I am grateful to Dr. Masaru Fujimoto for his technical suggestions for VAEM. I am also grateful to Prof. Koh Iba and Dr. Mimi Hashimoto-Sugimoto for providing GFP-PATROL1 transgenic plants, and discussions about PATROL1. I thank Prof. Seiichiro Hasezawa for his continuing support of my work. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 25711017.

1.准备幼苗<ol><li>消毒的种子。 <ol><li>和1μl10％的Triton X-100，以500微升无菌水:通过添加500微升的NaClO（5.0％有效氯）制备灭菌溶液。 </li><li>广场约 10株转基因A.拟南芥种子携带的GFP-PATROL1 8成 1.5毫升管中。 </li><li>加入1毫升70％的乙醇溶液，并通过反转五次拌匀。离开1分钟。 </li><li>观察种子沉到试管底部。在洁净层流柜，轻轻取出使用微量的70％的乙醇，并加入1毫升消毒液。颠倒五次拌匀，离开5分钟。 </li><li>洗净的种子。在无菌条件下仍然工作在一个干净的长椅，轻轻取出使用微量的解决方案，并加入1毫升无菌水。重复此五次。所述灭菌的种子可以被存储在无菌水中，在4℃下2天。 </li></ol></li><li>所以瓦特上的0.5％吉兰糖胶凝固1/2 MS培养基板（pH为5.8）9的消毒的种子。胶带盖到使用外科带的两层板。 </li><li>孵育板在黑暗中，在4℃冷库室，O / N。 </li><li>板转移到生长室设定在23.5℃，用100微摩尔米-2秒 -1白色灯12小时/ 12小时光照-黑暗周期，并培育7天。幼苗具有约1mm长子叶然后可以收获。 </li></ol> 2.天空跌落安装子叶标本注:在试样准备VAEM观测的一个重要因素是避免检体和盖玻璃之间的包含气泡。气泡通过使折射率差大大降低VAEM的图像质量。一个简单的方法，我们称之为"天上降"的安装，可以用来避免泡沫在A之间拟南芥子叶和盖玻璃。这应做之前立即观察。 <ol><li>放置30微升基础缓冲液[5mM的2-（N-吗啉代） -乙磺酸三（MES-TRIS）在pH 6.5，50mM的氯化钾，100μM的氯化钙 2]在载玻片上的中心（尺寸:76 ×26毫米，厚度:1.0-1.2毫米）。 </li><li>从7日龄幼苗用解剖剪刀取出一个子叶。浮在子叶与观察侧朝上的基础缓冲器下降（图1，步骤1）。 </li><li>放置30微升缓冲基底的上玻璃盖的中心（尺寸:18×18毫米，厚度:0.12-0.17毫米）（图1，步骤2）。打开玻璃盖倒挂平缓。表面张力将防止脱落缓冲器降。握住玻璃盖用镊子在一个边缘。将相对的边缘上的载玻片，使得缓冲器降是约高于子叶。 </li><li>与盖玻璃载玻片上的边缘仍然，并仍然保持与镊子相对边缘，调整的玻璃盖下的缓冲器下降的位置，使得它直接子叶样品（图1，步骤3）的上方。放开玻璃盖。安装在导致制剂无气泡样本的下降。 </li><li>擦去使用无绒毛组织中多余的缓冲区。立即观察的制备（见步骤3）。 注意:没有必要密封样品。 </li></ol> 3. VAEM观察与电影采集注:TIRF显微镜系统9在本研究中使用的描述如下:一个倒置显微镜配备有TIRF单元和TIRF物镜具有1.49的数值孔径。为激光入射角的计算机化的控制，控制盒被使用。绿色荧光蛋白（GFP）是激发488nm的光泵浦半导体激光器，和叔他荧光通过510-550纳米的带通滤波器检测，以防止自体荧光从叶绿体中。光纤输出功率实测最大值为13.0-13.5毫瓦。进行检测，电子倍增电荷耦合器件（EM-CCD）照相机头系统和一个C型相机放大率改变单元被使用。 <ol><li>校准根据制造商的说明将激光定心和聚焦。 注意:此步骤是为精确VAEM观测至关重要。强烈推荐的激光光路调整程序定期检查，适当的显微镜下，进行了。 <ol><li>确定照射在显微镜房间的​​天花板的物镜无光路的中心位置。