作者感谢欧盟 H2020 计划（项目 GAIN4CROPS，GA 编号 862087）。AgroCropFuture 农业生态学和未来气候中的新作物卓越中心由爱沙尼亚教育和研究部资助。我们要感谢 Evelin Loit-Harro 教授提供 T. aestivum 和 Z. mays 的种子，感谢 Paula Palmet 和 Vaiko Vainola 在准备叶横截面方面的帮助，以及 João Paulo de Silva Souza 在分析方面的帮助。所有图像均来自爱沙尼亚生命科学大学的显微镜部门，在各种项目下。

1. 植物生长条件
<ol><li>分别选择小麦 （Triticum aestivum L. Var. 冬小麦， “Fredis”） 和玉米 （Zea mays L. Var. Saccharata， “Golden Bantam”） 作为代表性的 C3 和 C4 植物。在环境受控的生长室中用种子培养这两种植物。</li>
	<li>将相对湿度保持在 55%，空气温度为 25 °C/18 °C（昼/夜），光照时间为 12 小时。将植物表面的光合光子通量密度 （Q） 保持在 1200 μmol/m2/s，以代表其自然生长条件。在 400 μmol/mol 的环境 CO2 浓度下种植所有植物。</li>
	<li>每2 天用 蒸馏水给植物浇水，并在幼苗出苗后 10 天用 Hoagland 溶液施肥。在给定条件下将植物生长 60 天，并使用完全展开的叶子进行显微镜分析。</li>
</ol>2. 手绘部分的准备和查看
<ol><li>从每株植物中选择 3 片成熟的非衰老叶子，并将它们保存在蒸馏水中。使用新的单面剃须刀片，进行一次平整切割，与叶子表面形成直角，以确保截面是垂直于叶子表面的横向横截面。</li>
	<li>将叶标本放在玻璃板上的一块牙蜡上，以稳定样品并避免滑倒。通过在叶标本上笔直向下移动刀片进行切割，以干净地切开细胞。将横截面保存在蒸馏水中。</li>
	<li>将样品放在水滴上并用玻璃盖玻片覆盖，以便显微镜观察和成像，从而将样品安装在载玻片上。避免积聚气泡。</li>
	<li>在复合光学显微镜下以 10 倍和 20 倍放大倍率观察载玻片。根据软件指南保存图像以及相关比例。</li>
</ol>3. 半薄片的制备
<ol><li>如步骤 2.2 所示，在玻璃板上从 Z. mays 和 T. aestivum 的叶子上从中肋区沿肋间区域切下约 3 毫米 x 5 毫米大小的切片。确保切割部分的长度沿着叶子的纹理延伸。</li>
	<li>将植物材料放入装有 1 mL 固定缓冲液（4% 戊二醛在 0.1 M 磷酸盐缓冲液中，pH = 6.9， 表 1）的注射器中。</li>
	<li>垂直握住注射器，去除空气，然后用手指放在尖端，泵送注射器以产生真空。重复直到样品位于注射器底部，表明渗透成功。</li>
	<li>将注射器的内容物转移到标记的玻璃瓶中，并在下一步之前在 4 °C 下储存 48 小时（表 2）。</li>
	<li>对于每个小瓶，用移液管轻轻去除固定剂并加入磷酸盐缓冲液，直到样品被覆盖。静置 30 分钟。重复此步骤 3 次。</li>
	<li>用移液管除去磷酸盐缓冲液，并加入四氧化锇 （CAUTION） 直至样品被覆盖。样品和其他有机物会变黑。使用双层手套。放置 1 小时。</li>
	<li>用移液管除去之前的溶液，并用蒸馏水覆盖样品。放置 30 分钟 重复此步骤 3 次，并戴上双层手套。</li>
	<li>用移液管除去前一种溶液，并用 50/50 蒸馏水-乙醇溶液覆盖样品。静置 30 分钟。使用以下系列的蒸馏水-乙醇溶液重复此步骤：30/70、20/80、10/90 和 2x，100% 乙醇。这些溶液中的任何样品都可以在 4 °C 下放置过夜。</li>
