本研究从QNano项目http://www.qnano-ri.eu这是由欧洲共同体研究基础设施下的容量FP7计划（批准号：INFRA-2010-262163）资金的支持。 我们也感谢研究委员会FORMAS提供财政支持。

注：在本协议中给出的所有参数都是有效的在这里指出的仪器和模型。其中一些参数（带*的文本）可能会有所不同，如果其他制造商或型号使用。 1，启动的FIB / SEM的 <ol><li>安装定制冷nanomanipulator（NM）尖不附加铜​​辫子的anticontaminator（AC）。代替确保发辫被连接到网管的其余部分上面的绝缘点，以防止充电过程中向上锐化步骤（1.2）。 <ol><li>关闭扫描电镜室，泵高真空和图像网管技巧。 </li><li>如果针尖是钝的，弯曲的，或通过先前的使用受到污染，削通过离子束的方法：选择多边形铣削图案沿着尖端的侧面，使研磨后，针尖将逐渐下降到1微米或更小。 </li><li>一旦该尖端部的两侧被研磨去，由90手动旋转°整个NM杆从扫描电镜室外面。 </li><li>调节多边形铣削模式相适应的旋转头，并从不同的角度重复铣削。 </li></ol></li><li>一旦前端已削尖小于1微米，收回网管和插入气体注入系统（GIS）的针;重新定位针是上面的​​工作距离（而不是通常的175微米）约1mm。 </li><li>如果使用的是铂前体，改变其工作温度为24-26℃（而不是通常的40°C）。需要这些步骤铂低温沉积13。 </li><li>打开扫描电镜室，并通过安装在冷冻样品台和交流准备的FIB / SEM的低温状态。 </li><li>网管切换到插入位置，并连接它的铜辫子交流。确保不小心碰网管技巧。该系统与冷却插入，以确保的铜编织的灵活性在低温下的损失不会妨碍网管招网管换货。 </li><li>吹扫管与干燥氮气冷却几分钟。 </li><li>泵的低温制备室和主样品室高真空。 </li><li>添加液氮杜瓦瓶的冷却两院。等待，直到达到所需的温度。 </li></ol> 2，样品冷冻 <ol><li><ol><li>将两片TEM网格上的扫描电镜转让持有的FIB样品。通过加强与螺丝刀相应的螺丝固定。 </li><li>装载样品存根适当的样品并添加试样的部分。取决于样品的种类，试样可以是固定的与深冷胶或用夹子。使用尽可能小的值，以确保最佳的冷冻。 </li><li>扫描电镜传送架安装到真空传输设备（VTD）。 </li></ol></li><li><ol><li>添加液氮进入减水剂站，抽空来获得氮气的雪泥。 </li><li>打开SLushing站和切入冻结SEM转让持有人。再次抽空至沸腾完成并获得烂泥了。但应注意的是，乙烷或丙烷或搪塑高压冷冻更适合的技术来获得样品的玻璃化。 </li></ol></li><li><ol><li>扫描电镜转印夹持器缩回到VTD的真空室和密封。 </li><li>发泄减水剂站和低温制备室气闸。 </li><li>匹配VTD密封的低温制备室和泵的气闸。 </li><li>当一个良好的真空水平达到，搞的气闸针打开VTD和外气闸舱的密封;插入扫描电镜转让持有人。有在显示的溅射和压裂样品位置的准备室的滑动触点标记。 </li></ol></li><li>如果有必要，可将样品：断裂与冷刀;通过设置一个较高的温度（通常为-100升华6，C）;受冷溅镀的方式涂上金/钯或铂（300 V，10毫安，60秒为2-3纳米金/钯帽）*。升华不应被用于玻璃化的样品，以避免其重结晶。 </li><li><ol><li>使用冷刀切开TEM网格插槽的保护盖。 </li><li>带来的冷阶段，在样品腔室，所述接收高度（16毫米*）。 </li><li>关闭在FIB / SEM的HT和打开内气闸。 </li><li>使用VTD的扫描电镜夹持器传输到样品室中。调暗灯光在室内可能有助于该步骤。 </li><li>一旦扫描电镜夹持器是在寒冷的阶段，通过按压并旋转脱开VTD。 </li><li>收回VTD杆一路到VTD真空室，并关闭气闸内，外气闸和VTD密封。外气闸现在可以排到取出VTD密封。不需要这最后一步，但建议作为VTD杆可以由事故很容易脱落，这可能导致损坏到VTD或气闸。 </li></ol></li></ol> 3。离子铣削 <ol><li>打开上两列的高度紧张，并设置适当的成像参数（加速电压：10千伏的电子束，30千伏的离子束，光斑直径：3，工作距离：5mm时，离子束电流：10-100 pA的成像，1-3 nA的铣削）*。 </li><li>使用电子束来找到感兴趣的特征以及获取的图像文件的样本的薄片的萃取前的状态。 </li><li>一旦感兴趣区（ROI）提取区域已被确定，通过离子束图案标记它，除非样品本身的地形使得即使在铂沉积容易识别的投资回报率。为提高精确度，可以使用由彼得森等人 14所述的方法中的标记要深，宽和足够远是被覆盖的低温沉积的Pt（这是不SEL后仍可见ective和将覆盖在样品表面的几个毫米2）。 </li><li>加热该前体气体至24-26℃，并插入在GIS针的样品表面上方约1毫米的高度（参见步骤1.3）。 </li><li>而用电子束成像，打开了几秒钟的气体阀。的冷冻铂沉积速率为100-500毫微米/秒或更多，这取决于在GIS针，样品的粗糙度和在用户的系统中的距离。明智的做法是运行一些测试的供词，以确定最佳参数。 </li><li>原料冷冻铂沉积是非常粗糙和不均匀。通过使用一个1000 pA的离子束在低倍率（例如2,000倍）治愈存款除以投资回报率。不像低温沉积，这种固化是现场的选择性，并应仅在ROI执行的。该第一低温沉积的目的是为了保护该样本的表面从离子束的损坏，并在离子减薄13，以减少结壳。 </li><li>样品倾斜52°，使得表面是垂直于离子束。磨客场2梯田战壕上的投资回报率的两侧。典型的尺寸为沟槽是20-30微米的平行于薄片将被提取，10-15微米的垂直方向（Y）和具有可变方向（X）倾斜的深度（Z），与​​最深点贴近的投资回报率。坡度应为45-55°。在一些乐器，梯田战壕只能研磨，顶部的最深点。在这种情况下，磨一个投资回报率下，然后将图像旋转180°和工厂，第二个在另一边。铣削的深度可以，如果该材料的溅射速率是已知的被选择。对于大多数冷冻水合样品，冰的溅射速率可以使用7。 </li><li>样品向后倾斜至0℃，并使用离子束切去薄片的两侧和底部，确保切割痕迹通过整个薄片（他们应该离开铣痕迹的山坡梯田磨在上一步骤ED）。只留下2薄片连接到样品的其余部分小的桥。 </li><li>将GIS针（这可能稍​​有移位样品）。操纵NM直至其尖端与所述薄片的物理接触，最好是在一侧。确保网管不会妨碍两个小连接桥的离子束视图。 </li><li><ol><li>打开GIS阀数秒并通过连续成像的电子束监视低温沉积。 </li><li>当Pt的额外1-2微米的层一直低温沉积，关闭阀门。 </li><li>治愈铂（见步骤2.6）只能在周围的地步NM是与薄片接触的几微米。 </li><li>使用高离子束流切割薄板自由。两个连接桥应被研磨去，以及可能形成的薄片与样品的其余部分之间的新接触点的任何过量的Pt。不要收回GIS针呢。 </li></ol></l我&gt; <li>小心机动网管从战壕中提取薄片，将其移动至少500微米以上的样品表面。只有在这一步，收回的GIS针。 </li><li>降低样品台上几个毫米并移动它，直到TEM网格中的一个处于图。移动对电网的连接区域进入工作位置，并插入GIS针。 </li><li><ol><li>小心机动网管带来附加的薄片与在TEM网格附件区物理接触。应该有鳞片，在TEM网格和网管之间没有压力或紧张。 </li><li>开了几秒钟，并冷冻存款的Pt的额外1-2微米的层的气体阀。 </li><li>固化的Pt（参见步骤2.6）只有在周围薄片和TEM网格之间的接触点的几个微米。 </li></ol></li><li>使用高离子束流切割薄板自由网管的。这可以通过研磨或相差网管尖或样品的一侧来完成。在T他第一种情况下，针尖将必须再次激化了在下一次使用之前，如在步骤1.2中描述。 </li><li>可选：在这个阶段，可以使用VTD采取SEM转让持有人，其存储O / N在充满液氮杜瓦。这种转移和O / N存储很可能造成薄片的表面上形成冰，如果冰晶已经存在和/或，如果液氮被暴露于潮湿的空气中;但这种污染会通过在一个相对短的时间的下一个步骤中被除去。