我们感谢迈克尔 VanNieuwenhze (印第安纳大学) 在图 2和图 4中使用的 FDAAs 的礼物。我们感谢布朗实验室的成员在准备这份手稿时的反馈。研究在褐色实验室在A. 农杆菌细胞成长和分裂由国家科学基金会 (IOS1557806) 支持。

1. A. 农杆菌菌株的生长
<ol><li>
培养A. 农杆菌菌株
	<ol>
<li>使用一个无菌木棒或吸管提示, 接种1毫升的 ATGN 生长培养基 (参见材料清单的食谱) 与一个单一的群体所需的应变。 注: 对于A. 农杆菌衰竭菌株, ATGN 应包含1毫米 IPTG 作为诱导剂, 以维持必需蛋白的生物合成。</li>
		<li>在28° c 的 ATGN 中, 以 225 rpm 的震动在夜间将A. 农杆菌菌株生长。</li>
		<li>使用分光光度计测量 600 nm (OD600) 的细胞的光学密度。将单元格区域性稀释到 od600 = ~ 0.2, 并继续增长, 直到达到 od600 = ~ 0.6 为止。对于耗尽菌株, 继续增长与诱导直到 OD600 = ~ 0.6 达到。</li>
		<li>使用野生和突变株的A. 农杆菌在 OD600 = 〜0.6 为目标特定的染色和/或延时显微镜 (见2和 3.1)。对于耗尽菌株, 冲洗出诱导剂 (见1.2 节)。</li>
	</ol>
</li>
	<li>
移除诱导剂用于表征A. 农杆菌蛋白耗竭菌株
	<ol>
<li>颗粒1毫升的指数培养 (OD600 = 〜 0.4-0.6) 通过离心在 7000 x g 为5分钟在台式离心机在室温下。</li>
		<li>清洗, 去除上清和重的新鲜介质中的颗粒, 没有诱导剂和颗粒的细胞如上所述 (1.2.1)。在新鲜培养基中共洗涤3次细胞。</li>
		<li>重最后的颗粒在新鲜的媒体和集中的细胞到 OD600 = 〜 0.8, 立即染色与目标特定染料 (第2和图 4BD) 或延时显微镜 (第3.2 和图 4A).或者, 通过在细胞的染色和成像之前, 在没有诱导剂的培养基中培养细胞, pre-deplete 细胞达到预期的时间。</li>
	</ol>
</li>
</ol>2. 针对A. 农杆菌细胞的靶向染色
<ol><li>
荧光 d-氨基酸细胞壁贴标 注: FDAAs 是无毒的荧光 d-氨基酸, 很容易被纳入到A. 农杆菌肽。这个简单的标记过程在活细胞中探测生长模式6。用于合成四 FDAAs 的协议可用38。<ol>
<li>颗粒500µL 的指数培养 (OD600 = 〜 0.4-0.6) 离心在 7000 x g 为 5 min 在台式离心机。重100µL 新鲜培养基中的细胞颗粒。</li>
		<li>在洗涤和浓缩细胞中加入5µL 5 毫米 FDAA, 在黑暗中孵育2分钟。 注意: 限制 FDAA 暴露在光线下。潜伏期的变化取决于菌株的生长速率。通常, 潜伏期应该是5-10% 倍的时间6。</li>
		<li>用磷酸盐缓冲盐水 (PBS) 三次, 通过离心和洗涤细胞颗粒将细胞颗粒化。</li>
		<li>重颗粒在 ~ 50 µL PBS。 注: 用于悬浮的 PBS 体积可能根据颗粒大小而变化。</li>
		<li>使用荧光显微镜立即应用 0.8-1 µL 细胞到琼脂糖垫和图像 (见3.1 和3.2 节)。<ol>
<li>另外, 通过用 ice-cold 70% 乙醇固定细胞来阻止进一步的标签合并。在1毫升的 ice-cold 70% 乙醇中重细胞团, 在冰上孵育15分钟。</li>
			<li>收集细胞的离心和重在一个小体积的 PBS, 以成像在以后的时间。储存细胞悬浮在4° c 和图像在48小时内。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