	<li>用移液管除去前一种溶液，并用 1/3 树脂-乙醇溶液覆盖样品。将样品放在搅拌板上，放置 2 小时。使用以下系列的树脂-乙醇溶液重复此步骤：1/2、1/1、2/1、3/1 和 2x，100% 树脂。这些溶液中的任何样品都可以在 4 °C 下放置过夜。</li>
	<li>用 100% 树脂覆盖平面包埋模具的型腔（16 个型腔，型腔尺寸：14 L x 4.8 W x 3 D （mm）），并将样品放在型腔的一端。为每个样品准备铅笔书写的纸质标签，并将它们放在腔体的另一端。</li>
	<li>用 100% 树脂完全填充所有腔体，如果样品和标签移动，请将其拉直。用嵌入膜覆盖模具，并按照丙烯酸树脂产品指南在烘箱中聚合。</li>
	<li>将带有样品的聚合块放在超薄切片机的样品架中。使用粗糙的玻璃刀修剪块，直到组织变得可见并去除多余的树脂。</li>
	<li>使用玻璃或金刚石刀横向 （1 μm） 切割半薄片。使用金属接种环从水面收集切片，并将它们放在载玻片上。在热板 （60 °C） 上干燥载玻片以将切片固定在玻璃上。</li>
	<li>用甲苯胺蓝（1% 水溶液）染色固定切片 5 秒，然后用蒸馏水冲洗并在热板上再次干燥。 注意：确保在通风橱下进行化学固定过程。应定期更换玻璃刀，以确保切割锋利，使切片没有撕裂或变形。</li>
</ol>4. 样品成像
<ol><li>新鲜叶切片的成像<ol><li>根据手册使用随附的软件设置复合光学显微镜。</li>
		<li>将载玻片放在载物台上。调整目镜并以 10 倍观察截面以获得概览，然后在 20 倍和 40 倍物镜下观察。</li>
		<li>在每个目标处，导航 Live 频道，专注于感兴趣的区域，并找到静脉周围的束鞘细胞。</li>
		<li>聚焦后，单击 冻结 按钮并通过按 比例 尺 按钮 工具 选项卡。将图像存储在 中。TIF 格式进行图像分析。</li>
	</ol></li>
	<li>树脂浸润切片的成像<ol><li>将自动光学显微镜与计算机上的随附软件相结合。</li>
		<li>加载透明通道并将载玻片放在显微镜载物台上。使用 40 倍物镜放大倍率聚焦切片并调整亮度以确保所有细胞结构都可见。</li>
		<li>使用导航器功能设置截面的位置区域，以确保整个截面都成像，然后单击 Stitch 按钮。</li>
		<li>单击 Done（完成 ），然后单击 Start（开始 ），以允许显微镜对整个切片进行成像。打开图像分析软件并打开显微镜拍摄的图块。</li>
		<li>使用 Align 功能，确保切片不重叠。生成图像后，使用 调整 标签。</li>
		<li>在存储之前在图像上打印比例尺。TIF 格式。</li>
	</ol></li>
	<li>使用 ImageJ 软件进行解剖测量<ol><li>打开 ImageJ 软件并通过将图像拖到窗口中来加载图像。</li>
		<li>使用“线条”工具，按如下方式校准比例：在比例尺上绘制一条线，选择 “分析”>“设置比例”，将已知距离更改为比例尺的长度，然后将长度单位更改为正确的单位。</li>
		<li>通过选择 Line Tool，在感兴趣区域画一条线，然后按 M 键来测量预期特征。</li>
		<li>对于面积测量，请选择 多边形选择工具 并在感兴趣的组织周围绘制。按 M 键结束。测量值显示为弹出窗口，可以将其复制到电子表格中以进行进一步的数据分析。</li>
	</ol></li>
</ol>

提高 C4 作物光合效率的显微镜分析

作者声明没有利益冲突。

在本文中，我们讨论了测量叶片解剖结构的定量和定性方法以及优化它们的方法。此外，该方法应用于代表性作物物种，以确定哪些解剖性状在区分 C3 和 C4 横截面时最有用。了解这些特征至关重要，因为被称为 C2 光合作用的杂交物种正在成为一种更有前途的研究途径。截至目前，只有一种作物物种 Diplotaxis tenuifolia （芝麻菜）被确定使用 C2 光合作用，但?...

