笔者要感谢埃利奥特教授M.迈耶罗维茨对他的支持，以及对稿件达特茅斯学院和安德烈什·科拉佐博士在加州理工学院生物成像设备对他们的帮助解决的技术问题的意见，以及安·拉万韦现场共聚焦成像。纳森尔·普鲁内的工作由卫生部通过格兰特R01 GM104244埃利奥特M.迈耶罗维茨美国国家研究院的支持。

1.媒体和菜准备<ol><li>准备通过填充的圆形塑料盒（约6厘米宽，2厘米深）至0.5cm，用2％的琼脂糖解剖菜肴。 </li><li> 准备成像菜肴。 <ol><li>对于直立共聚焦显微镜，填充矩形塑料铰接盒（大约7厘米长，4.5厘米宽，深3厘米）到0.5厘米，成像介质（参见1.3节，"成像介质"; 图1F）。 </li><li>对于倒置的共焦显微镜:填充小培养皿（约3.5厘米宽，深1厘米）正好与成像介质的边缘（参见1.3节，"成像介质"; 图1G）。 </li></ol></li><li> 准备成像介质。 <ol><li>对于单点成像，使用1％琼脂糖。 </li><li>时间推移的实验中，使用顶点生长培养基（不含维生素0.5×Murashige和Skoog基础盐混合物，1％蔗糖，0.8％琼脂糖，pH5.8下与钾氢氧化物ë溶液，补充有维生素[0.01％肌醇，0.0001％烟酸，0.0001％盐酸吡哆醇，0.001％盐酸硫胺素，0.0002％的甘氨酸]和细胞分裂素[500nM的N6苄基腺嘌呤]）15。 </li></ol></li></ol> 2.植物生长<ol><li>播种在MS平板（0.5或1x Murashige和Skoog基础盐混合物不含维生素，0.8％琼脂，用氢氧化钾溶液的pH值5.8）与适当选择灭菌的种子。在漫长的一天（16小时光照），或连续天代替板，16-22℃的条件下，持续两周。 </li><li>移苗到土壤上有足够的空间，让蓬勃发展。广场植物在短日照，16-22℃的条件下三个星期。 注:在移栽过早开花和不那么剧烈，并且更难解剖花序结果后直接生长的植物在一天或连续天的条件。同样，离开植株在短日康迪特离子为三周以上的结果在一天的长度无关的开花和不那么剧烈的花序。 </li><li>转移植物整天（16小时光照），或连续天，16-22℃的条件下，直到它们的花。茎尖最容易解剖时花序为2-10厘米长。 </li></ol> 3.茎尖解剖<ol><li>可选:用细磨刀石和轻油的下降，锐化体视显微镜下用镊子使它们刀锋般，没有点状。 </li><li>通过推动梗的基部与所述镊子直到断裂除去的长角果，老年花芽和次级花序从主花序。除去尽可能多的花朵尽可能不用放大（ 图1，比较A和B）。 </li><li>使用镊子，刺穿在解剖盘的琼脂糖的垂直孔，切断最后0.5厘米花序的，并在琼脂糖垂直把它粘。剩余的花芽必须琼脂糖表面之上。 </li><li>用无菌，去离子水填充成像菜，使茎尖完全浸没。放置解剖盘的立体显微镜下，并通过创建与1000微升移液管的水射流去除捕捉周围的茎尖的空气（ 图1，比较C和D）。 </li><li>下的立体显微镜，使用镊子除去未通过推动梗的基部或萌芽的直至断裂花梗（ 图1E）的顶部上被成像花芽。该梗在与干结干净突破，如花梗剩阻碍拆除年轻的花蕾是很重要的。 </li><li>如果成像只花芽阶段5和年轻（ 图2A），解剖阶段6-8花芽之前除去水。如果成像花芽台5及以上，则进入萼片烧蚀（参见第5.1节）。否则，亲CEED到步骤3.7。 </li><li>使用镊子，刺穿在成像盘的介质的垂直孔，并坚持解剖顶点直立在介质中，因此，只有枝条顶端分生组织和周围花蕾的介质的表面的上方。取决于哪个花芽（多个）要被成像时，可能需要稍微倾斜样品，如花芽不一定取向酷似阀杆（ 图2A）。 </li><li>前往如果需要染色的样品。