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?...

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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX-73</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TIRF unit</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX3-RFAEVAW</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TIRF objective lens&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">UAPON 100 &#215; OTIRF&#160;</td>
			<td>NA = 1.49</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser angle control box</td>
			<td style="width:71px;">Chuo Seiki</td>
			<td style="width:119px;">QT-AK</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Optically pumped semiconductor laser</td>
			<td style="width:71px;">Coherent</td>
			<td style="width:119px;">SapphireTM LP USB 488-20 CDRH Laser</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">510&#8211;550 nm band-pass filter</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">U-FBNA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EM CCD camera</td>
			<td style="width:71px;">Hamamatsu Photonics</td>
			<td style="width:119px;">ImagEM C9100-13</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C-mount camera magnification change unit&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">U-TVCAC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MetaMorph software</td>
			<td style="width:71px;">Molecular Devices</td>
			<td style="width:119px;">MetaMorph version 7.7.11.0</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">TIRF microscopy manual</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">AX7385</td>
			<td style="width:167px;">Instructions: Total Internal Reflection Illumination System (Printed in Japan on August 24, 2012)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Immersion oil</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">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?...

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

Research

JoVE Journal

Biology

8.9K 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。 