正如前面的步骤可能会采取几个小时才能完成，也可能是适当的话，因为下面的步骤之后，这种存储O / N是不推荐的，因为就没有办法通过升华除去冰除污染（这如果样品的玻璃化要保持不能被执行）。 </li><li>样品倾斜至52°，并使用离子束薄到透明电子7。建议在开始走高，roug她的电子束电流以除去大部分并进行精细抛光的表面具有较低的离子束电流，最终也降低加速电压。薄片的最终厚度应在100-200千伏TEM 100-200纳米或更低的超微结构分析或在300千伏的TEM高达500纳米的成像，这取决于样品的组合物。在减薄，样品的内部应力可能会导致薄片卷曲或弯曲。在这种情况下，薄的区域应被限制。发生这种情况，例如在图11和12。 </li></ol> 4，低温转移到TEM <ol><li><ol><li>同花顺干燥氮气的低温中转站了几分钟。 </li><li>添加液氮的TEM交流的杜瓦和冷冻电中转站。 </li><li>将冷冻透射电镜转让持有的冷冻中转站的相应插槽中，并填补其杜瓦为好。等到每个比赛onent已达到所需温度时（约15分钟）。如果可能的话，在冷冻透射电镜转让持有的控制器应连接在传输过程中监测温度。要认识到，在TEM架（其中所述温度传感器是位于）的前端将与低温转运站接触，因此，将冷却比TEM支架的其余部分更快的是很重要的。因此，建议在需要对整个冷冻转让TEM支架降温的时间被预先测量，并且该系统允许热化，至少要的时间量。 </li><li>补的低温杯用液氮和沉浸在它：在TEM样品夹持工具，螺丝刀和镊子，以便其尖端冷却到所需的温度。所有工具应适当绝缘的另一端，以便不引起冷灼伤到操作者的手。 </li></ol></li><li>匹配VTD到外气闸。把冷雄鹿e来输送高度（16㎜*）。关闭高压。 </li><li><ol><li>打开VTD密封，外气闸和内气闸。 </li><li>使用VTD杆推并顺时针旋转锁定到SEM转让持有人。 </li><li>扫描电镜转印夹持器缩回到冷冻制备室。 </li><li>使用冷刀关闭TEM网格的保护盖。这是必要的，以减少传输过程中可能的冰污染。 </li><li>使用VTD杆的样品移动到VTD的真空室。 </li><li>关闭气闸和密封。 </li></ol></li><li>泄了气闸外，拆下VTD。匹配VTD到冷冻中转站的扫描电镜口。同时用干燥氮气冲洗，使用站上的脚打开VTD的密封和SEM转让持有滑入冷冻中转站的杜瓦。 </li><li><ol><li>添加足够的液体氮，以使冷冻传送站的电平足够高时只是淹没样品。 </li><li>使用先前冷却螺丝刀打开盖子之一，松开相应的螺丝是保持TEM网格到位。 </li><li>使用先前冷却镊子挑TEM网格并把它放入透射电镜持有人。 </li><li>使用冷却hexring紧固TEM网格上的TEM持有人。 </li><li>关闭冷冻透射电镜转让持有的快门。将样品转移步骤是关键的和用氮气可能会阻碍减少小TEM样品的可见性。 </li><li>断开冷冻传送站从泵送系统和运输它附近的透射电子显微镜，用低温TEM传输支架的加热器控制器一起。 </li><li>启动TEM的后盾线泵的TEM气闸的涡轮分子泵。 </li><li>设置在TEM样品台到-70°倾斜*。 </li><li>设定最短泵送时间为气闸（30-60秒），用干燥氮气吹扫的仅一个周期。 </LI&gt; </ol></li><li>确保在低温传输TEM持有人的保护盖是关闭的。从冷冻中转站取出TEM持有人，将其插入倾斜的测角仪（液氮将洒出的TEM持有人杜瓦瓶）。泵送循环开始。一旦循环完成后，将测角器以向后倾斜至0℃，并在同一时间，保持在TEM支架，使得它不与测角器旋转。完全插入它的TEM里面。在此步骤中，低温转印TEM支架应该连接到其加热器控制器来监控温度。到样品架插入TEM的程序可以在不同的电信设备制造商之间变化。因此，建议联系TEM制造商，以获得相应的程序。 </li><li>重新加注冷冻透射电镜转让持有杜瓦。等待在TEM的真空，以达到可接受的水平。 </li></ol>