如果不染色和/或成像样品马上加水，并关闭成像盘，以防止脱水。如果使用倒置显微镜，将成像皿中的透明塑料盒并关闭它。 </li></ol><img alt="图1" src="/files/ftp_upload/55156/55156fig1.jpg" /> 图1:茎尖活共焦成像的制备。 （A - E）的解剖茎尖成像。花序前（A）和（B）之后去除角果和旧的花。 （C - D）茎尖浸入水中在解剖盘，与截留在尖端（C）的空气气泡和除去气泡（D）的后。 （E）在解剖盘茎尖，花芽比阶段5（F - G）较旧的解剖后在成像盘茎尖的视图中，直立（F）和反向的（G）的共焦显微镜的载物台。在（F），一个40X水浸渍透镜定位在所述顶点中的一个，具有浸在水中的透镜的尖端。在（G），茎尖置于倒置的40X水浸渍镜头上方，具有水柱连接的成像介质的透镜的尖端。在（F）的较小的面板显示HIG她在红色的矩形的区域中，用插在成像介质一茎尖的放大图;红色和蓝色线表示介质和水的表面上，分别。 （H - I），其允许在水-浸渍透镜的尖端加入更多的水定制设备的示例:从火花塞引导（H）由硅橡胶套筒，和一个临时套筒从的手指做出无粉乳胶手套（G）。比例尺= 0.5厘米（A）和（B），0.1厘米（C）和（D），并在100微米（E）。这个数字最初发表于14参考。 <a href="http://ecsource.jove.com/files/ftp_upload/55156/55156fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4.染色注:细胞壁或细胞膜可以用碘化丙锭或FM4-64染色，分别实现蜂窝分辨率在成像期间。可替代地，与标记到质膜荧光蛋白报告线可以用来6,16。 <ol><li>从成像盘移除水，并确保表面的介质是干，以避免稀释染料。 </li><li>下的立体显微镜，或者适用1mg / mL的碘化丙啶溶液，或80微克/毫升溶液FM4-64对解剖顶点用10μL移液管的20〜30μL。确保整个样品在染料覆盖，以保证均匀染色。 </li><li>如果使用碘化丙啶，或20分钟，如果使用FM4-64染色2分钟。 </li><li>用无菌，去离子水冲洗两次。 </li></ol> 5.萼片消融注:在第4阶段，萼片原基开始覆盖FM。随着年龄的增长，它们滤除从下层组织的荧光，并阻碍成像过程。萼片既可以使用金属销mounte手动移除上的销-台钳d或防止使用于新兴萼片原基激光烧蚀增长。可替代地，有可能在某些情况下使用突变体如apetala1-1，其中萼片缺失或由不覆盖FM而花的中心部分通常发展（ 图2F）17叶片状器官代替。 <ol><li> 上花芽阶段5和老年人使用的销保持虎钳的直金属针萼片消融（图2，E1-E2） <ol><li>放置解剖盘与体视显微镜下解剖的顶点，并设定放大倍数为最大。沉浸茎尖无菌，去离子水，或者用1000微升吸管水规律适用于茎尖而解剖萼片。 </li><li>随着针钳，定位在背面萼片顶端的针，切向相对茎尖。轻轻地从茎尖推离萼片直到它打破客场从花蕾。 </li><li>与正面的萼片同样继续，但推动萼片向茎尖。 </li><li>与侧萼片类似地进行，但沿径向相对于所述茎尖销定位，并从花蕾推萼片到一边，远离。 </li><li>沉浸在无菌，去离子水茎尖几分钟，以防止脱水。 </li></ol></li><li> 在阶段3-4萼片原基的激光烧蚀（图2，C5-D5） <ol><li>将带有在共聚焦显微镜阶段染，解剖顶点成像盘中。找到并集中在顶点仿佛准备图像（参见第6节，"成像装置"）。 </li><li>使用激光烧蚀系统软件，定义消融区，其对应于波峰和新兴的萼片原基的尖端便开始覆盖花分生组织（通常是前，在阶段3为背面和正面萼片，并且在后期3 /初阶段4为横向本身PALS）。 </li><li>设置激光功率和停留时间，以适当的参数，以消融足够的细胞，而不造成对下面的组织损伤太大。通常情况下，根据所使用的激光消融系统，适当的参数最初需要通过试错过程加以识别，以确保有足够的细胞去除，以防止萼片随后在FM增长，在不影响花的休息芽。使用过多的激光功率和住在损坏的花的中心时的效果，影响其生长和/或存活。一旦合适的参数已经确定，它们可再用于后续实验。 注意:如果初始消融证明不充分，也能够继续进行到在以下时间的第二烧蚀（ 图2，C4和D2）。 </li></ol></li></ol> 6.成像装置<ol><li> 使用正置显微镜。 <ol&gt; <li>与用无菌的去离子水的解剖顶点填充成像盘，使得介质的表面被覆盖在2.5-5毫米水（ 图1F）。完全浸没样品。 注意:当与一个直立显微镜成像，几个顶点可以被放置在相同的成像菜。然而，如果成像过程持续超过一小时，碘化丙啶趋于稀释，并且一些样品可能需要在成像之前重新染色。 </li><li>放置在显微镜载物台上（ 图1F）在成像盘。降低水浸渍透镜和提升载物台，使得透镜的尖端在水中骤降。小心前进以免粉碎解剖顶点或透镜浸入成像介质。 </li><li>如果气泡被截留在透镜的尖端，使与1000微升移液管的水射流将其删除。 </li><li>下落射荧光照明，位置解剖顶点到镜片字段的一个使用XY CONtroller。看通过目镜并专注于使用Z控制器样品。为了不粉碎样品谨慎。 </li><li>利用共聚焦显微镜软件，缩小在花芽进行成像，并继续成像你的样品。 </li></ol></li><li> 使用倒置显微镜。 <ol><li>使用1000μL移液管，把无菌的去离子水滴在透镜的尖端。 </li><li>保持成像盘倒置，并与1000微升移液管，无菌，去离子水滴加至解剖顶点。 </li><li>放置在显微镜载物台上（ 图1G）的成像盘倒置。谨慎行事，以免粉碎样品。 </li><li>下落射荧光照明，定位解剖顶点在使用XY控制器透镜的尖端。与Z控制器，小心地将阶段，直到样品达到的水在透镜的尖端下降。水柱应该形成betwEEN透镜和介质的前端。 </li><li>如果水柱没有形成，仔细一滴水添加到了1000微升吸管样品。临时套管添加到所述透镜，以便在其尖端，这有利于水柱（ 图1H和1I）的建立和维护添加更多的水。 </li><li>下落射荧光照明，看通过目镜并专注于使用Z控制器样品。谨慎行事，以免粉碎样品。 </li><li>使用共焦显微镜软件，放大在花芽要被成像，然后继续使样品成像。 </li></ol></li><li>如果执行时间推移实验，倒入水从成像盘的并关闭，以防止在脱水时间点之间。放置在漫长的一天（16小时光照），或连续天的样品中，16-22℃下条件下成像的菜。重染色的样品每个时间点之前。 注意:当细胞开发板的平花器官可以在花分生组织鸿沟分裂速度更快，电池一次或两次，每隔一天，和细胞系很容易追踪的时间点，每24小时。然而，如果需要的话，也可以像样品每六个小时。 </li></ol> 7.注意事项上的摄像参数注意:如何设置成像参数取决于所使用的共聚焦系统上不少。下面是其中一些参数可以与任何共聚焦显微镜使用的建议。有关的摄像参数更多的考虑，见讨论部分和参考文献14。 <ol><li>对于XY分辨率:使用1024x1024个有一个良好的细胞高分辨率。另外，使用512×512，以减少成像时间，在分辨率为代价。 </li><li>对于Z轴分辨率:设置步长到0.5-1.5微米。降低步长增加了Z轴分辨率，而且成像时间。 </li><li&gt;对于针孔:设置针孔1-1.5通风单元。增加针孔增加信号的强度，也可以从非焦平面的噪声。 </li></ol> 8.可视化共聚焦数据的<ol><li> 打开仍花发育的时间点的图像拍成电影。 <ol><li>在软件（ 如斐济）打开静止图像（以JPEG或TIF格式）。 </li><li>去图像&gt;栈&gt;图片到堆栈。开放的图像将在堆栈进行分组。 </li><li>如果图像的堆叠中的顺序不对应于时间顺序中，使用堆栈分拣机插件（插件&gt;栈&gt;栈分选）以重新排序。 </li><li>要打开堆栈成电影，转到文件&gt;另存为&gt; AVI。 </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+apetala-1+gene+during+Arabidopsis+floral+development.">Function of the apetala-1 gene during Arabidopsis floral development.</a> Plant Cell. 2 (8), 741-753 (1990).</li><li>Vernoux, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+auxin+signalling+network+translates+dynamic+input+into+robust+patterning+at+the+shoot+apex.">The auxin signalling network translates dynamic input into robust patterning at the shoot apex.</a> Mol. Systems Biol. 7, 508 (2011).</li><li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+plant+stem+cell+function+by+conserved+interacting+transcriptional+regulators.">Control of plant stem cell function by conserved interacting transcriptional regulators.</a> Nature. 517 (7534), 377-380 (2015).</li><li>Barbier de Reuille, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MorphoGraphX:+A+platform+for+quantifying+morphogenesis+in+4D.">MorphoGraphX: A platform for quantifying morphogenesis in 4D.</a> Elife. 4, 05864 (2015).</li><li>Barbier de Reuille, P. B., Robinson, S., Smith, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+cell+shape+and+gene+expression+in+the+shoot+apical+meristem+using+MorphoGraphX.">Quantifying cell shape and gene expression in the shoot apical meristem using MorphoGraphX.</a> Methods Mol. Biol. 1080, 121-134 (2014).</li></ol>