为了纪念中央位置，将彩色圆封在天花板上（ 图2，第1步，2）。 </li><li>照亮带有物镜（图2，步骤3，4）的上限。动第illuminated区域的中心位置（图2，步骤5）。聚焦激光（图2，步骤6）。微调聚焦激光中心（ 图2，第7步）的位置。 </li></ol></li><li>设置一个标本在显微镜的阶段，选择单元格观察用明视场照明。 </li><li>检查荧光蛋白可以在细胞中可以观察到，并设置的 Z轴位置上使用表面荧光照明（图3A）的细胞表面上。 </li><li>执行VAEM意见。 <ol><li>与控制器箱倾斜的激光束的进入角逐渐。同时，仔细查看实时图像。最初，该图像会模糊（图3B）。 </li><li>作为激光角度越大，VAEM图像将变得模糊少，最终产生一个清晰的图像（图3C 和 D）。在这一点上，停止increa唱激光角。如果荧光信号消失，减少到一浅角度。 </li><li>微调的激光入射的角度，以获得更好的形象。微调的 Z轴位置的也可以提高图像。如果有必要，调整光学参数（激光功率，波长和过滤器设置）和图像传感器参数[图像尺寸，曝光时间，增益，数字化仪和电子倍增（EM）增益。 注意:在上述的TIRF显微镜设备的情况下，有代表性的参数如下。镜头只是在镜头前的放大倍率:2倍;激光输出:1.0毫瓦;图像尺寸:512×512像素;曝光时间:100毫秒;增益:5×;数字化仪:11兆赫;和EM增益:100。 </li></ol></li><li>获取电影如使用商业软件显微镜多页TIFF图像文件。这里，电影是GFP标记点闪烁气孔细胞的表面上的。 注:代表图像采集条件为FOL低点。采集模式:流到RAM;帧数:600;和多页TIFF文件大小:〜302 MB。 </li></ol>绿色荧光蛋白标记点停留时间4 Kymograph分析定量使用斐济软件<ol><li>安装斐济（"斐济是仅有的ImageJ"）的软件10使用作者的指令（http://fiji.sc/Fiji）。 </li><li>运行斐济和使用斐济菜单"文件- 打开 "打开采集的多页TIFF文件。 </li><li>将感兴趣的站点线路，使用斐济的工具栏菜单中的" 直线选择工具 "。或者，用" 分段线路选择工具 "或" 写意线路选择工具 "。在此处呈现的结果，为100像素（对应于8.0微米）的直线放置的附属小区（图4A）上。 </li><li>请使用斐济菜单"图像-栈，动态一个重新分区图像kymograph </e米&gt;"（http://fiji.sc/Dynamic_Reslice）（ 图4B）。任选地，在一个复选框窗口"垂直翻转 "和"90度旋转"也可（在此处呈现的结果未使用）在kymograph图像的x轴和y轴表示线位置（相当于100像素8.0微米）和观测时间（相当于600个像素至60秒），分别如果kymograph图像不令人满意，位置和类型（直的，分割和多页图像上线的徒手）可被改变。作为线变化，kymograph图像动态变化，保存kymograph形象使用斐济菜单"文件- 另存为页TIFF"TIFF文件。 </li><li>测量在时间轴的方向的斑点的长度。 <ol><li>要进行降噪处理，应用高斯滤波器使用斐济菜单"过程滤镜-高斯模糊 "的kymograph图像。在"西格玛（拉德IUS）"参数是可调的（1个像素的半径被用于所提出的结果）。 </li><li>段由阈值信号的区域，利用斐济菜单"图像-调整-阈值 "。 注:有几个阈值的算法可供选择。在提出的结果中，"颜"算法被选择（图4C）。 </li><li>准备使用菜单来测量时间（Y）的斑点的轴长"分析，集测量"。检查" 设置测量"窗口中的" 矩形边框 "。理想的是，区域集应当尽可能小，以排除噪声。这里，最小面积设定在50个像素。测量时间（Y）的BLOB的使用菜单轴长度"分析-分析粒子"。要获得的结果值，证明了测量区域的信誉，勾选" 显示结果 "和" 添加到管理器&lt;/ em&gt;的"框。 </li><li>在"高度"的列值的时间（y）的a轴的长度为所测量的斑点（图4D）。保存使用结果表菜单"文件- 另存为 "的价值计算和处理团块图像的持续时间使用的统计数据包和/或电子表格软件（ 图4E）的数据集。 </li></ol></li></ol>