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作者什么都没有透露。

此协议是一个相当简单的适应于在室温材料科学中使用的标准FIB / TEM样品制备的低温下。该方法可产生游离的机械变形和刀痕（切片技术的主要缺点）的透射电子显微镜的样品，但如果样品表面是不均匀的，可能会出现结壳。这可以减少通过低温沉积盖层（在本工作使用Pt），固化的，直到它是光滑和没有特征13。样品具有非常不同硬度的组件可以被制备为良好，没有他们将制备过程中在应力下破裂的风险。内部应力仍可能导致薄的薄片弯曲或卷曲，在这种情况下，段的大小必须减小。相对于其他方法的缺点是改变的生物结构由于暴露于离子束和样品中的离子可以植入的可能性。这些缺点也出现在RT样本prepar通报BULLETIN在材料科学15。它们可以通过完成与在最低的加速电压为离子（500-1,000 V）的最终抛光步骤中的变​​薄会降低。这很温柔的抛光步骤会从鳞片去除损伤层。 由于低温沉积（步骤3.5，3.10和3.13）的性质，样品的大部分地区将被覆盖，从而阻碍了原始表面的看法。这可能使得难以跟踪ROI的，除非多个标记被用作在步骤3.3建议。 在步骤4.5和4.7来与空气接触的薄鳞片风险。这必须避免，因为它会导致空气中的水分而形成冰晶的样品的表面上，可能是为了掩盖重要的特征点。这些步骤应尽可能快地进行，但在同一时间在转移过程中一个误操作有可能导致样品的损失它自我。建议用户通过使用空TEM网格上之前，在实际样品试图实行这些步骤。 在材料科学，FIB表仪器已成为一个十年的商业化中的TEM样品制备的主要方法。因为它可以在几乎任何样品中使用，它消除了需要调整的制备技术，以样品的种类。我们坚信同样可能发生在低温下，由于这里详述的过程。其应用到更大的样本仍然受到以冷冻保存他们在一个玻璃化状态的能力，但如暴跌冻结或高压冷冻3,5技术可以被证明是最佳的解决这一问题。

在这个协议中一个cryo-FIB/SEM是用于生产的TEM样品从样品中的特定区域，通过SEM分析以高精度预先确定的。电子显微镜（扫描或透射）分析生物样品是用于研究和诊断的常规技术。 SEM是相当快速和容易使用和解释，但信息只从样品表面，并与在1.5纳米范围内的分辨率获得。 TEM具有更高的分辨率，但更难以实现，图像分析是较复杂的和而得到的体信息，样品必须减薄至电子透明度（小于约500纳米厚以下）。另外一个问题是，这些工具的真空要求很少通过含水样品的耐受性。在大多数情况下，生物样品必须通过化学固定（代水用，例如，聚合物）或干燥。在这两种情况下，显著改变试样的形态和结构都可能发生。低温透射电镜制备水 ​​合样品的诱导最少的化学变化和它产生的样本尽可能接近其天然状态，特别是如果冰的玻璃化，得到1-6。 的FIB被广泛用于制备TEM样品，它的许多优点7。仅举几例：在接近垂直入射采用高能离子最大限度地减少材料相关的微分磨粉率的影响;从散装样品提取的区域可以与亚微米精度来选择;极少量的材料被提取。一些最新的技术发展已经使用FIB也为TEM样品制备在低温下2,8-10成为可能。有超过主要用于软物质的样品，例如缺乏切片薄片的机械变形的冷冻超薄切片11,12的传统制备方法的诸多优势，没有刀痕，并有可能准备与软/硬接口或部件复合材料样品。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Strata DB 235&#160;</td>
			<td style="width:71px;">FEI</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">FIB/SEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omniprobe 100&#160;</td>
			<td colspan="2">Oxford Instruments&#160;</td>
			<td>nanomanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alto 2500</td>
			<td style="width:71px;">Gatan</td>
			<td style="width:119px;"></td>
			<td>cryo preparation chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cryo-holder model 626</td>
			<td style="width:71px;">Gatan</td>
			<td style="width:119px;"></td>
			<td>cryo transfer TEM holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tecnai F30</td>
			<td style="width:71px;">FEI</td>
			<td style="width:119px;"></td>
			<td>TEM</td>
		</tr>
	</tbody>
</table>

