我们感谢马普学会，人类前沿科学计划（HFSP）和勃林格殷格翰全宗的资助和支持。

1。材料准备<ol><li> FEP管的制备<ol><li>通过管冲洗液（在下文所述的步骤），并使用附有钝端套管（ 图2B）的注射器清洁FEP管（ 图2A）注1:使用手套，整个过程注2:我们建议切割管中的CA件。在长度1米和清洗那些并联。 <ol><li>首先冲洗管1 M氢氧化钠反复。然后将管转移到50ml离心管中填充用0.5M NaOH和ultrasonicate它10分钟。 </li><li>聚合物管转移到一个小盆地与双蒸2 O。冲洗管首先用双蒸2 O，然后用70％的乙醇。 </li><li>随后，FEP管，用70％EtOH中转移到一个新的离心管中。再次ultrasonicate此离心管10分钟。 </li></ol></li><li>切开洗净FEP管成片约3厘米（典型长度在斑马鱼的安装和成像使用），并在必要时抚平。存储清洗FEP管在新的50ml离心管中充满了双蒸2 O（ 图2C）。 </li></ol></li><li>的股票三卡因溶液的制备<ol><li>准备0.4％三卡因原液在97.9毫升双蒸2 O溶解400毫克三卡因（MS-222）后的粉末完全溶解后，用2.1毫升1M​​的Tris（pH值9.0），将pH调节至7.0。由轻保护的解决方案。店铺等分于-20℃下</li></ol></li><li> 3％甲基纤维素的制备<ol><li>制备3％的甲基纤维素溶液为如下所述的FEP管的内涂层。升温E3 11在玻璃瓶中，以约60-70℃，并加入甲基纤维素粉末的适当的量。添加搅拌棒搅拌，在4°C。一旦溶液冷却至30℃以下，加入三卡因溶液至最终Çoncentration 100毫克/升。保持在4℃下过夜搅拌。 </li></ol></li><li> 1.5％的制备及其在E3 0.1％琼脂糖<ol><li>溶解在E3中的玻璃烧瓶中定义的量的低熔点琼脂糖粉适量。在微波炉加热的混合物，并在一段时间曾经动摇它，直到琼脂糖溶液呈均匀，无任何遗留晶体。 </li><li>制备1ml等份的0.1％琼脂糖溶液在1.5ml反应管中。的等分试样可被储存在4℃。 </li><li>使用1.5％的溶液涂覆在塑料培养皿中。琼脂糖层的厚度应为约为2mm。后的琼脂糖固化，在该层的顶部加E3介质，以防止其干燥。该涂层菜可以储存在4°C </li></ol></li><li>斑马鱼的制备<ol><li>保持斑马鱼（ 斑马鱼 ）的成人和胚胎在28.5℃和处理它们按照既定的协议11,12。 </li><li>成立雌雄鱼在下午和使用分频器将它们分开。取出分隔第二天​​早晨时间鱼的交配。 </li><li>收集胚胎在充满E3盘中，用立体显微镜检查它们。 </li><li>在24 HPF（或者你感兴趣的发育阶段），选择斑马鱼胚胎的荧光表达和积极的因素转移到新菜，新鲜的E3。 </li><li>使用两个锋利的钳（ 图2D和2E）仔细dechorionate胚胎。使用第一个钳子抓住不放绒毛膜和第二镊子撕它打开，并把它关闭。 注1:用立体显微镜的帮助下执行此步骤。 </li></ol></li></ol> 2。多层安装<ol><li>涂FEP管<ol><li>附上一个钝端套管的注射器。