DAPI 或橙染色的 DNA 染色 注意: 为了观察 DNA 结构, 细胞被标记为 DAPI 或橙色 (死细胞染色)。虽然经典描述为 "死细胞染色", 橙色是 permeant, photostable, 并不会影响细菌细胞的生长, 使活细胞 DNA 成像10。在A. 农杆菌中, 橙色标记在活细胞中很有效, 适合于延时显微镜 (图 3)。相比之下, 成像 DAPI 染色细胞所需的紫外线 (UV) 光曝光是光 (图 3), 因此 DAPI 染色最适合于染色活细胞以立即成像或染色固定细胞。<ol>
<li>细胞 DAPI 染色<ol>
<li>1毫升的指数培养 (OD600 = 〜 0.4-0.6) 通过离心在 7000 x g 为5分钟的台式离心机。</li>
			<li>(可选) 在染色前固定细胞, 在1毫升的 70% ice-cold 乙醇中重细胞颗粒。在冰上孵育10-15 分钟, 用离心法收集细胞, 如2.2.1.1 所述。</li>
			<li>重1毫升 PBS 中的细胞团, 含有1µL 的 DAPI 库存溶液 (1 毫克/毫升; 请参见材料表)。用移轻轻地混合, 在黑暗中孵育5分钟。</li>
			<li>在1毫升 PBS 中离心 7000 x g 的5分钟和重的颗粒细胞去除过量的 DAPI。再清洗两次细胞, 重50µL PBS 或培养基中的颗粒。</li>
			<li>使用荧光显微镜立即应用 0.8-1 µL 细胞到琼脂糖垫和图像。见3.1 和3.2 节。 注意: 该协议不是最佳的延时显微镜观察染色体动力学 (图 3)。考虑使用橙色染色作为替代 (参见2.2.2 节)。</li>
		</ol>
</li>
		<li>活细胞的橙色染色</li>
		<li>1毫升的指数培养 (OD600 = 〜 0.4-0.6) 通过离心在 7000 x g 为5分钟的台式离心机。重细胞颗粒在1毫升 PBS 含有1µL 橙库存溶液 (见材料清单)。用移轻轻地混合, 在黑暗中孵育5分钟。</li>
		<li>通过离心和重1毫升的 PBS 去除多余的橙颗粒细胞。再冲洗两次细胞, 重50µL PBS 中的颗粒。</li>
		<li>使用荧光显微镜立即应用 0.8-1 µL 细胞到琼脂糖垫和图像。见3.1 和3.2 节。 注: 本协议适用于活细胞, 适合于定时显微镜观察染色体动力学 (图 3)。如果不方便立即图像, 细胞可以固定在 ice-cold 70% 乙醇 (见第 2.2.1.2)。</li>
	</ol>
</li>
	<li>
膜贴标 注: 亲油性苯乙烯荧光染料4-64 已被广泛用于观察细菌细胞的细胞膜。与许多细菌相比, 4-64 标记为a. 农杆菌细胞不统一标签, 而是经常出现 "马蹄形" 模式14,15。值得注意的是, 生长极几乎没有污点, 而老极点是强烈的标签。因此, 4-64 在A. 农杆菌中实现了细胞生长模式和膜结构的可视化。<ol>
<li>在1毫升的指数相细胞培养中添加 FM 4-64 到最后浓度8µg/毫升, 在室温下孵育5分钟, 在黑暗中。</li>
		<li>在1毫升的 PBS 中, 在台式离心机和重细胞颗粒中, 在 7000 x g 处离心5分钟的颗粒标记细胞。清洗细胞共三次, 去除多余的染料。</li>
		<li>重细胞在一小体积的 PBS 和当场的琼脂糖垫上立即图像。(见3.1 和3.2 节) 警告: 细胞必须立即成像, 以观察特征标记模式。</li>
	</ol>
</li>
</ol>3. A. 农杆菌细胞的成像
<ol><li>
琼脂糖垫制备 注:图 1包含一个典型的琼脂糖垫的图像序列 (面板 a) 和示意图 (面板 B), 用于延时显微镜。琼脂糖垫通常是根据需要准备的。<ol>
<li>3.1.1. 使用片作为指导, 并削减了22毫米 x 22 毫米的实验室胶片 (见材料清单), 通过运行手术刀周围的边缘。</li>
		<li>从实验室胶片的中心切出一个正方形, 留下一个2-5 毫米的边界, 作为琼脂糖垫的衬垫。丢弃中心切口。</li>