笔者有没有透露。

在这里，我们提供了一种活的发育拟南芥花蕾用共聚焦显微镜和浸水镜头成像的协议。虽然这可与竖直或倒置显微镜来完成，它是更容易和更快地使用前者。还值得一提的是，不同的共焦系统中的速度，灵敏度和能力来分离波长方面显著变化。最好的共聚焦显微镜现在允许图像几个不同的信道（ 例如 GFP，YFP，DsRed的和碘化丙啶; 图2G）的相同样品英寸一些共焦显微镜配备了光谱检测器，其可以从几个荧光团同时收集的荧光，并将其随后分离。当使用常规的检测器，但是，一组激光器和滤波器必须用于激发和荧光从不同的荧光团分离。一些荧光基团具有完全不同的发射光谱（ 例如 CFP的ðYFP），并且可以同时进行成像。其它的荧光团具有部分重叠的发射光谱（ 例如 GFP和YFP），并且通常需要单独成像，这增加了摄影时间。成像接近的荧光团还常常需要限制多少光谱的收集对于每个信道，以防止荧光从一个荧光团泄漏到另一信道。这使得采集到的信号，这可以通过增加激光功率和增益进行补偿的强度的损失。然而，延长成像时间和增加的激光功率可进行漂白和/或损坏样品。增加激光功率和/或增益也可能增加背景噪声。信号强度，分辨率和成像时间为每个样品的优化是通过试验和错误的过程中完成的，并涉及永久权衡。 该协议说明了如何执行花蕾上解剖茎尖的侧面开发的现场聚焦成像。它可以说，该茎尖没有连接到工厂的其余部分，事实上生长在含细胞分裂素可能影响SAM和花卉发展的媒介。然而，该介质通过试错的过程凭经验设计，保证了拍摄解剖顶端分生组织的正常plastochron产生新的花蕾，而这些花蕾一般在15发展。同样，萼片解剖似乎并不影响基因表达模式或花的其余部分的发展。其它协议的细节如何执行SAM的活共焦成像仍连接到设备的其余部分（ 例如，参考文献11）。这样的协议可以适于，并提供用于开发花，特别是研究细胞分裂素相关的处理时的成像有用的替代方案。他们提出几个缺点但是:茎伸长和曲折，并改变花芽的方向;一个第二成像时附着在植物的其他部分茎尖与正置显微镜工作得很好，它是更为复杂倒置显微镜这样做。 浸没透镜具有更好的数值孔径（NA）比"干"透镜（即从样品通过空气中分离即透镜），并因此提供所述样品，使用共焦显微镜时，这是关键的更精细的分辨率。使用水浸渍透镜因此提供了更好的NA比干透镜，同时防止茎尖从在成像过程期间脱水。虽然甘油和石油镜头具有更高的NA比水的镜头，它们需要使用盖玻片，这是非常不现实的一个样本茎尖，这也使在时间推移实验的规模日益扩大的。给定样品的大小（取决于花香阶段，栈可以是超过150微米厚），它也是重要的是使用一个透镜具有长的工作距离。我们通常使用20或40X透镜为1的NA和1.7-2.5毫米的工作距离。 共焦显微镜的软件提供了几种方法来可视化共聚焦数据，包括三维重建（ 图2，B1，B2和B4）和切片视图（ 图2，B3）。虽然最常用的三维视图是共焦Z-栈（ 图2，B1和B4），一些软件的最大强度投影（ 例如 ZEN和了Imaris）还提供透明性的观点，从较深的部位滤除信号样本（ 图2，B2），看着，表皮层表示发生过程的基因，或当它可以是有用的。信号强度既可以用单一颜色（ 图2，B1）进行编码，使用像素INTEnsity，或者使用彩色码（ 图2，B4）。 现场共聚焦成像，不仅提供了宝贵的定性的见解花发育，还可能提供发育生物学家提供了丰富的定量数据。不同的软件（ 例如了Imaris，斐济，MARS-ALT，MorphographX 15，20，21）允许数或表达一个或几个记者，或在不同细胞中的报道基因的表达水平的细胞的体积的定量。这样的软件也可用于执行自动细胞分割，跟踪细胞谱系和量化的增长。这种不同的软件提供类似的工具，但也不同于在许多方面。 MARS-ALT和MorphoGraphX被植物15，20，21而设计的，不象一了Imaris第二斐济。 MorphoGraphX仅允许表皮层的分割和分析，而以及了Imaris MARS-ALT允许整个样品的分割和分析。然而，MARS-ALT需要相同的样品的从三个不同的角度15的现有成像和了Imaris只需要一个单一的叠层。获得定量信息是我们进一步发展花卉的理解是至关重要的。

