></li><li>为了在光学杜瓦所需的高度对齐并保持热电偶结，用另一种5毫米直径的木榫。对准热电偶结，使得它伸出的销钉的底部之下10-20毫米。对准销钉的相对侧上的铜和康铜导线和12.5毫米宽的聚四氟乙烯（PTFE）管道工的胶带紧紧包住牢固就位持有这些导线。 </li><li>孔另一孔大于5毫米，直径在杜瓦顶部的软木塞，以容纳该第二定位销，并允许样品环附近的热电偶结的垂直高度调节略小。 </li><li>孔在软木塞的第三小孔在杜瓦的顶部以用作氮气汽化通气孔。 </li></ol><p类="jove_title"&gt; 4。在光学杜瓦温度依赖性低温光谱<ol><li>打开在CCD摄谱仪至少1小时的所有电子提前，以允许电子预热和珀耳帖冷却的CCD照相机以达到稳定的工作温度。 </li><li>预热期结束后，波长校准与已知的原子发射线或带一系列的CCD光谱仪。测量的低压原子发射灯发射已知的波长，并通过与已知的频带的波长频带活化相关成分的CCD的像素数的频带的频谱。 注意:在最现代的CCD摄谱仪，包括在此研究中使用的安道尔CCD摄谱仪，波长校准过程在软件自动化。 <ol><li>强度的测定石英卤素灯的3,200ķ操作的灯，它接近于3,200ķBLA的已知光谱曲线谱比较校准光谱仪CK的身体。 </li></ol></li><li>预对准激发和发射光学器件，以确保激发光撞击在环和来自样品的发射的光的样品被收集并且在对CCD-摄谱仪的入口狭缝撞击。 注意:这两步机械过程。第一步是在光学的初始粗对准以获得发射的光到所述光谱仪的入射狭缝，使得一个发光信号由CCD检测到。第二步骤包括由激发光束，所述杜瓦，样品本身在循环，和发射收集光学元件的位置的系统的调整的样本的发光信号强度的仔细的优化。 </li><li>一旦光学对准和优化为4.3所述，测量样本的一个77K的液态氮参考发光光谱。对于该光谱，确保了在铜线环路冷冻薄膜样品中，第ermocouple相邻环路，杜瓦，激发光束和发射收集光学装置都在其最终的优化位置和样品是完全覆盖用液氮。 </li><li>设置热电偶的参比端与包裹在聚四氟乙烯带，就像样品交界处的电线木榫。这保护了参比端，并防止不良导线接触。 </li><li>验证该0℃的参考热电偶结点浸渍在水 - 冰雪泥。检查参比端频繁冰的水平。建立参比端的小广口实验室杜瓦，以降低其冰融化的速度。 </li><li>在检查对文学电压77 K时测得的热电偶电压在一个T型铜 - 康铜热电偶上市77K的。这两个热电偶电压应该是非常接近达成协议。进行适当的修正压力（ 例如 ，使用克劳修斯-克拉珀龙EQUA灰）如果大气压力小于1个大气压。 </li><li>允许液体氮慢慢蒸发掉。 注:样品的温度缓慢上升（历时几个小时）作为样品和液氮水平液氮沸水的结果缓慢增加之间的距离h。该数字电压表来响应该温度上升，从而提供精确的温度测量，因为样品热电偶结完全浸入冷的氮蒸气。 </li><li>暂时打开或解除激励光并用CCD的光谱仪使用制造商的协议来获得的发射光谱。光谱数据采集过程中应优化，只需要几秒钟。 <ol><li>关闭或重新块激发光只是只要数据采集过程完成以最小化不想要的激发光诱导样本光化学和/或激发光产生的错误诱导样品加热。 </li><li>记录在光谱数据采集间隔的开始和结束的热电偶电压。热电偶电压不应该明显地（内0.0-0.1ķ 即相关联的温度变化ΔT），在其上的光谱数据是在给定温度下获得的非常短的时间间隔（〜1-10秒）期间改变。通过引用一个T型铜 - 康铜温度与电压表转换电压读数的温度。 </li></ol></li><li>让液氮汽化以继续，测量另一个频谱当样品温度提高了约5 K.使这些温度依赖性光谱测量过程中在光学，电子或激发光强度没有变化。 注:根据光学杜瓦的质量，这个热身过程需要5到每〜5K的时间间隔15分钟。 </li><li>强度，正确的CCD光谱数据集减去关闭暗帧的强度（ 即 ，由当入口狭缝被阻塞在CCD记录的光谱强度），占波长相关的光谱响应。减去每个样品谱基线暗计数强度计数的水平。 <ol><li>正确用于通过测量已知强度，诸如3200 K的钨卤标准灯的标准的强度，并且使用公知的强度比来测量的强度的波长依赖性的光谱响应以校正样品的测定强度在每个波长。 </li></ol></li></ol>

