Name: light sheet-based fluorescence microscopy of living or fixed and stained tribolium castaneum embryos
Uploaded: 2017-04-28T15:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos at JoVE.com

我们感谢斯文·普拉斯的技术支持。神经胶质蓝色的转基因品系是从格雷戈尔·布彻（哥廷根，德国）惠赠。这项研究是由卓越法兰克福的集群资助上午主在歌德的布赫曼研究所分子生命科学（BMLS，主管恩里科·施莱夫）部分授予EHKS大分子复合物（CEF-MC，EXC 115，扬声器沃尔克·多茨奇）法兰克福大学是由德意志研究联合会（DFG）为主。

1. 赤培养的畜牧注意:标准条件定义为在25℃和70％相对湿度在12个小时亮/ 12小时黑暗周期的培养温度。有关赤畜牧业的更多信息，相应的指南可64。此协议需要两个不同的面粉媒体​​，可在千克数量制备和储存数月。 <ol><li>之前用面粉和非活性干酵母工作，存储未开启的包装，在-20℃下放置24个小时，然后让它们升温至室温。这个过程消除了潜在的病原体。 </li><li>通过与710微米筛孔尺寸的筛子通过全谷物小麦粉和非活性干酵母，然后用5％的补充过筛全粒面粉（重量/重量）过筛非活性干酵母制备生长培养基中。 </li><li>经过405精面粉和非活性干酵母通过筛250＃181;米筛目大小，然后用5％的补充过筛405细小麦粉（重量/重量）过筛非活性干酵母制备产蛋平台。 </li><li>通过相应的赤文化通过 用筛 800 微米网目尺寸。丢弃剩余的生长培养基。转移幼虫，蛹和成虫一个大玻璃盘。 </li><li>如果没有后代的文化存在，大约80幼虫和/或蛹转移到一个新的玻璃瓶，建立一个新的子代的文化。如果没有幼虫和/或蛹存在，约有40名成人代替。添加50克生长培养基中并在标准条件下温育。 </li><li>收集在另一个新玻璃瓶大约300成年人（500-700毫克）以建立所述成像培养。添加50克生长培养基中并在标准条件下温育。允许成像培养在胚胎收集前的新鲜生长培养基静置至少24个小时。 </li><li>更换成像文化至少每两个我们的生长培养基EKS，它可与胚胎收集结合来完成（见3.2）。两三个月后，从子代文化的新成人完全更换成像文化。 </li><li>如果使用非纯合转基因线，通过消除非转基因动物策划后代培养频繁。理想情况下，产生纯合系如果相应转基因是不致死或导致不育时存在两个染色体上。纯合的培养物通常具有较高的平均荧光水平和整体实验条件更窄，因为只有一个基因型存在。 </li></ol> 2.显微镜的设置和校准注: 赤胚胎具有约600-660微米的前-后长度和大约300-330微米的直径的横向。大多数现代CCD照相机具有约9毫米×7mm的传感器芯片尺寸， 即直径为11.4微米，6.45微米的像素的像素距离。当用10倍放大倍率的物镜相结合，胚胎可以全盘被成像为0.645微米的像素间距。大多数现代SCMOS相机具有约16毫米×14mm的较大的传感器芯片尺寸， 即直径为21.3微米，6.5微米的像素的像素距离。当用20X放大率的物镜相结合，胚胎可以全盘被成像用的0.325微米的像素间距。 <ol><li>附加照明和检测物镜的显微镜。确保激光器和荧光过滤器相匹配的荧光团的吸收和发射光谱很好。 </li><li>样品室连接到显微镜。填充pH7.4的PBS或任何其他合适的成像缓冲器中的样品室中。 </li><li>开机和校准显微镜。综合校准程序和故障排除指南定制基于光片显微镜（更具体的实施DSLM）已经出版公关eviously 65。对于商用设备，按照制造商的说明书非常谨慎。误操作可能消耗介质的样品室，并导致破坏到仪器以及试样。 </li></ol> 3.转基因或胚胎WT收集<ol><li>通过用800微米筛目尺寸的筛通过成像菌落。