小心地插入套管的约3毫米成的清洁一端切一块，FEP管（ 图2F和2G）注1:使用手套，避免管的任意弯曲或挤压。 </li><li>浸FEP管的自由端插入到3％的甲基纤维素和填充管与所述溶液。 </li><li>慢慢地通过推动注射器松开甲基纤维素。弃去溶液。 </li><li> 。使用E3 注1重复步骤2.1.2和2.1.3:将涂覆管需要被用于立即安装。 </li></ol></li><li>转移到斑马鱼的琼脂糖<ol><li>升温0.1％琼脂糖之一等分至70℃的加热块。涡流反应管短暂，并将其转移到一个加热块设定为38℃（ 图2H）。 </li><li>就拿反应管从散热块，让它冷却下来简要介绍。加三卡因对133-200毫克/升和涡流的最终浓度再（ 图2I）。 </li><li>选择一个dechorionated斑马鱼胚胎和transfe·R它在反应管与预热的0.1％琼脂糖用玻璃巴氏吸管（ 图2J和2K）注1:尝试传输尽可能少的额外E3越好。 </li></ol></li><li>安装斑马鱼<ol><li>占用胚胎与注射器连接FEP管（ 图2L）。该管应完全用0.1％琼脂糖充满，与位于靠近管的两端的胚胎，并与头朝筒的开口注1:请一定要采取一些琼脂糖占用鱼之前。 注2:上拉非常平缓的注射器，以避免气泡注3:保持所述管在水平方向上，以避免其内容（ 图2M及2N）的泄漏。 </li></ol></li><li>堵管 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51119/51119fig2highres.jpg"SRC ="/ files/ftp_upload/51119/51119fig2.jpg"/&gt; 图2。多层安装的斑马鱼胚胎A）氟化乙烯丙烯的准备。前B电缆卷筒（FEP）管）附加钝端套管的注射器。C）洗净，切FEP管D，24个高倍视野E）Dechorionation的融化的0.1％琼脂糖一个反应管内，同时将其传递到散热块斑马鱼胚胎F）装上管钝端套管G）与套管和管连接的注射器。 高）等分。 一）涡旋式用玻璃吸管dechorionated胚胎的琼脂糖J）吸收K）转移的胚胎进入熔融琼脂糖胚胎。 升）进邻近琼脂糖入FEP管M，N）的FEP管内的斑马鱼胚胎。 <stroNG&gt; O）切割标本和套管，P之间的管子）通过使用琼脂糖包被培养皿的堵管Q）胚胎的插入管，R）在金属支架的最终样品的制备中。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51119/51119fig2highres.jpg" target="_blank">点击这里查看大图。</a> <ol><li>用剃刀刀片切割的管销套管（ 图2O）。 </li><li>慢慢坚持通过在塑料培养皿堵塞管（ 图2P）1.5％琼脂糖的整个一层含鱼管的末端注1:避免泡沫的形成，并尝试通过举办获得直塞表面。管完全垂直于琼脂糖表面注2:旋转筒稍微得到一个不错的插件。 </li><li>保持在1.5ml反应管中装入的样品与E3，以避免干燥。样品为n的OW准备进行成像。成像前，检查插头（ 图2Q及2R）顶部的鱼是否是坐在右边。确保同时添加三卡因以相同的浓度在安装介质成像室内部的介质。 </li></ol></li></ol>