<ol>
	<li>Konopka, C. A., Bednarek, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+dynamics+and+functional+redundancy+of+the+Arabidopsis+dynamin-related+isoforms+DRP1A+and+DRP1C+during+plant+development.">Comparison of the dynamics and functional redundancy of the Arabidopsis dynamin-related isoforms DRP1A and DRP1C during plant development.</a> Plant Physiol. 147 (4), 1590-1602 (2008).</li><li>Fujimoto, M., 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=Arabidopsis+dynamin-related+proteins+DRP2B+and+DRP1A+participate+together+in+clathrin-coated+vesicle+formation+during+endocytosis.">Arabidopsis dynamin-related proteins DRP2B and DRP1A participate together in clathrin-coated vesicle formation during endocytosis.</a> Proc. Natl. Acad. Sci. USA. 107 (13), 6094-6099 (2010).</li><li>Higaki, T., Sano, T., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+microfilament+dynamics+and+actin+side-binding+proteins+in+plants.">Actin microfilament dynamics and actin side-binding proteins in plants.</a> Curr. Opin. Plant Biol. 10 (6), 549-556 (2007).</li><li>Rosero, A., &#381;&#225;rsky, V., Cvr&#269;kov&#225;, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtFH1+formin+mutation+affects+actin+filament+and+microtubule+dynamics+in+Arabidopsis+thaliana.">AtFH1 formin mutation affects actin filament and microtubule dynamics in Arabidopsis thaliana.</a> J. Exp. Bot. 64 (2), 585-597 (2013).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+fluorescence+microscopy+in+cell+biology.">Total internal reflection fluorescence microscopy in cell biology.</a> Traffic. 2 (11), 764-774 (2001).</li><li>Konopka, C. A., Bednarek, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable-angle+epifluorescence+microscopy:+a+new+way+to+look+at+protein+dynamics+in+the+plant+cell+cortex.">Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex.</a> Plant J. 53 (1), 186-196 (2008).</li><li>Wan, Y., Ash, W. M., Fan, L., Hao, H., Kim, M. K., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable-angle+total+internal+reflection+fluorescence+microscopy+of+intact+cells+of+Arabidopsis+thaliana.">Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana.</a> Plant Methods. 7 (27), (2011).</li><li>Hashimoto-Sugimoto, M., 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=A+Munc13-like+protein+in+Arabidopsis+mediates+H+-ATPase+translocation+that+is+essential+for+stomatal+responses.">A Munc13-like protein in Arabidopsis mediates H+-ATPase translocation that is essential for stomatal responses.</a> Nat. Commun. 4, 2215 (2013).</li><li>Higaki, T., Hashimoto-Sugimoto, M., Akita, K., Iba, K., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+environmental+responses+of+PATROL1+in+Arabidopsis+subsidiary+cells.">Dynamics and environmental responses of PATROL1 in Arabidopsis subsidiary cells.</a> Plant Cell Physiol. 55 (4), (2014).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat. Methods. 9 (7), 676-682 (2012).</li><li>Chebli, Y., Kaneda, M., Zerzour, R., Geitmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+wall+of+the+Arabidopsis+pollen+tube-spatial+distribution,+recycling,+and+network+formation+of+polysaccharides.">The cell wall of the Arabidopsis pollen tube-spatial distribution, recycling, and network formation of polysaccharides.</a> Plant Physiol. 160 (4), 1940-1955 (2012).</li><li>Fujimoto, M., Suda, Y., Vernhettes, S., Nakano, A., Ueda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol+3-kinase+and+4-kinase+have+distinct+roles+in+intracellular+trafficking+of+cellulose+synthase+complexes+in+Arabidopsis+thaliana.">Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.</a> Plant Cell Physiol. 56 (2), 287-298 (2015).</li><li>Li, X., Wang, X., Yang, Y., Li, R., He, Q., Fang, X., Luu, D. T., Maurel, C., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+of+PIP2;+1+dynamics+and+partitioning+reveals+multiple+modes+of+Arabidopsis+plasma+membrane+aquaporin+regulation.">Single-molecule analysis of PIP2; 1 dynamics and partitioning reveals multiple modes of Arabidopsis plasma membrane aquaporin regulation.</a> Plant Cell. 23 (10), 3780-3797 (2011).</li><li>Li, R., Liu, P., Wan, Y., Chen, T., Wang, Q., Mettbach, U., Baluska, F., Samaj, J., Fang, X., Lucas, W. J., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+membrane+microdomain-associated+protein,+Arabidopsis+Flot1,+is+involved+in+a+clathrin-independent+endocytic+pathway+and+is+required+for+seedling+development.">A membrane microdomain-associated protein, Arabidopsis Flot1, is involved in a clathrin-independent endocytic pathway and is required for seedling development.</a> Plant Cell. 24 (5), 2105-2122 (2012).</li><li>Staiger, C. J., Sheahan, M. B., Khurana, P., Wang, X., McCurdy, D. W., Blanchoin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+filament+dynamics+are+dominated+by+rapid+growth+and+severing+activity+in+the+Arabidopsis+cortical+array.">Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array.</a> J. Cell Biol. 184 (2), 269-280 (2009).</li><li>Ueda, H., 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=Myosin-dependent+endoplasmic+reticulum+motility+and+F-actin+organization+in+plant+cells.">Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells.</a> Proc. Natl. Acad. Sci. USA. 107 (15), 6894-6899 (2010).</li></ol>