		<li>将胶片垫片放在玻璃滑梯上 (75 mm x 25 mm), 用氨水和无酒精清洗剂 (见材料清单) 和热量进行清洗, 直到胶片稍微融化在玻璃上 (图 1A中的顶部图像)。要熔化实验室胶片, 请将热块的边缘设置为70° c 或在火焰上轻轻地熔化。</li>
		<li>用0.075 克琼脂糖 (见材料清单) 在5毫升的培养基中用小烧瓶制备琼脂糖溶液。在微波中加热溶液, 周期性旋转, 直到琼脂糖溶解, 溶液清晰。保持琼脂糖溶液在55-70 ° c, 并使用在48小时内建设多个琼脂糖垫。</li>
		<li>吸管介质含有 1.2-1.5% 琼脂糖进入垫片的中心。 注: 介质的体积通常是 50-60 µL, 但会因垫片大小而异。将介质添加到冷滑块中会导致琼脂糖固化得太快, 因此幻灯片应保持在温暖的表面上。热块的边缘设置到70° c 工作很好。水琼脂糖或缓冲琼脂糖溶液, 如磷酸盐缓冲盐水 (PBS) 可以使用, 而不是培养基中含有琼脂糖的应用, 其中持续细胞生长是不需要的。对于成像损耗的应变, 诱导剂可以存在或缺席的媒体。诱导剂的存在可以作为一种控制, 而诱导因子的缺乏将会暴露出衰竭表型。</li>
		<li>将片在垫圈上均匀地分布琼脂糖。</li>
		<li>将滑块放置在阴凉、平面上, 固化为〜2分钟. 避免 overdrying 琼脂糖垫, 因为这将导致在琼脂糖垫上的皱纹表面和池的细胞悬浮时, 适用于表面。</li>
		<li>小心地把片从琼脂糖垫上滑下来。 注意: 不要急于迈出这一步。琼脂糖垫很容易撕裂, 不均匀的琼脂糖垫可以使成像困难。</li>
		<li>允许琼脂糖垫的空气干燥1-2 分钟的室温, 直到表面的垫似乎干燥。使用手术刀, 取出一小条琼脂糖创建一个气囊 (中间图像,图 1A);空气袋通常是〜2毫米 x 7-10 毫米和A. 农杆菌细胞往往生长最好的靠近空气袋 (图 1C)。 注: 对于固定细胞的成像或在不监测生长的条件下, 不需要空气袋。</li>
		<li>点 0.8-1 µL 的细胞在琼脂糖垫和覆盖新的片。轻轻地将片放在琼脂糖垫的顶端, 将细胞分布在琼脂糖垫的表面。</li>
		<li>使用熔融 VALAP 密封片的边缘 (请参阅材料表中的配方;图 1A, 底部图像)。没有密封沿所有的边缘和角落的片可能会导致干燥的琼脂糖垫, 这将导致细胞在成像过程中漂移。注: 片的密封只需要长期的延时成像。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56497/56497fig1.jpg"> 图 1: 琼脂糖垫准备.(A) 琼脂糖垫制备协议的图像序列。图1是一张带有实验室胶片衬垫的幻灯片。在图2中, 琼脂糖垫和空气袋是可视化的。最后, 图像3显示完整的琼脂糖垫与细胞下的 coverglass 和密封 VALAP。(B) 提供了用于延时显微镜的琼脂糖垫示意图。在示意图上标注了琼脂糖垫的主要功能。(C) 空气袋促进了在琼脂糖垫上的A. 农杆菌的生长。显示了在琼脂糖垫上生长的野生. 农杆菌细胞的图像20小时。图像拍摄的位置越来越远离空气袋。从图像最接近的边缘到空气袋的距离显示在每个图像的上方。缩放栏 = 10 µm.<a href="//cloudflare.jove.com/files/ftp_upload/56497/56497fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="2"><li>
成像 注: 差分干扰对比度、相位对比度和荧光成像采用倒置显微镜, 配备基于硬件的自动、自动化阶段、标准过滤器、LED 光源、60X 油浸目的 (1.4 NA) 的相位对比或差分干扰对比 (DIC), 和1K 电子倍增电荷耦合器件 (EMCCD) 相机。显微镜应该放在一个温度控制的房间里。或者, 在成像过程中, 可以使用舞台加温或腔室来保持一致的温度。<ol>