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Pharmacol. 33 (2), 313-317 (1975).</li><li>Preibisch, S., Saalfeld, S., Schindelin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Software+for+bead-based+registration+of+selective+plane+illumination+microscopy+data.">Software for bead-based registration of selective plane illumination microscopy data.</a> Nat. Methods. 7, 418-419 (2010).</li></ol>

作者什么都没有透露。

如光片显微先进的显微技术，使我们在数天的过程中具有高时空分辨率来记录图像数据，还需要样品安装技术来提高。我们提出多层安装的斑马鱼发育的轻质板材镜的详细协议。该方法实现起来非常简单，我们用它在我们的实验室每天。 该FEP管我们的安装策略的主要组成部分是由聚合物氟化乙烯丙烯（FEP）的管子。我们决定使用，主要是因为它的折射率1.338的指数，这是类似的水，因此非常适合用于光片用显微镜浸水的目标和要求含水介质样品FEP。用0.8毫米内径和0.4壁厚合适的显影斑马鱼非常良好的管，可以用适合于1.6毫米典型样品架被用来毛细血管，是我们对所提出的安装方法的第一选择。我们完全在我们的家庭建造的设置，也可以在商业系统"Lightsheet Z.1"卡尔蔡司显微镜测试了它们，结果发现全氟乙丙烯管提供长期稳定，对光学性能只有很小的影响，并有非荧光10。它们，然而，不适合于应用程序，需要一个不同的折射率， 例如 ，清除标本样品。 可在各种直径和厚度的管，以及全氟乙丙烯制成的箔。此外，FEP可在260℃熔化，从而提供了定制的样品安装支持几乎无限的可能性。聚合物管趋向于提供非无菌，这就是为什么在清洁协议包含几个步骤，以确保最佳的光学质量。用甲基纤维素的涂层确保了日益增长的斑马鱼胚胎的任何部分枝到聚合物中。 0.1％琼脂糖的决定是基于增长速度，不动在不同浓度的琼脂糖和甲基纤维素10的广泛的测试。琼脂糖浓度0.1％以上，已经表现出对24 HPF斑马鱼的生长速度造成严重影响。此外，我们使用低剂量的麻醉药物三卡因的。三卡因块的动作电位，从而抑制大脑和肌肉之间的信号传输，并抑制肌肉收缩16。为133-200毫克/升的浓度，确保24-72 HPF斑马鱼之间有足够的固定化。然而，三卡因和其他麻醉药物，我们测试显示剂量依赖的副作用如心脏水肿。另外三卡因的所需剂量随胚胎的发育阶段。因此，我们建议降低三卡因浓度需要被成像的发展阶段的量。为了确保最佳performanCE和防止形成有毒的副产物，新鲜的或解冻三卡因原液使用，应避光和高于30°C加热不在FEP管的嵌入还允许安装到中占据特定需求的容易变形。对心脏的时间推移成像，我们建议用3％甲基纤维素和100 mg / L的三卡因，而不是0.1％琼脂糖和200 mg / L的安装。甲基纤维素是高于0.1％的琼脂糖因而其本身提供了更高的固定性和少三卡因需要被使用，以确保胚胎的禁闭更粘稠。 三卡因是在温度高于30℃敏感，也可通过介质中的成像腔稀释，导致性能和胚胎可能抽搐下降。因此，请务必使用三卡因亦在其中安装的试样浸入成像介质。如果您的实验需要使用更高的温度下，我们要么经常通过使用灌注系统或手动用两个时间点之间的管和注射器建议在成像室交换的媒介。 关键步骤样品的安装过程中的关键步骤是该试样的摄入量入管和琼脂糖插头的插入。吸气定义了胚胎的初始位置，这是麻烦的事后改变。多个标本应提供给安装和安装质量应用立体显微镜进行监测。如果样品取向不理想，改变拟定的标本，避免活塞的后续动作的速度可能会帮助，因为这往往扭曲的胚胎。在1.5％琼脂糖插头是必要的，以避免在0.1％琼脂糖的泄漏。管内的插塞的表面应是平坦的，以开发过程中不影响样品取向。为了达到良好的冲浪能手，不同厚度的琼脂糖层的需要进行测试和管需要被放入琼脂糖垂直于表面。旋转管一点点，拔出管子从盘仔细释放插头。的安装质量应与前成像立体显微镜进行检查。 多视点成像光表镜的一个主要优点是多视点成像，旋转试样，从多个角度采集的z栈，并随后注册并融合他们的能力。已建立的方法来注册的Z堆叠在3D空间中是使用了样品周围作为受信标记17荧光珠基于珠子的登记。然而这种方法依赖一个牢固的安装介质，如1.5％琼脂糖，因而似乎是与此处多层安装呈现不兼容。但几种可供选择的解决方案根据不同的应用工作。首先，将荧光珠可以掺入琼脂糖插头，其具有将在视场的采集过程。第二，舞台的位置可以用与荧光珠1.5％琼脂糖柱的实际实验之前进行校准。第三，替代标记物，如荧光细胞核，可用于识别的Z堆叠的相对于彼此的位置。 蒸发典型光片镜设置使用一个开放的样品室，这必然导致介质的蒸发。由于介质是必不可少的试样的生存，以限制所述安装介质的蒸发和对光学元件和样品之间的正确的折射率时，建议采取以下一种措施，以防止蒸发或更多。首先，在腔室中的介质，可以不断地通过灌注系统进行交换。第二，该介质可以被手动重新填充。第三，该腔室可盖上盖子或柔性箔。 <p类="jove_step"&gt;氧气供应该聚合物的FEP的开发是为了承受大的各种化学品，因此是健壮的，化学惰性和不渗透到气体。在多层安装氧气的情况下只能通过琼脂糖塞和琼脂糖 - 空气界面渗透，但它仍有待证明如果比较时安装在1.5％琼脂糖无支撑聚合物管中的氧供给多层安装是严重降低。然而，由于安装在1.5％琼脂糖只能用约两小时之前斑马鱼的形态受到影响多层安装似乎是长期成像的整体优越的解决方案。 成像早期发展阶段现在多层安装已经成为我们对斑马鱼的年龄超过24 HPF时间推移光片镜检标准的嵌入技术。除此之外，我们还可以使用聚合物管早期胚胎成像。电子mbryos留在他们的绒毛膜和被安装在一个FEP管1毫米的内径和充满E3。斑马鱼可以自由发展绒毛膜内，利用光片镜以及仍然可以成像。 展望所提出的多层样品安装释放出的光薄片显微镜与斑马鱼实时发育生物学的全部潜力。它可以很容易地用于诸如光学投影层析成像（OPT）和其他微生物等显微技术所采用。我们认为，标准尺寸的全氟乙丙烯管只是对根据特定需求的样品安装技术的第一步。