在这个视频文章，方案给出了监测和测量GFP-PATROL1点对拟南芥的气孔复杂的动态行为。如图所示，VAEM观察是一个强大的工具，用于植物细胞表面的实时成像。下这里使用的GFP-PATROL1监测实验条件下，有用于视频捕捉1分钟的样品中很少荧光光漂白，因为高度敏感的EM-CCD的允许使用在VAEM光学相对弱激发激光的。激光定心和聚焦，每次实验开始前，对于成功VAEM观察重要。用户应该由专业的工作人员从选择的显微镜公司使用VAEM前培训。 VAEM表明GFP-PATROL1具有动态定位与不动的点状隔室上闪烁质膜。荧光标记质膜的质子泵AHA1误升在patrol1突变保卫细胞和辅助细胞，ocalizes如先前报道8,9。因此，VAEM-可视GFP-PATROL1点动力学可表示输送质子泵入质膜。 GFP-PATROL1这种动态行为很难想象使用常规电反射镜型共聚焦激光扫描显微镜，表示VAEM在植物细胞表面露出实时蛋白动力学的有用性。 GFP-PATROL1观测是在植物细胞生物学VAEM应用的一个例子。 VAEM将适用于众多的事件在细胞质中的植物细胞的薄皮层的监视（例如，酶分泌细胞壁合成或修饰11,12，质膜蛋白动力学7,13,14，皮质微管动力学4，微丝4,15的重排和细胞器移动6）。种类繁多的植物细胞的科学家应该获益适合从VAEM技术。 生物图像数据的定量评价近来已成为重要的是，在显微镜设备和计算技术进步推动的。含512（x）的×512（y）的600×（t）的像素VAEM电影数据可以在只有几分钟容易地获得。图像分析有助于不仅在测量荧光标记的蛋白质的寿命或运动，而且在从大型，多维图像数据集挖掘的生物信息。甲kymograph是一个基本的，但有价值的，办法来确定靶蛋白运动从VAEM电影数据，如图由蛋白质的连续募集到质膜2。所述kymograph允许的GFP-PATROL1点&#39;的停留时间的可视化，以及它们对质膜低蠕动， 如图4，反之，一个kymograph已经用于评估更复杂的运动，如胞质流效果有限。欲了解更多complicated运动，其他的图像分析方法，如光流16将是有益的。