大多数植物体的形成后embryonically从位于或接近尖端干细胞的群体 - 或者顶端分生组织 - 芽和根的。所有植株的地上结构派生从茎尖分生组织（SAM），它连续地产生在其侧面侧生器官:在营养生长期的叶，花分生组织（FMS），它转变到生殖阶段之后。反过来外长发育成花朵。虽然拟南芥SAM产生侧生器官一次一个，以迭代的，螺旋形图案，FMS产生内的四个轮生四种类型的花器官，以部分同步的方式，与多个发育程序同时解开。不同的花器官身份的规范基础的遗传网络已经部分破译（综述见参考文献1，2）花DEVELOPME的，但很多方面个nt，诸如花器官定位和边界的旋涡之间的定义，仍然知之甚少。 植物发育早期的分子遗传学研究主要依赖于技术，如原位杂交和GUS记者分析基因表达。虽然这些方法都提供了丰富的信息，并极大地促进了我们的植物生长和花卉发展的理解，也有很大的局限性:他们缺乏良好的细胞高分辨率，不允许在同一个易于观察多个基因的表达模式样本，并且重要的是，可以仅适用于死的，固定的组织。在激光共聚焦显微镜的最新进展已克服这些限制，并提供发育生物学家提供了强大的工具来研究这个过程潜在的植物形态。特别是，共聚焦显微镜允许在他们的生活形成组织和器官的观察，这为critical全面理解一个典型的动态过程，如发展。 活共焦成像已被广泛用于分析由SAM植物的地上生长和生产横向器官（ 例如参照图3，4，5，6），但与的少数报道的例外（ 例如参考文献7，8， 9，10），它并没有被广泛地应用到花发育的研究。对于SAM的活共焦成像协议可（ 如参考11，12），并提供如何图像显影花蕾周围的SAM了良好的基础。然而，成像花蕾提出具体的挑战:例如，花芽很快变得比SAM更大，和在阶段4，萼片开始覆盖FM（阶段如参考文献13所述），和调光从下面的组织（ 图2A）的荧光。在这里，我们提供详细的协议说明了如何使用任一垂直或倒置显微镜和水浸渍透镜显影拟南芥花芽执行实时共焦成像。我们以前出版的参考14此协议。

<table><tbody><tr><td>Forceps&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>72701-D</td><td>Dumont Style 5 Inox, for apex dissection</td></tr><tr><td>Pin Vise</td><td>Ted Pella, Inc.</td><td>13560</td><td>80 cm long, for sepal ablation</td></tr><tr><td>Straight Stainless Steel Needles&amp;nbsp;</td><td>Ted Pella, Inc.</td><td>13561-50</td><td>0.1 mm Diameter, 1.2 cm long, for sepal ablation</td></tr><tr><td>Round plastic boxes</td><td>Electron Microscopy Sciences</td><td>64332</td><td>transparent, 6 cm diameter, 2 cm deep, to use as dissecting dishes</td></tr><tr><td>Rectangular hinged boxes</td><td>Durphy Packaging Co.&amp;nbsp;</td><td>DG-0730</td><td>transparent, 2-7/8&amp;rdquo; long, 2&amp;rdquo; wide, 1-1/4&amp;rdquo; deep, to use as imaging dishes with an upright microscope</td></tr><tr><td>Tissue Culture Dishes, Polystyrene, Sterile</td><td>VWR</td><td>25382-064</td><td>transparent, 3.5 cm diameter, 1 cm deep,to use as imaging dishes with an inverted microscope</td></tr><tr><td>P10 and P1000 pipettes&amp;nbsp;</td><td></td><td></td><td>with corresponding filter tips</td></tr><tr><td>Stereomicroscope</td><td></td><td></td><td>with an 80-90X maximum magnification</td></tr><tr><td>Laser ablation system</td><td></td><td></td><td>for sepal ablation</td></tr><tr><td>Laser scanning confocal microscope system</td><td></td><td></td><td></td></tr><tr><td>20 or 40X water-dipping lens</td><td></td><td></td><td>with a NA of 1 and a working distance of 1.7-2.5 mm</td></tr><tr><td>Agarose</td><td></td><td></td><td></td></tr><tr><td>Sucrose</td><td></td><td></td><td></td></tr><tr><td>Murashige and Skoog basic salt mixture</td><td>Sigma-Aldrich</td><td>M5524</td><td>without vitamins</td></tr><tr><td>Myo-inositol</td><td>Sigma-Aldrich</td><td>I7508</td><td>vitamin</td></tr><tr><td>Nicotinic acid</td><td>Sigma-Aldrich</td><td>N0761</td><td>vitamin</td></tr><tr><td>Pyridoxine hydrochloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P6280</td><td>vitamin</td></tr><tr><td>Thiamine hydrochloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T1270</td><td>vitamin</td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8790</td><td>vitamin</td></tr><tr><td>N6-Benzyladenine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B3408</td><td>BAP, a cytokinin</td></tr><tr><td>Propidium iodide solution</td><td>Sigma-Aldrich</td><td>P4864</td><td>1 mg/mL solution in water, to stain the cell walls</td></tr><tr><td>FM4-64</td><td>Invitrogen</td><td>F34653</td><td>dilute in water to 80&amp;nbsp;&amp;mu;g/mL, to stain the plasma membranes</td></tr></tbody></table>