光合作用是将光能转化为化学能的基本过程，是陆地营养网络的基石。大多数植物遵循光合作用的 C3 途径，其中主要的光合作用产物是三碳化合物甘油酸酯 3-磷酸盐。C3 光合作用是在 20 多亿年前在大气中进化而来的，其中 CO2 含量丰富，O21 含量较低。在这些条件下进化的关键光合酶核酮糖 1,5 二磷酸羧化酶/加氧酶 （Rubisco） 在当前低 CO2 高 O2 条件下是次优的，因为它与 O2 竞争性反应，启动光呼吸2。光呼吸是一种浪费的途径，它消耗能量而不是产生能量并释放 CO2 作为副产品。因此，在叶绿体中 Rubisco 周围保持高 CO2 浓度以防止氧合至关重要 3,4。由于 C3 植物无法浓缩 CO2，因此 CO2 从环境空气中大量吸收到叶绿体中，抑制光合作用并影响植物生长和生物质生产 2,5,6。
在 C3 植物中，光合作用受到 CO2 通过气孔进入、通过叶肉扩散以及光合酶的生化活性的限制。CO2 的进入首先受到气孔导度的限制，气孔导度受空气温度和湿度等环境条件控制。然后 CO2 通过叶子的内部气相和液相扩散到 Rubisco7。在 C3 植物中，光合作用的所有阶段都发生在叶肉细胞的叶绿体中，植物需要保持 CO2 从大气中持续流入叶绿体。叶绿体中 CO2 的可用性取决于气孔开放性、叶肉结构以及单个细胞和叶绿体特性，这使得植物容易受到环境限制的影响，最终影响光合作用，如低水可用性和高温 7,8,9,10，特别突出了它们对气候变化条件的脆弱性11。
鉴于 C3 途径的低效率带来的挑战，以及维持最佳 CO2 水平和对环境因素的敏感性的限制，在某些植物中，另一种途径，即 C4 光合作用途径，已经进化出来。特征性地，C4 植物具有两条空间上分离的生化途径;磷酸烯醇式丙酮酸羧化酶在叶肉细胞中发生初始 CO2 固定，磷酸烯醇式丙酮酸羧化酶对 CO2 的亲和力高于 Rubisco，并且也缺乏氧合活性。形成的 C4 产物进一步转运到束状鞘细胞，在那里脱羧，CO2 再次被 Rubisco（C3 光合作用）释放和固定12,13,14。PEP 羧化酶对 CO2 的亲和力更大，束鞘细胞的厚细胞壁允许 CO2 在束鞘细胞中浓缩，因此，C4 植物通过空间分离 CO2 固定和卡尔文循环来最大限度地减少光呼吸。C4 途径的采用展示了大自然对环境限制的适应性反应，为在不断变化的气候条件下提高作物生产力和复原力的潜在策略提供了见解15。
C4 植物叶结构的专门解剖结构的特征是叶脉被含有叶绿体的扩大的维管束鞘细胞包围，并且叶肉细胞呈放射状排列，外环围绕束鞘细胞。叶肉细胞靠近束鞘细胞，这使得代谢物能够在两种细胞类型之间快速连续地运输。该细胞的排列是典型的 C4 植物，称为 Kranz 解剖学16。在 C3 物种中，叶肉细胞的特化和处置可能会有所不同，但束鞘细胞明显较小，并且有一些叶绿体或根本没有叶绿体。特异性 Kranz 解剖结构允许将 CO2 集中在 C3-羧化酶 Rubisco 所在的束鞘细胞中的叶绿体中，从而有效阻碍光呼吸 4,17,18。尽管其看似复杂的排列，但这些变化在被子植物的进化中独立地多次发生，表明它是一个相对可行的进化途径 19,20,21，并且各种分类群已被证明处于 C 3 和 C4 碳代谢之间的中间阶段，称为 C3-C 4 或 C 2， 具有浓缩和重新吸收 CO2 22,23,24,25 的能力。
许多 C4 植物是具有高度经济重要性的作物，对 C3 作物（如水稻）进行基因工程改造以提高其气候适应力并确保产量一直是过去几十年来感兴趣的话题26,27。然而，工程工作需要详细了解 C4 特化解剖结构及其如何控制光合作用 2,28。
建立 C4 Kranz 解剖结构是实现将 C4 光合作用工程化为 C3 作物的雄心勃勃目标的先决条件25。然而，目前对 Kranz 解剖学的调节和快速筛选其关键解剖特征的方法的理解是有限的，因此很难识别杂交物种。先前的研究表明，调节 C3 和 C4 植物光合效率的关键性状包括脉间距离、束鞘复合体的直径和束鞘细胞的大小14,29。这些性状可以使用徒手切片轻松筛选，并使用半薄切片进行定量分析。在这里，我们描述了通过徒手交叉和光学显微镜评估允许 C3 和 C4 解剖区分诊断的性状的方法，即束鞘面积、静脉间距离和静脉频率。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Disodium hydrogen phosphate dihydrate (Na2HPO42H2O) pure</td>
			<td>PENTA, CZ</td>
			<td>10028-24-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding Film, 7.8 mil Thick, 8 x 12.5, (203 x 318mm)</td>
			<td>ACLAR, US</td>
			<td>10501-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, abs. 100% a.r.</td>
			<td>Chem-Lab NV, BE</td>
			<td>CL00.0505.1000</td>
			<td>Danger: Highly inflammable liquid and vapour.</td>
		</tr>
		<tr>
			<td>EVOS Invitrogen FL Auto 2 Imaging System</td>