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The authors have nothing to disclose.

低温冷光光谱该设备的发展产生了必然的。它是含有感兴趣的发色团，并与氧气过饱和溶液可以加载，冷冻，并定位成用于光谱所有在其中样品温度定义良好，稳定，缓慢多变杜瓦/低温恒温器设计，可以是至关重要的。几乎所有的商业低温恒温器需要更多的时间与样品装入比这些实验的限制将使。同样重要的是有一个小型便携式杜瓦/低温恒温器，让轻便装样/在楼上的化学实验室其次是运输楼下?...

在极低温领域，发光分子的电子荧光光谱和荧光寿命的温度依赖性研究提供了丰富的关于从这些国家产生的这些分子的电子激发态和光化学和光物理现象的信息。克罗斯的开拓性随温度变化的光物理调查和同事在钌（II），铑（I），和铑（III）1,10-菲咯啉的配合物，2,2&#39;-联吡啶，和其它配体示出良好的随温度变化的光谱的内在动力阐明发射电子激发态的歧管的结构，对称性，能量学和化学的行为。1-6 然而，要做到随温度变化的低温光谱以及是不是一件小事。这是太容易根据光谱审讯样品不被热EQU<html> ilibrated从而跨越热梯度表现一个宽的温度范围。所得测量光谱是，实际上，排放在温度范围内的叠加。此外，即使在这个温度范围内的平均温度可能是从放置在或靠近于样品温度探头（ 例如 ，热电偶或电阻温度装置）的读出完全不同的。因此，要做到温度依赖性低温光谱正确地需要建立下试样温度是已知的实验条件下，稳定的，均匀的，并且，在时机成熟时，可调节的。这些条件可以与由CCD摄谱仪的极为有限的装置，激发源，光学杜瓦和热电偶下简单，直接的实验方案（参见图1）工作来实现。 "SRC ="/文件/ ftp_upload / 54267 / 54267fig1.jpg"/&gt; 图1.发光光谱仪设置为低温光谱如在此顶视图中所示的系统包括:（一）CCD探测器，（B）光谱仪，（三）入口狭缝和过滤器，（D）的发光收集光学（E）激光或弧光灯激励源，（F）的激发光，（G）熔融硅光学杜瓦在XYZ翻译安装，（H）热电偶样品交界处，（我）的样品，（J 。）热电偶参考结:0°C = 273.15ķ冰/水浴，（k）的数字电压表。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54267/54267fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 样品和错误平均样品温度不希望的热梯度几乎一定会导致如果一个样本的一侧被放置在与低温"冷手指"表面，而样品的另一侧的物理接触是在真空。最实用的方法，以确保完整的示例是在均一可测量温度T是完全沉浸样品和在温度T低温液体的温度探针（ 例如 ，液态氮或液态氦），或在温度T a低温蒸气（ 例如，冷氮气或冷氦蒸汽）。变温度低温恒温器实现通过平衡与电阻加热冷冻剂流量以达到所需的低温样品温度的恒温样品环境7-9的热交换气体可以使用，以确保样品的温度是均匀的。我们的想法是在与交换气体而这又是在与低温恒温器热平衡热平衡的样品。低温恒温器的设计已经出现，实现在各种温度下的样品的热平衡只是通过调节的液面上方的样品高度h制冷剂在存储杜瓦，10个样本被激发，并通过光纤电缆或透镜系统检测发光。在给定的样品/探针高度h，致冷剂蒸气温度为T（h）和该温度的增加为h的增加（ 即 ，杜瓦在蒸气提供了一个平滑的热梯度的dT / DH&gt; 0）。上述的制冷剂气体实际上变成液体交换气体。为H定位一个小样本和温度探头确保在T（h）上的样品的热平衡。以提高样品温度，h的增加。为了降低样品的温度，h的降低。这样的低温恒温器的温度下限为H = 0的液体冷冻剂的温度。此温度下限还可以通过降低压力可以降低。在一个大的存储杜瓦（ 例如 ，一个100升液氦杜瓦或10升液氮杜瓦），冷却剂蒸发RA德是一系列光谱测量由此允许在样品高度h的液态制冷剂上方的调整成为样品温度的已知的调整的时间帧期间可以忽略不计。 在这个实验室从过渡金属配合物的发光的氧诱导淬火的温度依赖性的光谱研究导致了小的熔融二氧化硅的自适应光学杜瓦用于与在77-300 K范围之外液氮变温分光调查（见图2）。 <img alt="图2" fo:display-align="middle" src="/files/ftp_upload/54267/54267fig2.jpg" /> 图2.熔融石英光纤杜瓦设置为变温（77-300 K）低温发光光谱，光杜瓦下图说明了完整的变温系统。 （一）。液氮，（二）TRansparent（4.0厘米）杜瓦，（C）铜样品环，（四）热电偶结，（E）镀银杜瓦地区unsilvered光纤接入区，（F）鳄鱼夹，（G）木榫， （H）距离液氮水平和样品之间，（我）内部和外部杜瓦壁之间抽真空区，（J）软木塞，（K）氮气排气孔（L。）热电偶丝，（M 。）热电偶导线分开并固定带PTFE密封带木榫。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54267/54267fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 熔融二氧化硅是不发光，并提供高的光传输从近紫外，透过可见光，并输出到近红外（〜200-2,000纳米）。