<li>荧光显微镜 注意: 对于活细胞的荧光成像, 有必要限制图像采集的次数, 并优化曝光时间, 以最小化漂白和光。对于每个染料的成像, 建议找到一个最佳曝光时间, 提供足够的荧光检测, 但不导致漂白或光。在图 2-4中, 所有荧光图像都使用了200毫秒的曝光时间。对于图 3中显示的延时序列, 每10分钟获取3小时的图像。<ol>
<li>将浸入式油放在所需的目标上, 并将倒滑梯放在舞台上的滑动架上。使用聚焦旋钮使细胞进入焦点。</li>
			<li>获取相 (或 DIC) 和所期望的荧光过滤器的图像。 注: 在图 2中使用的污点的最大激发和发射波长,图 3和图 4如下所示: 哈达 (405/460), 纳 (450/555), 多 (555/570), 4-64 (515/640), DAPI (360/460), 橙色 (547/570)。</li>
		</ol>
</li>
		<li>延时显微镜 注意: 在延时显微镜下, 以下功能是计算机控制的: x、y、z 位置、百叶窗和荧光过滤器。一个基于硬件的自动系统是最佳的, 以保持重点在延时成像。或者, 可以使用基于软件的自动循环。<ol>
<li>将浸入式油放在所需的目标上, 并将倒滑梯放在舞台上的滑动架上。使用聚焦旋钮使细胞进入焦点。</li>
			<li>可选: 获取多个 (x、y) 位置。随机选择10个单元格, 接近于琼脂糖衬垫的空气袋。</li>
			<li>在所需的时间间隔内, 在相或 DIC 中设置时间序列获取图像。 注意: 对于图 4中所示的延时序列, 在 14 h 中每10分钟就获得了 DIC 图像和30毫秒曝光。当在延时显微镜中使用荧光成像时, 调整采集间隔和曝光时间, 以最小化漂白和光。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

此协议包含一系列用于调查a. 农杆菌野生、突变体和耗尽菌株的过程。值得注意的是, 《议定书》一节所列的所有程序都可以很容易地适用于其他细菌菌株, 并对培养基、温度和生长速率进行了额外的修改。
使用靶向染料是一个很有价值的工具, 可以为细菌细胞中的细胞周期事件提供详细的描述。在这里, 肽插入模式观察使用荧光 d-氨基酸, 血脂领域是可视化与 4-64, 核被...

通过细菌细胞周期的进展需要协调许多过程, 包括细胞膜和细胞壁生物合成, DNA 复制和分离, 和细胞分裂。要充分了解细菌细胞生物学的复杂性, 就必须研究这些重要事件;然而, 这是一项不平凡的任务, 因为当这些通路的关键成分被 mutagenized 时, 细胞的生存能力就会受到影响。荧光显微镜结合靶向染料是一个强有力的方法, 以探索这些关键的过程中野生和突变菌株。
肽专用染料包括荧光抗生素 (万古霉素-fl, bocillin) 和荧光 d-氨基酸 (例如, 7-素-3-羧酸 acid-3-amino-d-alanine, 哈达; 4-氯-7-nitrobenzofurazan-3-氨基-d-丙氨酸纳达;tetramethylrhodamine-3-amino-d-alanine;多)。在革兰氏阳性菌中, 使用致死浓度的荧光抗生素类似物来探测肽生物合成的位置是一个有效的策略, 以揭示肽插入模式1,2, 3,4。虽然荧光万古霉素标记已被用来获得洞察肽插入模式固定革兰阴性细菌5, 外膜一般提供了渗透屏障, 防止使用荧光抗生素作为活体细胞肽生物合成的探针。相比之下, 短脉冲的荧光 d-氨基酸或 d-氨基酸与双正交功能基团共价标签地区最近肽插入在广泛的活细菌细胞6,7。用合成 d-氨基酸观察到的肽插入模式包括点状和间隔 (大肠杆菌和枯草芽孢杆菌)、极性和间隔 (农杆菌介导和李斯特菌), 仅间隔 (金黄色葡萄球菌), 和顶端 (链霉菌 venezuelae)6,7。这些观察表明, 细菌表现出不同的细胞壁生物模式, 而使用合成 d-氨基酸作为探针来检测生长模式是许多细菌中的一个有价值的策略。