cryo-electron microscopy specimen preparation by means of a focused ion beam

这里，我们提出用于制备黑曲霉孢子低温TEM样品的协议，但它可以很容易地适应任何数量的微生物或解决方案。我们利用一个定制的冷冻中转站和改进的低温扫描电镜制备室2。孢子从培养取，切入式冷冻在液氮雪泥和在低温SEM观察来选​​择感兴趣的区域。薄的薄片，然后使用FIB萃取，附着于TEM网格并随后减薄至电子透明度。网格转移至低温TEM支架和成TEM高分辨率研究。由于引入了冷却nanomanipulator尖和一个冷冻中转站，该协议是一个简单的适应经常使用的纤维蛋白原制剂的TEM样品的低温。因此，它具有需要少量的修改现有的工具，设置和程序的优点;但我很容易实现;它具有广泛的应用范围，原则上相同，对于低温TEM样品制备。一个限制是，它需要的标本中熟练运用的关键步骤，以避免或尽量减少污染。

在这项工作中，我们利用了：双束FIB / SEM配备了nanomanipulator和低温制备室;用低温传输持有人的TEM;原型冷冻中转站。在低温制备室的anticontaminator（AC）叶片和nanomanipulator（NM）的前端由加坦进行了修改。相对于一个标准的低温​​制备室中，AC叶片都较大，为网管尖端有较大的散热片。此外，交流配有夹子，用于连接铜辫子与网管尖热交换。的FIB / SEM的气动装置进行了修改，以允许NM是，甚至当试样腔室通风保持插入。但应注意，在该工作中所使用的参数最适合于上面列出的设备;可能需要这些参数与其它类型的设备中工作时进行调整。要使用此协议的工作，正常的预防措施低温，液态氮和真空系统应遵循。 该方法已经过测试，在不同类型的样品具有良好的效果，从溶液或含有纳米粒子的聚合物基体，以单细胞生物线虫。的各个步骤的程序的例子示于图1-12上A.曲霉孢子沾有四氧化锇和高锰酸钾。孢子是通过SEM第一成像（ 图1），以确定现场提取。在这种情况下，任何孢子的横截面是足够的，但它可以放置ROI提取与亚微米的精度，例如，切片的特定单元格在从细胞膜的特定距离。一旦所感兴趣的特征已被确定时，冷冻的Pt沉积的第一步骤的实现（ 图2），以保护样本从离子束铣削的损坏。将样品倾斜至52°进行的第一个步骤ÖF中的铣削（ 图3）：在薄片的两侧的两个沟溅射。然后将样品向后倾斜，并进一步研磨以仅留下2将其连接到大容量小的桥（ 图4）。将冷却的nanomanipulator被带入与薄片接触 ​​（ 图5）和另一低温沉积的Pt的焊料与它们在一起（ 图6）。小连接桥然后研磨相差和网管移动TEM网格的连接区域（ 图7），在那里它被焊接与最终低温沉积的Pt（ 图8）附近的薄片。网管然后从该薄片（ 图9），这是减薄至电子与离子束透明度（ 图10和11）分开。该薄片是最后转移到TEM（ 图12），其中高解析度成像，光谱，成像和其他技术的CAn为使用。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig1highres.jpg" src="/files/ftp_upload/51463/51463fig1.jpg" /> 图1：A的孢子冷冻SEM图像尼日尔 ，铂沉积之前。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig2highres.jpg" src="/files/ftp_upload/51463/51463fig2.jpg" /> 图2中 ，相同的区域在图1中的Pt的沉积之后，但在固化之前。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig3highres.jpg" src="/files/ftp_upload/51463/51463fig3.jpg" /> 相同的区域在图2的图3。冷冻SEM图像，倾斜52°，铂沉积和固化之后，随着沟槽铣削正在进行（见步骤3.7）。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig4highres.jpg" src="/files/ftp_upload/51463/51463fig4.jpg" /> 图4的薄片，准备提列。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig5highres.jpg" src="/files/ftp_upload/51463/51463fig5.jpg" /> 图5。冷nanomanipulator尖使得与薄片接触 ​​。 <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig6highres.jpg" src="/files/ftp_upload/51463/51463fig6.jpg" /> 图6。第二 ​​个铂低温沉积是用来焊接在一起nanomanipulator和薄板。 <i毫克ALT =“图7”FO：内容宽度=“5英寸”FO：SRC =“/ files/ftp_upload/51463/51463fig7highres.jpg”SRC =“/ files/ftp_upload/51463/51463fig7.jpg”/&gt; 图7。冷nanomanipulator用于薄片传送到TEM网格的附着面积。 <img alt="图8" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig8highres.jpg" src="/files/ftp_upload/51463/51463fig8.jpg" /> 图8。超低温沉积用于再次向薄片附着到TEM网格。 <img alt="图9" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig9highres.jpg" src="/files/ftp_upload/51463/51463fig9.jpg" /> 图9的薄片被切断游离的nanomanipulator的，它现在可以存储或减薄至电子透明度。 图10。变薄的中间步骤，在横截面可见几孢子。 <img alt="图11" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig11highres.jpg" src="/files/ftp_upload/51463/51463fig11.jpg" /> 图最终减薄后的试样的11的Cryo-SEM图像;其他大部分孢子只好被碾碎了，因为薄片已经开始卷曲。 <img alt="图12" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig12highres.jpg" src="/files/ftp_upload/51463/51463fig12.jpg" /> 图12。的薄片的复合冷冻透射电子显微镜照片。铝存根的一部分已被列入在薄片（黑色箭头）。