收集成虫在一个新的玻璃瓶，加入10 G蛋铺设介质。孵育产蛋培养在25℃下在光1个小时，70％相对湿度。 </li><li>通过产蛋培养通过用800微米的筛目尺寸的筛子从产蛋培养基，其含有30-100左右分离胚胎成人。大人转移到一个新的玻璃瓶，并添加老任或50克鲜生长培养基。 </li><li>如果观察应在均一胚阶段（例如数据集DS0002）开始，孵育15个小时产蛋介质在25℃和70％相对湿度。 ŤO起动在germband缩回（例如数据集DS0001和DS0003）的开始，在32℃和70％相对湿度下孵育胚胎23小时。如果其他发育阶段期望，请参见相应的文献64，65和适应孵育时间和/或温度是必要的。 </li></ol> 4.胚胎Dechorionation 注:绒毛膜是蛋壳的外层和由蛋白质和多糖的。强烈吸收光的，但对正常发育不是必需的，因此可以化学方法除去。 <ol><li>通过用300微米筛目尺寸的筛通过产蛋平台。收集在100微米筛孔尺寸的细胞过滤的胚胎，并丢弃剩余的产蛋平台。 </li><li>准备一个6孔板如下:填充A1，A2，A3和B3的井用10mL pH 7.4的PBS和B1和B2井用9mL PBS。然后小心地加入1mL的次氯酸钠到B1和B2。观察的步骤4.3到立体声显微镜下4.8的进展。次氯酸钠小心钠有腐蚀性。 </li><li>插入细胞滤网进入A1以及（PBS pH 7.4）中和洗胚胎在温和搅拌下一分钟。较大的面粉颗粒应该从胚胎中分离。 </li><li>转移细胞滤网给B1阱（次氯酸钠在pH 7.4的PBS）中并剧烈摇动板30秒。剩下的面粉颗粒应该从胚胎完全分离。 </li><li>转移细胞过滤到A2以及（PBS pH 7.4）中和洗胚胎在温和搅拌下1分钟。 </li><li>对于dechorionation，转移细胞滤网到B2阱（次氯酸钠的PBS，pH 7.4）中。大力摇晃板，直至绒毛膜破裂，并从胚胎中分离。绒毛膜看起来像一个薄的半透明膜具有撕裂边缘，而dechorionated胚胎有一个普通的剪影。 </li><li>转让日È细胞过滤到A3以及（PBS pH 7.4）中和洗胚胎在温和搅拌下1分钟。如果任何绒毛膜片段洗涤后仍附着于胚，重复步骤4.6。 </li><li>存储在B3以及（PBS pH 7.4）中的胚胎。存储了好几个小时不伤害胚胎，但请记住，继续发展。 </li><li>对于非纯合转基因系，除去任何非荧光胚胎如果可能的话。用于固定和WT或转基因胚胎的染色，继续执行步骤5。对于转基因胚胎的实时成像，则继续步骤6。 </li></ol> 5.卵黄膜透化，固定，染色和注:卵黄膜是蛋壳的内层。它是不受用于荧光染料，因而有染色前被化学透。 <ol><li>填充固定/透化溶液（1.5mL 4％（在PBS pH值6.9重量/体积）的多聚甲醛的闪烁小瓶ND 1.5毫升正庚烷）。移植胚胎用画笔小瓶。用铝箔盖的小瓶，以保护从光的荧光基团并以每分钟50转温育30分钟在摇动平台上。注意多聚甲醛是有毒的，腐蚀性的，正庚烷是易燃和有毒。 </li><li>除去固定液和治疗胚胎三次在3mL 0.1％（V / V）的Triton X-100的PBS，pH 7.4 15分钟，随后一次15分钟在3mL 1％（V / V）的Triton X-100中以每分钟50转在摇摆平台上PBS pH 7.4中。小心的Triton X-100是腐蚀性的。 </li><li>除去透化溶液，并添加0.5毫升的染色溶液的小瓶中。在表2中提供了示例性染色溶液。染色的胚胎在4℃下过夜，以每分钟50转在摇动平台上。对于表2中提出的染料，清洗是没有必要的。 </li></ol> 6.制备安装琼脂糖的<ol><li>添加1克低熔点琼脂糖至100mL pH 7.4的PBS和热，以便在800 W.微波炉2-3分钟将该混合物如果小琼脂糖颗粒仍然存在，重复加热过程在30秒的时间间隔，直到颗粒完全溶解。安装琼脂糖不需要每次要新鲜制备的，该琼脂糖固化可以通过加热上述再液化。这个过程可以了太多的水已经蒸发之前重复5-6次。 </li><li>允许琼脂糖冷却至大约40-45℃，然后填充两个1.5毫升反应管与加热的琼脂糖和保持这些管在35℃下在热块。 </li></ol> 7.胚安装和插入到样品室<ol><li> 选项1:琼脂糖柱（ 图3A-C，第一列;表3中，第二列） <ol><li>填充1.0毫升的注射器用蒸馏水和使用小型橡胶软管将其附加到玻璃毛细管开口中的一个。填充毛细管中途用蒸馏水。 </li><li>挑胚胎与漆刷，将其放在其他的玻璃毛细管开口的内侧。快速地进行下一步的胚胎干燥前。 </li><li>坚持毛细管进入液体琼脂糖慢慢吸取液体琼脂糖几微升旁边的胚胎移植到毛细管。然后迅速增加牵引力，使胚胎拖动随着琼脂糖。最后，琼脂糖的几μL应该高于和胚胎的下方。将毛细管静置几分钟，让琼脂糖巩固。 </li><li>缩短用金刚石玻璃刀毛细管至约5毫米的长度。剩余的片段应完全充满琼脂糖和胚胎应该是第二四分位数内。 </li><li>插入钢瓶与销到样品室，然后贴在销的顶端毛细管片段。琼脂糖柱应该伸出几毫米˚FROM与包含胚胎部分毛细管片段。 </li></ol></li><li> 选项2:琼脂糖半球（ 图3A-C，第二列;表3中，第三列） <ol><li>吸取液体琼脂糖的200μL成1.0毫升的注射器，然后用它来填充从琼脂糖顶部的钢管，直到在底部开口半球形式。半球直径应等于管子的直径。直到琼脂糖已经凝固等待几分钟。 </li><li>挑胚胎用画笔并重新排列其上的刷子的前端，以使前端变得可访问。浸琼脂糖半球成液体琼脂糖用薄膜来覆盖它。 </li><li>将其前端胚胎直立琼脂糖半球的极点。打开钢管周围并更正刷胚胎的位置。如果必要的话，加入2μL琼脂糖胚胎/半球边境稳定的胚胎。理想LY，不到一半的胚胎表面的应该在琼脂糖覆盖和侧面应尽可能地陡。 </li><li>插入钢管半球和胚胎慢慢进入样品室。 </li></ol></li><li> 选项3:蛛网保持器（ 图3A-C，第三列;表3中，第四列） <ol><li>挑画笔胚胎，并把它放在一边。 </li><li>覆盖5-8μL液体琼脂糖蛛网保持器的长孔，然后除去大部分的琼脂糖的直到只有一个薄膜琼脂糖保持。 </li><li>放置胚胎到琼脂糖膜并调整其位置。小心地将胚胎移动到长孔的x轴中心，并与所述长孔的细长轴对准其细长前后轴线。 </li><li>插入与所安装的胚胎的蛛网保持器慢慢到样品室。因此，它不与激发和发射干扰蛛网挡位置光， 即 45°相对于x轴和z轴。 </li></ol></li></ol>基片8的光荧光显微<ol><li>沿胚胎应多少方向（和沿其方位）被成像决定。四个方向（沿取向0°，90°，180°和270°）典型地提供了良好的折衷覆盖和能量负载之间。 </li><li>建立荧光通道的数量。每个信道的主要参数是激光线，荧光滤光器，激光功率和曝光时间。对于长期实时成像，即综合能源负荷不超过胚胎的耐受性是很重要的。 25-50毫秒的曝光时间确保了快速成像，而100和300μW之间的激光功率不应该影响胚胎的生存能力。对于固定的样品，暴露时间和激光功率可以至少加倍。 </li><li>对于实时成像检测，配置的时间间隔。样稿赤的勒特胚胎发生在室温（23±1℃），并在32-35°的小不到三天C 64需要大约7天。根据应该观察到的形态发生的事件定义的时间间隔。对于短的时间间隔，考虑降低曝光时间和激光功率（见8.2）。 </li><li>而不将其暴露于激光中沿着x轴和y轴进入视场的中心透射光模式胚胎定位。通过在90°360°旋转胚胎以检查可能在安装过​​程中发生的任何损伤胚胎。 </li><li>适当地转动胚胎绕y轴与显微镜轴，例如与x轴的横向轴线， 即与z轴的背腹轴对准胚轴。当使用蜘蛛网架技术，对准也许是不可能的。 </li><li>在荧光模式，定义的z堆FOr各自方向。添加25-50微米，如前面空间缓冲液和胚胎的后面。包括缓冲，典型的Z-堆叠覆盖周围350-400微米为赤胚胎。还定义的z间隔，这应该是横向间隔的四倍， 即 ，在间隔沿x和y轴66。 </li><li>如果两个或多个胚胎在并行成像中，重新启动在8.4与下一个胚胎。 </li><li>对于长期成像，确保样品室填充有足够的pH 7.4的PBS或其它成像缓冲器。还覆盖所述样品腔室开口。 </li><li>启动成像过程。对于长期活体成像，监测沿在所有胚胎所有频道的所有方向正确采集。偶尔检查在接下来的日子里，该胚胎存活，并在适当的位置。 </li></ol> 9.胚胎检索和质量控制<ol><li>一旦成像完成时，删除与该样品保持器胚胎从样品室中。从实时成像检测，只有胚胎必须被检索。固定并染色的胚胎可以被丢弃。 </li><li>如果进行了活体成像测定法中，将胚胎转移到物体滑动。对于琼脂糖柱技术，提取从用手术刀，微刀或刀片琼脂糖周围胚胎。对于琼脂糖半球技术中，与平面侧到对象幻灯片转移半球。胚胎切除是没有必要的。对于蛛网保持器技术中，轻轻地从用画笔琼脂糖膜卸除胚胎。孵育在标准条件下的饱和湿度的气氛小玻璃皿直到孵化的幼虫的对象滑动。 </li><li>孵化后，幼虫转移到24孔平板的各个孔中和500mg生长培养基添加到每个孔中。在标准条件下孵育。成像幼虫的总开发时间和形态比较非成像WT和转基因larvAE（见讨论）。在表征WT发展分析，形态未见明显畸变理想情况下应明显。 </li><li>一旦幼虫发育到健康的成年人，为他们提供相同的背景应变的适当WT伙伴。两周后，检查是否有后代生产。也可以考虑通过交配它们间削去检查后代的生育能力。只有当所成像的胚胎发育成产生育子代肥沃的成年人中，成像过程可以被认为是非侵入性的。 </li></ol> 10.图像数据处理注:对于图像数据的处理，开源图像处理软件ImageJ的67或它的衍生物斐济68推荐的。这两个版本以及全面的文档在www.imagej.net被发现。对于LSFM数据的标准文件格式是标记的图像文件格式（TIFF）。内在容器选项允许STO图像栈如Z-堆叠或时间序列能够在ImageJ的或经由FIJI拖和降低而打开的单个文件内ž最大突起的愤怒。 <ol><li>如果必要，可旋转的z堆栈对准与y轴（图像→变换→旋转）胚胎的前后轴。请注意，旋转参数必须为每个方向单独设置，但不适合的时间间隔和荧光通道。 </li><li>裁剪沿z栈x轴，y轴和z轴，使得只有最小的背景（20 - 40个像素沿x轴和y轴，5 - 沿z轴10的平面）保持（用于x - 轴和y轴，使用矩形选择工具，然后图像→作物;对于z轴，使用图像→堆栈→工具→切片守护者）。请注意，裁剪参数为每个方向单独设置，但不适合的时间点和荧光通道。 </li><li>计算的Z最大投影每个Z堆叠（图片→栈→Z项目，选择最大强度）。强度投影计算是数据简化程序，除去一个空间维度。 </li><li>对于长期实时成像数据，可以考虑使用执行处理步骤为所有方向上，所有时间点和所有荧光通道65 10.1至10.3的ImageJ的或斐济脚本。理想情况下，串连每通道和方向所有的Z最大突起，并将它们保存在一个TIFF容器中的时间序列。可替代地，BigDataViewer 69插件斐济可用于通过太字节大的数据集导航。 </li><li>根据不同的转基因系和成像模态，时间堆叠的动态强度的调整可能是理想的，由于光漂白和/或荧光团表达的波动。无论是ImageJ-或斐济固有漂白校正功能（图像→调整→漂白校正，选择直方图匹配）或奉献，泰德软件65建议。 </li><li>如果胚胎被记录和/或沿多个方向，并在多个荧光通道，这些方向的水平组合（图像→堆栈→工具→组合）和信道合并（图像→颜色合并→频道）被推荐。 </li><li>登记和沿着多个方向70最终与去卷积71组合获得的，Z-栈的融合，使质量均匀的和各向同性的分辨率优良的三维图像的计算。这两个插件都是开源的，预装在斐济，但他们需要的地标标本周围存在（荧光微球， 如珠）。 </li><li>用于大规模定量数据提取和分析的开源软件框架也可，例如用于细胞核72或CEL的分割和跟踪升形状识别73。 </li></ol>