理解胚胎发育过程中的复杂事件和相互作用，研究在发育生物学是越来越多地从单个细胞和器官的显微移动到体内成像整个生物体。传统显微镜技术通常不能提供的空间和时间分辨率以上的很长一段时间，以跟随快速的事件常常造成脱色或示例1中的光毒性效果。我们和其他人发现光片显微镜（ 图1）是理想的技术在活体成像斑马鱼2-4。样品用薄板的激光的，正交的检测轴，该照明提供快速的光学切片的高空间分辨率，以及最小化的光毒性5。同时，由于这种独特的光学布置，使用的培养皿或培养腔室的传统样品的安装技术是不适合的。在一个典型的光片显微镜三个平移马达和旋转马达保持样本，以使得它可以被精确地定位，打开，并从任何方向看。在过去的标本进行光片显微镜常常被嵌入在1-2％琼脂糖玻璃毛细管6,7内。在这种情况下，与嵌入标本凝固的琼脂糖圆筒被挤压出来的玻璃毛细管进入充满水状介质用于成像的样品室。琼脂糖的折射率接近的水，因此是非常适合于在体内光片镜，有利于水校正照明和检测光学系统。样品被限制在刚性的琼脂糖和可成像很好的时间很短，但形态变化和胚胎的生长成像数小时7,8时被强烈地受到损害。虽然对于其它应用的解决方案已被开发如图9所示 ，非侵入性的长期的时间推移斑马鱼<html>光片显微成像的潜力不能使用传统的1.5％琼脂糖包埋利用。 因此，我们开发了一个新的安装策略，斑马鱼胚胎10 在体内光片镜。样品被安装在0.1％琼脂糖有由光学透明的聚合物氟化乙烯丙烯（FEP）的管内200 mg / L的三卡因。嵌入式胚胎正常发展，由于介质的粘度低。在同一时间，该FEP管限制所述介质和样品在实验的持续时间。在这里，我们提供了新的安装技术和现在的数据反映其优于传统协议的详细协议。我们证明，我们的方法斑马鱼发育，可连续成像的高空间和时间分辨率在一段超过两天。 5英寸"FO:SRC ="/ files/ftp_upload/51119/51119fig1highres.jpg"SRC ="/ files/ftp_upload/51119/51119fig1.jpg"/&gt; 图1:光片显微镜的原理。安装在制造氟化乙烯丙烯（FEP）的试管A斑马鱼可以翻译并通过检测目标的焦平面旋转。激光（光片）的一个薄片被照明的目标投射到检测目标的焦平面上并照亮试样的仅一个薄的光学部分。所发射的荧光信号被检测物镜收集并记录在摄像机芯片。

<table border="0" cellpadding="0" cellspacing="0" width="1097">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="52">
			<td height="52" style="height:52px;width:268px;">Fluorinated Ethylene Propylene (FEP) tubes</td>
			<td style="width:143px;">Bola</td>
			<td style="width:113px;">S 1815-04</td>
			<td style="width:573px;">0.8 / 1.6 mm inner / outer diameter, other sizes available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Omnifix-F Solo 1 ml Syringe</td>
			<td style="width:143px;">B. Braun Melsungen AG</td>
			<td style="width:113px;">9161406 V</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sterican Single-Use Cannula, blunt</td>
			<td style="width:143px;">B. Braun Melsungen AG</td>
			<td style="width:113px;">9180109</td>
			<td style="width:573px;">0.8 x 22 mm, other sizes available, outer diameter of cannula has to fit inner diameter of FEP tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml Polypropylene Centrifuge Tube</td>
			<td style="width:143px;">Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M NaOH</td>
			<td style="width:143px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 M NaOH&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% EtOH</td>
			<td style="width:143px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Methylcellulose</td>
			<td style="width:143px;">Sigma</td>
			<td>M0387</td>
			<td>supplied as powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">E3 medium (for zebrafish embryos)</td>
			<td style="width:143px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">1.5 ml Reaction Tube</td>
			<td style="width:143px;">Eppendorf</td>
			<td style="width:113px;">3810X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Agarose, low gelling temperature</td>
			<td style="width:143px;">Sigma</td>
			<td>A9414</td>
			<td>supplied as powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Petri Dish</td>
			<td style="width:143px;">Greiner</td>
			<td>633180</td>
			<td>plastic, 94 x 16 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Tricaine</td>
			<td style="width:143px;">Sigma</td>
			<td>E10521</td>
			<td style="width:573px;">synonyms:&#160; Ethyl 3-aminobenzoate methanesulfonate, MS-222, TS 222, Tricaine methanesulfonate&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">10x/0.3 W Microscope Objective Lens</td>
			<td style="width:143px;">Leica</td>
			<td style="width:113px;">HCX APO L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">EMCCD Camera</td>
			<td style="width:143px;">Andor Technology</td>
			<td style="width:113px;">iXon 885 EMCCD</td>
			<td></td>
		</tr>
	</tbody>
</table>