Watch this Scientific Journal Video about Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam at JoVE.com

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

低温电子显微镜，或扫描（SEM）或透射（TEM），被广泛地用于具有高的水含量1的生物样品或其它材料的特性。的SEM /聚焦离子束（FIB）来识别的样品中感兴趣的特征，并提取用于转移到低温TEM薄，电子透明薄片。

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 ...

cryo-electron-microscopy-specimen-preparation-means-focused-ion

Research

JoVE Journal

Biology

26.4K 次观看.  Uppsala University. 低温电子显微镜，或扫描（SEM）或透射（TEM），被广泛地用于具有高的水含量1的生物样品或其它材料的特性。的SEM /聚焦离子束（FIB）来识别的样品中感兴趣的特征，并提取用于转移到低温TEM薄，电子透明薄片。 

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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups. This technique uses HistoGel (Thermo Scientific) with conventional laboratory equipment.
The use of cell block sections is a valuable ancillary tool for evaluation of non-gynecologic cytology. They enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion. Most importantly, they allow for the evaluation of ancillary studies such as immunocytochemistry, in-situ hybridization tests (FISH/CISH) and in-situ polymerase chain reaction (PCR). Traditional cell block preparation techniques have mostly been applied to non-gynecologic cytology specimens, typically for body fluid effusions and fine needle aspiration biopsies.
Liquid based cervicovaginal specimens are relatively less cellular than their non-gynecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve. In addition, the histotechnologist sectioning the block cannot visualize the level at which the cells are at the highest concentration. Therefore, it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing. As a result, the area of the cell block with the cells of interest may be missed, either by cutting past or not cutting deep enough. Current protocol for Shidham's method addresses these issues. Although this protocol is standardized and reported for gynecologic liquid based cytology specimens, it can also be applied to non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc for improved quality of diagnostic material in cell block sections.

Shidham's method for preparation of cell blocks with AV-marker from cytology specimens containing individually scattered cells and small cell groups.

个别细胞散为主细胞学标本的细胞块的制备

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

考试<em&gt;果蝇</em幼虫气管终端细胞光镜

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

Do&#039;s and Don&#039;ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

该做什么和不该做什么低温电子显微镜的：入门样品制备和高质量的数据采集为大分子三维重建

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 &#181;m x 1,000 &#181;m) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of &#8804;5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROIs) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours.

Here, we present a protocol for efficiently combining serial block face and focused ion beam scanning electron microscopy to target an area of interest. This allows for efficient searching, in three dimensions, and locating rare events in a large field of view.

使用串行块面和聚焦的Ion光束扫描电子显微镜进行有针对性的研究

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.