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>

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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。 

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

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

科研

生物学

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

膜蛋白的实时测量：受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

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 ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

闪存和冻结：一种新的技术来捕获膜动力学与电子显微镜

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...