作者什么都没有透露。 作者投稿 FS和EHKS设想的研究。 FS产生转基因AGOC线。基于光的片材荧光成像用FS和SK进行。 FS和EHKS与SK输入写的稿子。

品质管制 在活体成像试验中，准备和记录程序必须是非侵入性的， 即，既不机械和化学处理（收集，dechorionation，安装到样本保持器），也没有观察时的集成能量负载应该影响样品的可行性。对于研究表征WT发展，它是从其中胚生存的录制过程中，成功地检索并发展成一个健康的成年人实验建议只使用数据。成人不应该表现出任何形态畸变，宜肥沃，且其...

在赤拟谷盗赤拟谷盗 ，属于大家族暗中甲虫（拟步甲科），具有农业和生命科学领域内历史悠久，是果后的第二个最好的研究模型昆虫的模式生物飞行果蝇 。在过去的四个十年里，它成为发育遗传学的强大和受欢迎的昆虫的模式生物，在进化发育生物学，并在过去的二十年中，在各种原因的胚胎形态: 果蝇和赤同属Holometabola，但约300万年前的1，2，3，4分歧。而果蝇胚胎发育通常被认为是高度衍生， 赤示出devel的更祖模式即在昆虫物种5，6，7，8，9的一个相当大的比例发现opment。首先， 赤呈现出非渐开头的发展， 即，其口器和天线胚胎发生10，11，12，13，14，15中已出现。其次， 赤如下短胚芽发展， 即腹部段germband伸长16，17，18，19在从后部生长区依次加入的原理。第三， 赤开发和后来降级两个额外的胚胎外膜即羊膜，它涵盖了胚胎只腹侧，和浆膜，其完全包住胚胎20，21，22。两种膜起到至关重要的形态发生23以及抗微生物24，25和26干燥保护作用。第四，embryonically显影腿是在幼虫生命阶段完全功能和蛹变态27，28，29，30，31时用作原基为成人腿。 由于其体积小，适度的要求，在实验室赤的培养是相当简单的。野生型培养物（WT）菌株或转基因品系通常由周围100-300成人和可以保持一升玻璃瓶（足迹80厘米2）往三四十厘米高（约50克）与由全谷物小麦的生长培养基内面粉补充有非活性干酵母。供水是没有必要的。这样，即使小型实验室，以保持小型或中型商用虫孵化器内的几十个甲虫文化。 赤的后面发育阶段（幼虫后约4龄，蛹和成虫）很容易从生长培养基通过筛分分离。同步胚胎，恒温产蛋介质上短期内得到成年人。对于快速发展，甲虫培养物保持在32℃（每代约四周），而贮存保持在22-25℃通常执行（每一代约十周）。 在过去的十年中，许多标准TEChniques已经逐渐适应和赤优化，总结在新兴的模式生物书32。非常重要的是先进遗传方法例如胚胎33，幼虫34,35或亲本36，37基于RNA干扰的基因敲除，与任一piggyBac转 38，39或米诺斯 40转座系统和CRISPR /基于Cas9基因组种系转化工程41。此外， 赤基因组被测序大约十年前42，并且现在是在第三轮基因组的装配释放43，其允许高效且全基因组鉴定和基因44的系统的分析</s向上&gt;或其它遗传元件45，46。