此前10，其中液态制冷剂上面的示例高度决定样品温度描述的大型存储系统杜瓦手术的基本概念，成功地延续上小规模使用这种小型光学杜瓦。然而，代替机械调整上述静止液体冷冻剂水平高度的样品高度h来调节样品温度T，相对于该杜瓦本身样本位置是固定的（ 图2）。缓慢沸水在光学杜瓦液氮的历时几个小时逐渐增加下落的液体氮水平（ 图3）以上的样品的距离h。 <img alt="图3" fo:display-align="middle" src="/files/ftp_upload/54267/54267fig3.jpg" /> 图3. 关闭了光学杜瓦的样本区域 。温度:样品浸入液氮中以高度h 0，得到-T 0 = 77 k;样品浸在冷氮蒸气的水平H 1 &lt;H 2 &lt;H 3 &lt;/ SUB&gt;液氮水平之上给予样品温度牛逼1 &lt;T 2 &lt;T 3。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54267/54267fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 这允许在样品温度随时间（长达数小时）缓慢的，受控的增加，同时保持两个样品和温度探头，一铜 - 康铜热电偶结，在与冷的氮蒸气的热平衡。发光光谱跨越可见光和近红外区域中每光谱只有几毫秒（或每秒几百谱）用CCD配备发光光谱其间样品温度几乎是恒定（ΔT&lt;0.1 K）的每个光谱获取数据集收购。在温度〜5K的光谱之间的典型等待时间间隔是〜5-15分钟。 Moreo版本，样品加热或通过激发光的样品的光化学降解的影响被最小化，因为激发光仅被允许撞击样品每光谱几秒钟。在简单性，可移植性和样品加载速度的利益，光纤电缆未使用。样品直接与无论是从一个汞弧灯的365nm的频带或405纳米线的二极管激光器的激发。从样品中发出的光从在杜瓦发光样品通过聚光透镜由聚焦透镜拾取直接撞击在摄谱仪的入口狭缝。被调查的钌，铑配合物的样品中氧饱和溶液光谱研究〜10 -3 -10 -4 M溶质的薄膜制成。的解决方案是由在小铜导线环的表面张力保持（〜在形成3毫米环直径选自0.0150。直径的铜线）。然后，热电偶接头高度adjusteð所以它等于样品高度（h 热电偶 = H 样品 ），并在靠近试样回路， 如图2和3。温度通过测量热电偶样品结并用高阻抗数字电压表和比较的温度下对电压表为一个T型铜/康铜热电偶0℃的水/冰热电偶参考结之间的电压差来确定。在导线环捕获的薄膜样品溶液是闪光灯在光学杜瓦液氮通过快速浸入冷冻。然后将冷冻的解决方案允许随时间非常缓慢升温，其余冻结，而其发光光谱是作为温度的函数测量的。发光强度与温度数据根据以下模型进行分析。 在温度的样品的总发光强度&lt;青霉&gt; T被给定为从氧化和unoxygenated络合物所引起的强度之和: <img alt="公式（2）" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq2.jpg" /> 。 （1） 从配合物没有氧气的发光强度假定为与温度无关。然而，氧化配合物的发光强度会随着温度的升高，由于氧气淬指数递减。这可以通过以下形式的Arrhenius方程来描述<img alt="公式3" fo:vertical-align="middle" src="/files/ftp_upload/54267/54267eq3.jpg" /> 。 （2） 在等式（2）中，E a是淬火活化能，k是玻尔兹曼常数。最大发光强度将在低温区观察到（参见图5），其中t<html>这里是热能不足以克服淬火活化屏障（ 即，从复合物到氧气能量转移）。如果式（2）代入公式（1），则表达式<img alt="公式5" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq5.jpg" /> （3） 被获得。在等式（3）， <img alt="公式6" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq6.jpg" />是从在低温区域的氧化复合物所引起的强度。方程的重排（3）收益率<img alt="公式7" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq7.jpg" /> 。 （4） 取方程的两边的自然对数（4）给出的表达7eq8.jpg"/&gt;。（5） 从等式（5）可以明显看出的图<img alt="公式9" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq9.jpg" />与<img alt="10式" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq10.jpg" />会给用直线<img alt="11式" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq11.jpg" /> ，从该发光猝灭活化能得到<img alt="12式" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq12.jpg" /> 。 （6）