multilayer mounting for long-term light sheet microscopy of zebrafish

光薄片显微镜是理想的成像技术来研究斑马鱼胚胎发育。由于最小的光毒性和漂白，它特别适合用于长期延时成像在许多小时到几天。然而，适当的样品安装策略是必要的，同时提供隔离和样品的正常发育。多层安装，使用低浓度的琼脂糖在光学透明管一个新的嵌入技术，现在克服了这个限制，并释放出光片显微镜进行实时发育生物学的全部潜力。

斑马鱼血管系统开发的多天成像我们用1 DPF 的Tg（kdrl:GFP）13斑马鱼表现出与多层安装策略，长期光片显微镜的优点。其目的是对图像中的血管系统中的整个斑马鱼的发育超过2天的疗程。在斑马鱼胚胎中，安装在0.1％琼脂糖一个甲基纤维素包衣的聚合物管内。这里，斑马鱼未被安装介质的限制，并且可以正常地发育。它的头提出，尾部延伸和血管发芽不受阻碍地出现（ 图3）。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51119/51119fig3highres.jpg" src="/files/ftp_upload/51119/51119fig3.jpg" /> 图3。斑马鱼血管发育的多日的成像。一个multila的血管揭掉安装1 DPF 的Tg（kdrl:GFP）13斑马鱼的发展不受阻碍地在光薄片显微镜在两天的课程。 488 nm激光片光源激发的荧光，并使用10X/0.3 W的目标和EMCCD相机检测到信号。被收购的四个相邻的位置，每10分钟的Z-堆栈，随后缝合与斐济15。示的时间点的子集的最大强度投影。比例尺:500微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51119/51119fig3highres.jpg" target="_blank">点击这里查看大图。</a> 斑马鱼早期胚胎发育的成像在这里，我们展示FEP管的能力，作为对斑马鱼早期胚胎发育过程中受精后的第一天灯片镜的安装支持。 8 HPF 的Tg（H2A:GFP）14斑马鱼胚胎被安装其完整绒毛膜一个E3填充管内与我新新人类创新直径为1毫米。而导致绒毛膜轻微夹紧。胚胎发育过程中所有的发育过程是不受安装并可以使用光片显微镜得到最佳可视化。在这个例子中，我们成像的样品在12小时的过程中（ 图4）。在早期胚胎发育的典型形状的变化，如蛋黄，并沿中线组织增厚的增大伸长率，将使用不绒毛膜在1.5％琼脂糖传统安装时受到压迫。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51119/51119fig4highres.jpg" src="/files/ftp_upload/51119/51119fig4.jpg" /> 图4的成像斑马鱼早期胚胎发育 的Tg（H2A:GFP）。通过早期胚胎发育14斑马鱼通过。 488 nm激光片光源激发GFP和荧光信号检测与10X/0.3 W的目标和EMCCD相机。 Z-堆栈从两个角度分别获得每3分钟超过12小时的课程。示的时间推移的一个子集的归一化最大强度投影。可见运动模糊在收购过程中胚胎抽搐19 HPF结果，因为没有麻醉剂被使用。比例尺:150微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51119/51119fig4highres.jpg" target="_blank">点击这里查看大图。</a>

Watch this Scientific Journal Video about Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish at JoVE.com

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

斑马鱼的发展可以遵循以上光片镜天，当胚胎嵌在光学透明的聚合物管与低浓度琼脂糖。

多层安装于斑马鱼的长期光表镜

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

multilayer-mounting-for-long-term-light-sheet-microscopy-of-zebrafish

Nanyang Technological University

Research

JoVE Journal

Biology

18.7K 次观看.  Max Planck Institute of Molecular Cell Biology and Genetics. 斑马鱼的发展可以遵循以上光片镜天，当胚胎嵌在光学透明的聚合物管与低浓度琼脂糖。 

视频: Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

光固化牙科粘合树脂安装电极或微透析探针在慢性实验中的应用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

制备果蝇 S2细胞，光学显微镜

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

设置简单的轻表为显微镜<em&gt;在托托</em的&gt;成像<em&gt; C。线虫</em&gt;发展

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

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) ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

植物根系的轻质板材荧光显微镜生长在一个凝胶表面

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

LarvaSPA，一种用于长期延时成像的安装果蝇幼虫的方法

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...