			<td>Thermo Fisher Scientific, US</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flat Embedding PTFE Mold with Metal Frame, 16 cavities</td>
			<td>PELCO, US</td>
			<td>10501</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial 2 ml</td>
			<td>VWR Life Science, US</td>
			<td>548-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 50% solution</td>
			<td>VWR Life Science, US</td>
			<td>23H2856331</td>
			<td>Danger: Fatal if inhaled. Toxic if swallowed. Causes severe skin burns and eye damage. May cause respiratory irritation. Wear protective gloves, protective clothing, eyes and face protection.</td>
		</tr>
		<tr>
			<td>Histo diamond knife</td>
			<td>Diatome, US</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LEICA EM UC7</td>
			<td>Leica Vienna, AT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LR White resin hard grade</td>
			<td>Electron Microscopy Sciences, US</td>
			<td>14383</td>
			<td>Danger: Causes skin irritation. Causes severe eye irritation May cause respiratory irritation. May cause drowsiness or dizziness Wear protective gloves, protective clothing, eyes and face protection.</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Normax, PT</td>
			<td>5470308A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse E600 and Nikon DS0Fi1 5 MP</td>
			<td>Nikon Corporation, JP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide (OsO4)</td>
			<td>Agar Scientific Ltd, GB</td>
			<td>R1019</td>
			<td>Danger: Fatal if swallowed, in contact with skin or if inhaled. Causes severe skin burns and eye damage Wear double protective gloves, protective clothing, eyes and face protection.</td>
		</tr>
		<tr>
			<td>Pipette and pipette tips</td>
			<td>Thermo Scientific, FI</td>
			<td>KJ16047</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate dihydrate (NaH2PO4 . 2H2O) pure</td>
			<td>PENTA, CZ</td>
			<td>13472-35-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 ml</td>
			<td>Ecoject, DE</td>
			<td>20010</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue, general purpose grade</td>
			<td>Fisher Scientific, GB</td>
			<td>2045836</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing structural traits in triticum aestivum and zea mays for c3 and c4 photosynthetic differentiation using free-hand and semi-thin sections