live confocal imaging of developing arabidopsis flowers

植物生长发育的研究长期依赖于使用死，固定的组织，缺乏适当的细胞高分辨率的实验技术。在激光共聚焦显微镜的最新进展，与众多荧光团的发展相结合，克服了这些问题，并打开的可能性，同时学习几种基因的表达，具有良好的细胞高分辨率，活样本英寸现场共聚焦成像提供植物生物学家提供了强大的工具来研究开发，并已被广泛用于研究根系的生长和侧生器官对茎尖分生组织的侧翼形成。然而，它并没有被广泛地应用到花发育的研究，部分原因是由于特定于成像花卉，如生长在花分生组织的萼片，和下面的组织筛选出荧光挑战。在这里，我们提出了一个详细的协议对现场进行直播共焦成像，阿拉伯发展idopsis花芽，即使用竖直或倒置显微镜。

图2呈现的表达不同的荧光报告基因活拟南芥花芽共焦Z-堆叠的不同视图，以及与任一碘化丙啶（ 图2，A-C5和G）或FM4-64（ 图2，E1-F）到染色提供清晰的细胞高分辨率。大多数共聚焦系统允许两个荧光团的具有非重叠发射光谱如GFP或YFP与任一碘化丙锭或FM4-64（ 图2A - 2F）一起成像。最好的共焦系统还能够将多个荧光团与同一样品中的部分重叠的发射光谱，例如GFP和YFP，DsRed的和和碘化丙啶（ 图2G）中分离出来。 的时间推移实验三个例子显示在图2，并显示后，无论是用激光烧蚀系统中执行的萼片消融花芽发育正常（ 图2，C1-C5和D1-D5）或手动销虎钳和金属销（ 图2，E1-E2）。注意，在图2中所示的花芽，E1-E2是从超人-1突变体的植物，这就是为什么心皮原基不会在芽的中心观察到的，在阶段7激光烧蚀花芽的新兴萼片原基在图2中所示，C1-C5防止它们覆盖FM，这仍然可以在阶段5（ 图2，C5）进行成像。相反，在图2所示的花芽的情况下，D1-D5，激光烧蚀只延迟萼片增长，但萼片最终增长了第5阶段（ 图2，D5到覆盖大部分FM的</STRONG&gt;）。相反，如果激光烧蚀过程中使用过多的激光功率和停留时间，损害蔓延到花的中央部分，并影响其生长，有时会导致整个花芽随后死亡。 <img alt="图2" src="/files/ftp_upload/55156/55156fig2.jpg" /> 图2:现场花蕾共聚焦Z-栈的可视化。 A和B2是透明度图。 B1和B4-G是最大密度投影; B3是正交的切片视图。 （A - E2）鲜花表达金星记者对APETALA3基因（绿色）;细胞壁用碘化丙啶染色（红色，除了B4，绿色）。 （A）花序;数字表明花香阶段;在阶段4萼片和5个花滤除金星记者的荧光，这通常FO有效值的环。请注意，一些花芽出现较花序倾斜。 （B1 - B4）的阶段5花芽的同一共焦Z堆叠的四种不同的观点:最大密度投影与金星信号强度在绿色（B1）与像素强度编码（B1和B4）或用火颜色代码（B4;颜色代码在面板的底部示出）;透明度图（B2）和正交的切片视图（B3;切片的XY，XZ和YZ方向在面板被指示）。 （C1 - C5）4天的个体花芽从第3阶段至第5阶段的时间推移;激光消融（标记为白色性状）第1天进行3天就足以防止从萼片覆盖花芽的中心在阶段5（D1 - D5）4天的个体花蕾的从时间推移阶段3至第5阶段;在第1天，第2和3进行激光消融不足以防止萼片从覆盖花芽的中心。 （E1 - E2）个体花芽人工剥离背面和正面萼片（E1）之后，并且所有萼片（E2）;白色箭头指示剩余萼片;白色星号表示从除去萼片产生的疤痕。 （F）阶段7 apetala1-1花表达DR5-3xVenusN7记者18（绿色）;质膜用FM4-64（红色）染色;蓝色箭头指示的叶状结构是取代萼片和不覆盖的花芽。 （G）第4阶段花芽表达为DORNROSCHEN-LIKE 7（绿色），一个金星记者SUPERMAN（红色）的荧光GFP报告和DsRed的记者CLAVATA3 19（青色）;细胞壁用碘化丙啶（灰色）染色。 D:天; ST:阶段。比例尺= 25微米;规模是在B1，B2和B4相同，在C1-C5和D1中-D5。该图是从参照图14进行修改。 <a href="http://ecsource.jove.com/files/ftp_upload/55156/55156fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Live Confocal Imaging of Developing Arabidopsis Flowers at JoVE.com