另外，四个其它鞘翅目物种的基因组可用于比较遗传方法47，48,49，50。与测序基因组关联，两次大规模的遗传分析已经执行， 即 ，插入诱变屏幕51和基于干扰系统的RNA基因敲除画面52，53。 与宽视场，共焦或基于光的片材显微镜（LSFM）荧光实时成像允许观察赤的胚胎形态作为时间多维上下文（ 表1）的函数（ 即 ，形态发生）。在宽视场和共聚焦荧光显微镜，该EXCIT通货膨胀和发射光通过相同的物镜引导。在这两种方法中，整个试样被照亮每记录二维平面。因此，试样承受非常高的能量水平。在LSFM，只有在焦平面中的荧光团是兴奋由于通过使用两个垂直布置的物镜（ 图1）照明和探测的去耦。 LSFM有两种法服实现-该单个平面照明显微镜（SPIM）和数字扫描的激光基于片材的荧光显微镜（DSLM， 图2） -和在传统的方法提供了几个重要的优点:（ⅰ）固有光学切片能力， （ⅱ）良好的轴向分辨率，（ⅲ）强烈降低光致漂白的水平，（ⅳ）非常低的光毒性，（v）的高信噪比，（ⅵ）相对高的采集速度，（ⅶ）成像沿多个方向和（viⅱ）更深的组织穿透由于低数值孔径照射物镜54，55，56的使用。 LSFM已经在赤已成功地应用于记录几乎整个胚胎的形态发生57和分析外胚膜破裂的原则，在背封23的开端。为了提高LSFM的吸引力在赤社区和一般昆虫科学，这是非常重要的，建立标准作业程序，改进方法，协议和资源的水平池在显微镜变成了一个易于在发育生物学实验室-use标准工具和生物学问题留在人们关注的焦点。 该协议开始于赤的基本&lt;/ em&gt;的培养， 即维修，繁殖和胚胎收集。接着，两个实验策略中示出:（ⅰ）的定制的转基因品系实时成像和（ii）该染色用荧光染料（ 表2）固定的胚胎的成像。 （i）所述琼脂糖柱，（ii）所述琼脂糖半球和（iii）所述新的蛛网持有人:随后，具有稍微不同的目的三个安装技术详细（ 图3和表3）进行说明。该协议然后解释与LSFM获取数据的步骤。影像学技术和关键因素进行了概述。最后，胚胎检索解释并提供了基本的数据处理建议。在代表性结果，从活体成像数据的两个新的定制和胶质-蓝58个的转基因株系被示出和各个成像数据集作为可下载资源提供。此外，图像该染色用各种荧光染料的固定胚胎的数据表示。讨论集中在质量控制方面，实时成像方式的电流限制和协议的其他物种的适应性。 该协议是针对配有样品室和用于标准化样品架54，59，60，这是典型的直径由金属，塑料或玻璃筒形元件可旋转的夹持机构基于光的片材荧光显微镜写入在毫米范围内。该协议也适用于这两种法服实施方式中， 即 SPIM和DSLM，以及用于与两个或更多照明和检测臂61，62，63设置。代表结果表明在两个光谱信道的数据，绿色（ILlumination用488nm的激光，检测通过五十零分之五百二十五通过七十零分之六百零七带通滤波器的带通滤波器）和红色（照明用561纳米的激光，检测），但是协议可以扩展到三个或四个光谱信道。