<table><tbody><tr><td>Diode laser 405 nm</td><td>Generic</td><td></td><td>Generic pencil-type laser pointer for luminescence excitation: 5 mW at 405&amp;nbsp;nm</td></tr><tr><td>Quartz optical dewar</td><td>Custom fabrication</td><td></td><td>3.5 cm id. x 25.0 cm length with 4.5 cm unsilvered region for optical access</td></tr><tr><td>Programmable 5 1/2 digit DMM</td><td>Keithley</td><td>Model 192</td><td>High impedence DMM for reading thermocouple voltages</td></tr><tr><td>Copper thermocouple wire</td><td>Omega Engineering</td><td>SPCP-010</td><td>0.010 in. diameter bare copper thermocouple wire</td></tr><tr><td>Constantan thermocouple wire</td><td>Omega Engineering</td><td>SPCC-010</td><td>0.010 in. diameter bare Constantan (copper/nickel) thermocouple wire</td></tr><tr><td>Polychromator/Spectrograph</td><td>Jarrell-Ash</td><td>82-415</td><td>0.25 m Ebert monochromator with back slit assembly removed to enable operation as a polychromator</td></tr><tr><td>CCD camera</td><td>Andor</td><td>DV-401-UV</td><td>Thermoelectrically cooled (-35 &amp;#176;C) CCD camera for detecting emitted light</td></tr><tr><td>Copper wire for sample loop</td><td>Generic</td><td></td><td>0.0150 in. diameter bare copper wire for sample loop</td></tr></tbody></table>

a simple dewar/cryostat for thermally equilibrating samples at known temperatures for accurate cryogenic luminescence measurements