Live Confocal Imaging of Developing Arabidopsis Flowers

现场共聚焦成像提供生物学家提供了强大的工具来研究开发。在这里，我们提出了一个详细的协议制定拟南芥花的活共焦成像。

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

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14.8K 次观看.  California Institute of Technology. 现场共聚焦成像提供生物学家提供了强大的工具来研究开发。在这里，我们提出了一个详细的协议制定拟南芥花的活共焦成像。 

视频: Live Confocal Imaging of Developing Arabidopsis Flowers

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Lhgra 介导的反激活在拟南芥诱导、细胞类型特异性表达中的表达

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

科研

遗传学

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

表征在拟南芥胚方法

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

发育生物学

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

不同植物种的射击顶端共聚焦活图

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

细胞细胞成像的微管细胞和微机械操作的 阿拉比多普西斯拍摄 阿皮克·梅里斯特姆

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

生物学

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

一种用于阴南芥叶片安装的通用方法

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

住发展拟南芥花的共焦成像

摘要

探索更多视频

Lhgra 介导的反激活在拟南芥诱导、细胞类型特异性表达中的表达

表征在拟南芥胚方法

不同植物种的射击顶端共聚焦活图

细胞细胞成像的微管细胞和微机械操作的阿拉比多普西斯拍摄阿皮克·梅里斯特姆

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

细胞特异性分析

从长期来看，高解析度激光共聚焦时间流逝成像

测量时空的Ca

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

一种用于阴南芥叶片安装的通用方法

住发展拟南芥花的共焦成像

摘要

探索更多视频

Lhgra 介导的反激活在拟南芥诱导、细胞类型特异性表达中的表达

表征在拟南芥胚方法

不同植物种的射击顶端共聚焦活图

细胞细胞成像的微管细胞和微机械操作的 阿拉比多普西斯拍摄 阿皮克·梅里斯特姆

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

细胞特异性分析

从长期来看，高解析度激光共聚焦时间流逝成像

测量时空的Ca

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

一种用于阴南芥叶片安装的通用方法

细胞细胞成像的微管细胞和微机械操作的阿拉比多普西斯拍摄阿皮克·梅里斯特姆