与 C3 机制相比，C4 光合作用的效率更高，源于其将 CO2 浓缩在束鞘细胞中的能力。C4 光合作用的有效性和内在水分利用效率与叶肉和束状叶细胞的份额、束状叶鞘的大小和密度以及束状鞘状细胞的大小、密度和细胞壁厚度直接相关。可以使用常规光学显微镜对徒手和半薄切片进行这些性状的快速显微镜分析，通过识别和检查特定细胞类型提供有关 C4 作物光合效率的宝贵信息。此外，还显示了影响解剖测量和细胞类型诊断的手绘和半薄片制备错误，以及如何避免这些错误。这种显微镜方法提供了一种有效的方法来收集对环境变化的光合适应的见解，并有助于快速筛选作物以适应未来的气候。

图 1A 显示了用于新鲜切片和光学显微镜检查的叶子切片的正确方向。使用单面剃须刀和牙科蜡片切割新鲜切片的方法如图 1B 所示。生成的部分如图 1C 所示。
图 2 显示了 C3 植物 T. aestivum 和 C4 植物 Z. mays 的代表性叶子的徒手切片。在叶脉周围可以看到束鞘?...

作者聚焦：了解气候适应型作物植物结构-功能关系的创新方法

评估 C3 和 C4 叶横截面之间的解剖学差异有助于了解光合作用效率。本文描述了徒手和半薄叶横截面的制备和检查，以及作物物种 Triticum aestivum 和 Zea mays 的准备注意事项。

使用徒手和半薄切片评估 小麦和 Zea mays 的 C3 和 C4 光合作用分化的结构特征

The enhanced efficiency of C4 photosynthesis, compared to the C3 mechanism, arises from its ability to concentrate CO2 in bundle sheath cells. The ...

assessing-structural-traits-triticum-aestivum-zea-mays-for-c3-c4

Research

JoVE Journal

Biology

Assessing Structural Traits in Triticum aestivum and Zea mays for C3 and C4 Photosynthetic Differentiation Using Free-hand and Semi-thin Sections

732 次观看.  Estonian University of Life Sciences. 我们的研究重点是植物结构、功能关系。我们的目标是更好地了解不同植物功能类型的光合作用的局限性，以及如何改善它们以用于气候适应型作物。新技术和软件成本高昂，而且通常无法获得，这需要一定程度的试验和创新才能在对不同的植物功能类型进行成像时实现相同的分辨率。鉴定区分 C3 和 C4 叶的解剖结构，特别是在显微镜检查的准?...

视频: 作者聚焦：了解气候适应型作物植物结构-功能关系的创新方法

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

科研

生物学

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

评估电子晶体结构生物学研究的小膜蛋白的二维结晶试验

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

评估抗小鼠动脉功能使用压力Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

与气孔导度，以叶功能性状

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies.

细胞分裂和扩张的运动学分析:量化增长和采样产业开发区的细胞学基础<em&gt;玉米</em&gt;叶

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

免疫荧光DNA修复蛋白相互作用的可视化

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

使用自由浮动组织切片进行基于组织学的染色

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

Arbuscular mycorrhizal fungi are symbionts in the roots of plants. Their role is to sustain host development and maintain the nutritional equilibrium in the ecosystems. The colonization process is dependent on several factors like soil ecology, the genetic diversity of the fungi and host, and agronomic practices. Their synchronized action leads to the development of a complex hyphal network and leads to the secondary development of vesicles and arbuscules in the root cells. The aim of this research was to analyze the efficiency of the mycorrhizal patterns (MycoPatt) method for the positioning of fungal structures in the roots of Festuca rubra and Zea mays. Another objective was to explore the fungal colonization strategy as revealed by mycorrhizal maps of each species. The acquisition and assemblage of multiple microscopic images allow mycorrhizal colonization assessment in both corn and red fescue plants to provide information on the realistic position of the developed structures. The observed mycorrhizal patterns highlight the variable efficiency of each plant in terms of developing connections with soil symbiotic fungi, caused by applied treatments and growth stage. Mycorrhizal detailed maps obtained through the MycoPatt method are useful for the early detection of plant efficiency in symbiotic acquisition from the soil.

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

The protocol here describes the methods for the assessment of the arbuscular mycorrhizal colonization patterns and strategy in roots for two species: Zea mays and Festuca rubra. The use of the MycoPatt method permits the calculation of parameters, the conversion of mycorrhizal structures into digital data, and the mapping of their real position in roots.

菌根图谱作为探索红费斯图卡和玉米根部定植模式和真菌策略的工具

Arbuscular mycorrhizal fungi are symbionts in the roots of plants. Their role is to sustain host development and maintain the nutritional equilibrium in ...

使用徒手和半薄切片评估 小麦和 Zea mays 的 C3 和 C4 光合作用分化的结构特征

使用徒手和半薄切片评估小麦和 Zea mays 的 C₃ 和 C₄ 光合作用分化的结构特征