The plant cell surface, including the plasma membrane and its immediately adjacent cytoplasm, is the main region of a plant cell&#8217;s perception and integration of biotic and abiotic cues from the extracellular environment. In response to these cues, cell surface components including plasma membrane proteins and the cortical cytoskeleton undergo dynamic changes, on a time scale of seconds to minutes1-4. Thus, real-time and high-resolution imaging of fluorescent proteins on the cell surface can illuminate a plant&#8217;s responses to environmental cues at the molecular level.
Confocal laser scanning microscopy is a powerful tool for determination of fluorescently-tagged protein localization3, however, it is often difficult to monitor the real-time protein dynamics because of its relatively long capturing times. An emerging technique for real-time monitoring of proteins in the plant cell is variable angle epifluorescence microscopy (VAEM), which is an adaptation of equipment usually used for total internal reflection fluorescence (TIRF) microscopy. In TIRF microscopy, the fluorescence-excitation light source is an evanescent light field that is generated when the entry angle of the laser is shallow enough to totally internally reflect light at the glass&#8211;water interface. The penetration depth of the evanescent light field is around 100 nm. TIRF microscopy is an outstanding tool for single molecule imaging, such as the detection of exocytosis in animal cells5. However, evanescent light cannot reach plasma membranes or the cortical cytoplasm in plant cells, because they have thick cell walls. Recently, TIRF microscopy equipment has been adapted by plant cell biologists, observing that a laser, if angled slightly more deeply than when being used to induce total internal reflection phenomena, could excite the surface of plant cell samples, resulting in high-quality plant cell imaging6,7. The excitation illumination depth is varied by adjusting the entry angle of the laser; therefore, this technique is described as VAEM. This optical system is also called variable angle TIRF microscopy (VA-TIRFM) because there is a possibility that total reflection may take place at the cell wall-periplasm interface7, however, the term VAEM is used in this article, as per the first report in plants6.
The goal of this protocol is to demonstrate practical procedures for using VAEM to visualize fluorescently-tagged protein dynamics on plant cell surfaces. Additionally, an image analysis protocol to quantify the residence time (duration of presence) of molecules is described for VAEM movie analysis. GFP-PATROL1 dot blinking on stomatal complex cells in Arabidopsis thaliana cotyledons is used as an example. PATROL1 was identified by forward genetic approaches as a causal gene of a stomatal response defect mutant in A. thaliana8. PATROL1 is a plant homolog of MUNC-13, which is a priming factor in synapse vesicle exocytosis8. In response to environmental cues, such as light or humidity, it is thought that PATROL1 reversibly regulates the delivery of a proton pump to plasma membranes in the stomatal complex. Stomatal complexes each comprise a pair of guard cells8 and subsidiary cells9, and they require a proton pump for stomatal movement. In these cells, GFP-tagged PATROL1 localizes to dot-like structures that remain on the plasma membrane for less than 1 min9.

<table><tbody><tr><td>Inverted microscope</td><td>Olympus</td><td>IX-73</td><td></td></tr><tr><td>TIRF unit</td><td>Olympus</td><td>IX3-RFAEVAW</td><td></td></tr><tr><td>TIRF objective lens&amp;nbsp;</td><td>Olympus</td><td>UAPON 100 &amp;times; OTIRF&amp;nbsp;</td><td>NA = 1.49</td></tr><tr><td>Laser angle control box</td><td>Chuo Seiki</td><td>QT-AK</td><td></td></tr><tr><td>Optically pumped semiconductor laser</td><td>Coherent</td><td>Sapphire&lt;sup&gt;TM&lt;/sup&gt; LP USB 488-20 CDRH Laser</td><td></td></tr><tr><td>510&amp;ndash;550 nm band-pass filter</td><td>Olympus</td><td>U-FBNA</td><td></td></tr><tr><td>EM CCD camera</td><td>Hamamatsu Photonics</td><td>ImagEM C9100-13</td><td></td></tr><tr><td>C-mount camera magnification change unit&amp;nbsp;</td><td>Olympus</td><td>U-TVCAC</td><td></td></tr><tr><td>MetaMorph software</td><td>Molecular Devices</td><td>MetaMorph version 7.7.11.0</td><td></td></tr><tr><td>TIRF microscopy manual</td><td>Olympus</td><td>AX7385</td><td>Instructions: Total Internal Reflection Illumination System (Printed in Japan on August 24, 2012)</td></tr><tr><td>Immersion oil</td><td>Olympus</td><td>Immersion Oil Typr-F</td><td>ne = 1.518 (23 degrees)</td></tr></tbody></table>

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.