<table><tbody><tr><td>full grain wheat flour</td><td>Demeter e.V.</td><td>1.13E+08</td><td>US: whole wheat flour, UK: whole meal flour</td></tr><tr><td>405 fine wheat flour</td><td>Demeter e.V.</td><td>1.13E+08</td><td>US: pastry flour, UK: soft flour</td></tr><tr><td>inactive dry yeast</td><td>Flystuff / Genesee Scientific</td><td>62-106</td><td></td></tr><tr><td>Phosphate-buffered Saine (PBS), pH 7.4</td><td>Thermo Fisher Scientific</td><td>10010-023</td><td></td></tr><tr><td>sodium hypochlorite, ~12% active Cl</td><td>Sigma Aldrich</td><td>425044-250ML</td><td>Caution: sodium hypochlorite is corrosive</td></tr><tr><td>low-melting agarose</td><td>Carl Roth</td><td>6351.2</td><td></td></tr><tr><td>6-well plate</td><td>Orange Scientific</td><td>4430500</td><td></td></tr><tr><td>24-well plate</td><td>Orange Scientific</td><td>4430300</td><td></td></tr><tr><td>glass capillaries, internal &amp;Oslash; 0.46 mm</td><td>Brand GmbH + Co KG</td><td>7087 09</td><td></td></tr><tr><td>SYTOX Green</td><td>Thermo Fisher Scientific</td><td>57020</td><td>Staining solution preparation is explained in Table 2</td></tr><tr><td>YOYO-1 Iodide</td><td>Thermo Fisher Scientific</td><td>Y3601</td><td>Staining solution preparation is explained in Table 2</td></tr><tr><td>BOBO-3 Iodide</td><td>Thermo Fisher Scientific</td><td>B3586</td><td>Staining solution preparation is explained in Table 2</td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Thermo Fisher Scientific</td><td>A12379</td><td>Staining solution preparation is explained in Table 2</td></tr><tr><td>Alexa Fluor 546 Phalloidin</td><td>Thermo Fisher Scientific</td><td>A22283</td><td>Staining solution preparation is explained in Table 2</td></tr><tr><td>sieve, 800 &amp;micro;m mesh size</td><td>VWR International</td><td>200.025.222-051</td><td></td></tr><tr><td>sieve, 710 &amp;micro;m mesh size</td><td>VWR International</td><td>200.025.222-050</td><td>for growth medium preparation (step 1.1)</td></tr><tr><td>sieve, 300 &amp;micro;m mesh size</td><td>VWR International</td><td>200.025.222-040</td><td></td></tr><tr><td>sieve, 250 &amp;micro;m mesh size</td><td>VWR International</td><td>200.025.222-038</td><td>for egg laying medium preparation (step 1.2)</td></tr><tr><td>glass dish, &amp;Oslash; 100 mm &amp;times; 20 mm</td><td>Sigma Aldrich</td><td>CLS70165102</td><td></td></tr><tr><td>cell strainer, 100 &amp;micro;m mesh size</td><td>BD Biosciences</td><td>352360</td><td></td></tr><tr><td>paint brush, head &amp;Oslash; 2 mm</td><td>VWR International</td><td>149-2121</td><td></td></tr><tr><td>syringe, 1.0 mL</td><td>B. Braun Medical AG</td><td>9166017V</td><td></td></tr><tr><td>scintillation vials</td><td>Sigma Aldrich</td><td>M1152-1000EA</td><td></td></tr><tr><td>paraformaldehyde (PFA)</td><td>Sigma Aldrich</td><td>158127</td><td>Caution: paraformaldehyde is toxic and corrosive</td></tr><tr><td>n-heptane &amp;ge;99%</td><td>Carl Roth</td><td>8654.1</td><td>Caution: n-heptane is flammable and toxic</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100-100ML</td><td>Caution: Trition X-100 is corrosive</td></tr></tbody></table>

light sheet-based fluorescence microscopy of living or fixed and stained tribolium castaneum embryos