根据一个小的熔融二氧化硅光学杜瓦，热电偶组件的简单液氮杜瓦/低温恒温器装置，以及一个CCD摄谱仪的设计和操作进行说明。指此杜瓦/低温恒温器被设计的实验需要快速样品装载，快速采样冷冻，样品的快速对准，准确和稳定的样品的温度，和小尺寸和杜瓦/低温恒温器低温单元的便携性。当与CCD摄谱仪的快速数据采集率耦合，这杜瓦/低温恒温器是能够在一系列在77-300 K范围之外已知的，稳定的温度下的支撑上的发光样品低温发光光谱测量。发光在铑（Ⅲ）的过渡金属配合物中的氧淬灭的温度依赖性研究是作为调查可能与该杜瓦/低温恒温器的类型的一个例子。在该装置的情况下，用于低温spectros稳定的温度拷贝意味着热与任一液态氮或气态氮在已知可测量温度是由CCD的分光测定的短时间尺度（〜1-10秒）期间不发生变化（ΔT&lt;0.1 K）的平衡的发光试料。杜瓦/低温恒温器的工作原理是取正的热梯度的dT / DH以上在杜瓦其中h是液氮水平以上的样品的高度液氮水平的发展的优点。在几个小时中h的缓慢增加，在样品温度T超过这个时间期间随之缓慢增加液氮结果的缓慢蒸发。一个迅速获得的发射光谱有效地捕获在一个恒定的，热平衡温度样品。

在上述装置中的发光化合物三 （4,7-二甲基-1,10-菲咯啉） ​​合铑的77-200数区域依赖于温度的发光猝灭研究中获得的代表性结果（III）的[Rh（4- ，7-我2 -1,10-phen）的3] 3+，溶解在氧饱和的甘油在表1中列出并在图4,5和 7作图。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><t...

Watch this Scientific Journal Video about A Simple Dewar/Cryostat for Thermally Equilibrating Samples at Known Temperatures for Accurate Cryogenic Luminescence Measurements at JoVE.com

A Simple Dewar/Cryostat for Thermally Equilibrating Samples at Known Temperatures for Accurate Cryogenic Luminescence Measurements

A simple liquid nitrogen Dewar/cryostat apparatus comprised of a small fused silica optical Dewar, a thermocouple, and a charge-coupled device (CCD) spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, freezing, and alignment, accurate and stable sample temperatures, and small size/portability.

一个简单的杜瓦瓶/低温恒温器的热的样品配平在为准确低温发光测量已知温度

The design and operation of a simple liquid nitrogen Dewar/cryostat apparatus based upon a small fused silica optical Dewar, a thermocouple assembly, and ...

a-simple-dewarcryostat-for-thermally-equilibrating-samples-at-known

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

9.5K 次观看.  Concordia University. A simple liquid nitrogen Dewar/cryostat apparatus comprised of a small fused silica optical Dewar, a thermocouple, and a charge-coupled device (CCD) spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, freezing, and alignment, accurate and stable sample temperatures, and small size/portability.