在这个视频文章，为VAEM A中观察GFP-PATROL1的协议提供了拟南芥子叶气孔复杂的细胞。天空降安装是一个简单的制备方法，可以帮助减少在 A中的VAEM制剂气泡的发生拟南芥子叶（ 图1）。 Overtilting条目激光和/或标本VAEM z定位将提供的图像不清晰。如果出现这种情况，则建议立即从样品上方的位置重新开始，作为判断通过荧光照明。后的VAEM观察几天的经验，应该有可能获得VAEM电影成功和常规，如这里所示的GFP-PATROL1的斑点闪烁之一。使用kymograph分析在图4中，185的GFP-PATROL1点从12个独立的副卫细胞在A中的停留时间呈现拟南芥子叶进行了测量。拟合密度功能显示，大部分GFP-PATROL1点的子公司小区居住的人，为2至10秒，大约3.5秒（ 图4E）的峰值，这表明快速胞吐/质子泵内吞作用的子公司细胞表面。 <img alt="图1" src="/files/ftp_upload/53437/53437fig1.jpg" width="700" /> 图1示意图的天空下降安装方法:步骤1:试样拟南芥子叶浮在基底缓冲器的载玻片上的下降。步骤2:将一滴基础缓冲器滴被放置在盖玻璃上。步骤3:玻璃盖与降上下翻转并且液滴被置于上述载玻片上的标本。步骤4:盖玻璃被释放，并且降落在到试样。 （上）鸟瞰图。 （底部）横向和放大图<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53437/53437fig1large.jpg" target="_blank">。PLE</a>ASE点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53437/53437fig2.jpg" width="700" /> 图2.校准激光的定心和聚焦步骤 1:在显微镜左轮手枪的空位置被选择为激光光发送到显微镜上面的天花板。步骤2:中央位置用在天花板上的着色圆圈密封标示。第3步:客观选择。步骤4:检查通过物镜照射的激光光线的位置。步骤5:照明被调整到标记的中心位置。步骤6:激光聚焦。第七步:将聚焦激光移动到标记的中心位置<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53437/53437fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3.比较荧光显微镜和可变角度荧光显微镜（VAEM）的。（A）激光在荧光显微镜的光路示意图。 （B）中的GFP-PATROL1在拟南芥的7天龄的幼苗的子叶表面代表萤光图像。比例尺为5微米。激光在VAEM的光路的（C）的示意图。注意，激光的条目是倾斜的，并且它照亮试样的表面与可变深度。同一区域的（D）的VAEM图像，如图（B）中。比例尺为5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53437/53437fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图4. Kymograph分析，以量化GFP-PATROL1点的停留时间。（A）VAEM图像直线放置做出kymograph。比例尺为5微米。 （二） 沿 （a）所示的A线kymograph图像。 （二）（ 三）二值图像的基础上甄子丹的算法，在斐济的软件生成。黄线显示​​GFP-PATROL1信号的自动检测到的区域。 （四）截图斐济的结果表"分析粒子"的功能。 （E）的子公司细胞GFP-PATROL1点的停留时间柱状图。红线显示密度函数。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53437/53437fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy at JoVE.com

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

该协议的目的是演示如何监测荧光标记蛋白动力学上的植物细胞表面与可变角荧光显微镜，表示闪烁GFP标记PATROL1，一个膜运输蛋白质的点，在所述气孔络合物在细胞皮质拟南芥。

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 ...

real-time-imaging-plant-cell-surface-dynamics-with-variable-angle

Nanyang Technological University

Research

JoVE Journal

Biology

9.0K 次观看.  The University of Tokyo. 该协议的目的是演示如何监测荧光标记蛋白动力学上的植物细胞表面与可变角荧光显微镜，表示闪烁GFP标记PATROL1，一个膜运输蛋白质的点，在所述气孔络合物在细胞皮质拟南芥。 

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

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

科研

生物工程

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

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. ...

发育生物学

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

长期共焦成像根和下胚轴发育的简单室

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

生物学

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

使用钙氟在苔藓中细胞壁动力学的 3-D 延时成像

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. 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 ...

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 ...

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 ...

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 ...

通过共聚焦成像对 ERL1 动力学进行时空研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

作者聚焦：研究气孔谱系细胞中膜运输事件的基于图像的方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

基于图像的方法研究气孔谱系细胞中的膜运输事件

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...