在赤拟谷盗赤拟谷盗已成为发育遗传学及进化发育生物学的一个重要昆虫的模式生物。 赤胚胎与基于光的片材的荧光显微镜观察具有优于常规宽场和共聚焦荧光显微镜多个优点。由于基于光的片材显微镜的独特性质，活标本的三维图像可以记录具有高信号噪声比和比上几个周期显著降低光致漂白，以及沿多个方向的光毒性天。随着四年多的发展方法和数据的不断增加，时间似乎是适当的建立光片技术在赤社区的使用以及在昆虫社会大众的标准作业程序。这个协议描述了适合˚F三个安装技术或不同的目的，提出了两种新的定制的转基因赤线适合于长期实时成像，表明5个的荧光染料来标记固定胚胎的细胞内结构以及用于记录数据的及时评估提供了关于数据的后处理的信息。代表性的结果集中在长期实时成像，光学切片并沿多个方向相同的胚胎的观察。相应的数据集提供的可下载资源。最后，协议讨论了用于实时成像测定，电流限制和所概述的程序到其它昆虫物种的适用性的质量控制。 该协议主要针对谁寻求成像解决方案，超越标准的实验室设备发育生物学家。它促进了连续尝试关闭技术上导向的实验室/社区，其发展和完善万分之一之间的差距复制方法论，生命科学实验室/社区，这需要技术上的挑战"插件和播放的解决方案。此外，它支持移动的生物学问题成为人们关注的焦点的公理化方法。

这个协议描述了用于生活的荧光成像或固定和染色赤胚胎LSFM实验框架。由于光漂白和光毒性，其光学切片能力的直接结果的低水平，LSFM是特别适合于长期实时成像。 新颖AGOC {ATub&#39;H2B-mEmerald}＃1转基因线下的α-微管蛋白1启动子74的控制表达histone2B-mEmerald融合蛋白。它示出了一个增强子捕获状表?...

Watch this Scientific Journal Video about Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos at JoVE.com

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

成像昆虫胚胎与基于光的片材的荧光显微镜的形态已成为现有技术的状态。该协议概述并比较适合赤拟谷盗胚胎三个安装技术中，引入了两个新的定制的转基因系非常适合于实时成像，讨论必需的质量控制和指示当前实验的限制。

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

light-sheet-based-fluorescence-microscopy-living-or-fixed-stained

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

10.5K 次观看.  Buchmann Institute for Molecular Life Sciences (BMLS). 成像昆虫胚胎与基于光的片材的荧光显微镜的形态已成为现有技术的状态。该协议概述并比较适合赤拟谷盗胚胎三个安装技术中，引入了两个新的定制的转基因系非常适合于实时成像，讨论必需的质量控制和指示当前实验的限制。 

视频: Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

<meta charset="utf8"></head><body>斑马鱼胚胎的精细多视图光片显微镜实验方案

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

<meta charset="utf8"></head><body>作者聚焦:用于高分辨率多视图光片显微镜的斑马鱼胚胎安装策略 — 成像和图像重建技术

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

早期斑马鱼胚胎的光片显微镜成像和安装策略

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

用光片荧光显微镜观察运动轴突导航和小鼠胚轴突树枝状的定量化

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

神经科学

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 ...

生物学

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures&#160;similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet&#160;fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

光片为主的生活的荧光显微镜或固定和染色

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诱导生活青蛙和斑马鱼胚胎缺氧

早期斑马鱼胚胎的光片显微镜成像和安装策略

早期斑马鱼胚胎的光片显微镜成像和安装策略

早期斑马鱼胚胎的光片显微镜成像和安装策略

用光片荧光显微镜观察运动轴突导航和小鼠胚轴突树枝状的定量化

设置简单的轻表为显微镜

样品室光片荧光显微镜中多个昆虫胚胎的同步实时成像