视频: A Simple Dewar/Cryostat for Thermally Equilibrating Samples at Known Temperatures for Accurate Cryogenic Luminescence Measurements

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

低成本的低温光学显微镜舞台制作相关的光/电子显微镜

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

科研

生物工程

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

Measuring the Densities of Aqueous Glasses at Cryogenic Temperatures

We demonstrate a method for determining the vitreous phase cryogenic temperature densities of aqueous mixtures, and other samples that require rapid cooling, to prepare the desired cryogenic temperature phase. Microliter to picoliter size drops are cooled by projection into a liquid nitrogen-argon (N2-Ar) mixture. The cryogenic temperature phase of the drop is evaluated using a visual assay that correlates with X-ray diffraction measurements. The density of the liquid N2-Ar mixture is adjusted by adding N2 or Ar until the drop becomes neutrally buoyant. The density of this mixture and thus of the drop is determined using a test mass and Archimedes principle. With appropriate care in drop preparation, management of gas above the liquid cryogen mixture to minimize icing, and regular mixing of the cryogenic mixture to prevent density stratification and phase separation, densities accurate to &#60;0.5% of drops as small as 50 pL can readily be determined. Measurements on aqueous cryoprotectant mixtures provide insight into cryoprotectant action, and provide quantitative data to facilitate thermal contraction matching in biological cryopreservation.

A protocol for determining the vitreous phase densities of micro- to pico-liter size drops of aqueous mixtures at cryogenic temperatures is described.

在低温下测量水性玻璃的密度

We demonstrate a method for determining the vitreous phase cryogenic temperature densities of aqueous mixtures, and other samples that require rapid ...

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 &#197;. 
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. 
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

金刚石光源下光束线I23真空长波长晶体学的样品制备和转移实验方案

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

生物化学

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

使用高分辨率X射线吸收光谱在低温下制备生物样品以进行形态分析

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...

化学

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native conditions, suspended in vitreous ice. This technique allows for the high-resolution visualization of proteins and other biomolecules without the need for crystallization, offering significant insights into their function and mechanism. Recent advancements in single-particle analysis, coupled with improved computational data processing, have made cryo-EM an indispensable tool in modern structural biology. Despite its growing adoption, cryo-EM faces persistent challenges that can limit its effectiveness, particularly uneven particle distribution. This issue often leads to poor resolution and reduced accuracy in reconstructed protein structures. This article outlines a simple, practical approach to address this challenge, using the small heat-shock protein from Methanocaldococcus jannaschii (MjsHSP16.5) as an example. The method optimizes sample preparation to minimize preferential adsorption, ensuring more homogeneous particle distribution and higher-quality protein cryo-EM structures. This technique offers valuable guidance for researchers aiming to overcome similar challenges in structural studies.

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

优化低温电子显微镜的样品制备

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

低温电子显微镜样品制备借助于聚焦离子束

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

生物学

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

低温样品加载到魔法角度旋转核磁共振光谱仪中，可保持细胞生存能力

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. 
It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

This protocol demonstrates how to image biological cryo-preserved samples using cryo-structured illumination microscopy. We demonstrate the methodology by imaging the cytoskeleton of U2OS cells.

低温结构照明微观数据收集来自低温保存细胞

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than ...

Regulating Temperature in the Lab: Preserving Samples Using Cold

Preservation of laboratory samples, specimens, and reagents using extreme cold is routinely performed in biomedical research labs. This video will discuss some of the methods for keeping laboratory samples cold and will explain the correct cooling method to use for each experimental requirement.
For example, cooling agents, such as ice and dry ice, are typically used when keeping samples cold during experiments. This video discusses the physical properties of the most commonly used cooling agents, as well as safety precautions for working with them.
When it comes to keeping samples cold in between experiments, cooling equipment, including laboratory grade refrigerators and freezers can be used to preserve samples for extended period of time. Also discussed in this video are types of samples and reagents that can be stored in the commonly-available laboratory cooling equipment.
Finally, the concept of cryopreservation is introduced as a process through which tissues, cells, and biomolecules are cooled to sub-zero temperatures, thereby effectively stopping all sample-degrading biological activity. Several methods of cryopreservation are discussed that minimize or eliminate the formation of damaging ice crystals.

Preserving Samples Using Cold: Cooling Agents and Instruments, and Cryopreservation

实验室中的温度控制:冷却保存样品

Preservation of laboratory samples, specimens, and reagents using extreme cold is routinely performed in biomedical research labs.  This video will ...