标记细菌染色体的染料包括脱氧核糖核酸 (脱氧核糖核酸) 具体小槽黏合剂 (46-diamidino-2-吲;DAPI) 和高亲和力菁染料 (绿色和橙色; 见材料清单)。DAPI 染色固定细胞协助计数细菌从环境样品8, 而 DAPI 染色的活细胞被用来表明细菌生存能力9。相比之下, 如橙和绿等菁染料经常被描述为膜 impermeant "死" 细胞污渍, 以枚举不细胞9。值得注意的是, 当这些试剂被用来探测细胞生长过程中细菌核的形态时, DAPI、橙和绿都被证明是膜 permeant, 能够标记活细胞10。在活着的大肠杆菌细胞中, DNA 的 DAPI 染色出现弥漫性, 这是由于荧光从细胞质和 DAPI 染色的细胞重复暴露到紫外线 (UV) 光 perturbs 核结构10。染色大肠杆菌或B. 枯草芽孢杆菌与橙色显示, 这种染料是膜 permeant 和提供持久荧光后, 结合到活细胞 dna, 而不影响细胞生长, dna 复制, 或染色体分离10.这些观察表明, 在许多细菌的细胞生长过程中, 可以使用菁核 DNA 染料来监测其形态。
磷脂特定的 stryl 染料, 如 n-(3-triethylammoniumpropyl)-4-(6-(4-(二乙胺) 苯基) hexatrienyl) 吡啶敌 (4-64; 见材料清单) 是阳离子化合物和副优先与负电荷磷脂, 如心和甘油11。当4-64 被用来标记不同细菌的细胞膜时, 观察到了截然不同的模式。在大肠杆菌中, 4-64 在极点、沿侧壁的带区中以及在晚期 pre-divisional 细胞的分裂部位12中富集。在枯草芽孢杆菌中, 4-64 标记使脂质螺旋的可视化显示为13。在农杆菌介导中, 4-64 标记外膜, 并在一个特征 "马蹄形" 模式中观察到生长极没有标记14,15。这些观察表明, 这些细菌表现出不同的脂质分布, 由于存在的脂肪领域, 导致细胞不对称。4-64 标记模式的变化, 如弥漫标记, 泡或囊泡, 内陷, 或膜萎缩, 可以在特征突变, 影响分布或生物合成的脂质的信息。
除了染色细胞, 确定参与基本过程的蛋白质的功能是必要的。基本蛋白质的定性是技术上的挑战, 因为它是不可能删除的基本基因和研究表型的后果。因此, 出现了消耗蛋白质的替代方法。例如, 一个基本的基因可以被置于一个可诱导的启动子的控制之下, 而不是它的本地启动子。诱导促进剂对小分子反应灵敏, 如;锌16, 异丙基β d-1-thiogalactopyranoside (IPTG)17,18,19,20,21, 糖22, 酸 17,23,和木糖23, 因此靶基因的转录停止, 当诱导剂被移除时, 感兴趣的蛋白质就会耗尽。消耗必需蛋白质的替代方法包括合成开关24 , 它使用小分子 RNA 相互作用阻碍靶基因的转录, CRISPR 干扰25,26阻断靶基因的转录和诱导蛋白的降解27,28 , 它使用肽标签对蛋白质进行 ClpXP 蛋白酶降解。耗尽菌株在细胞丧失生存能力之前只提供了很短的时间来表征, 因此, 在蛋白质耗竭过程中, 细胞的显微成像是一种强有力的表征方法。事实上, 活体细菌细胞显微镜使研究人员能够深入了解基本的生物学过程, 包括细胞形状维护、分泌和条块分割的机制29。
a. 农杆菌是细菌植物病原体30和自然基因工程工程师31,32。因此, 与致病性相关的机制, 包括宿主-病原体相互作用33,34,35, 分泌36, 和主机转换30,31, 37已被广泛调查。要设计防止.. 农杆菌介导的疾病或增强植物转化的策略, 必须更好地了解a. 农杆菌的生存过程。使用目标特定的染料和最近开发的蛋白质耗尽策略. 农杆菌18提供了一种方法来调查基本过程。
在这里, 详细的协议, 显微镜分析的野生, 突变体, 蛋白质枯竭菌株的A. 农杆菌提供。前两个协议描述了如何准备细胞和标签与目标特定的染料。第三个协议为准备琼脂糖垫 (图 1) 和成像细菌细胞 (图 2、 图 3、 图 4) 提供了 step-by 步骤的指导。这些协议也可能适用于其他的细菌与其他的适应, 以考虑不同的媒体条件, 生长速率, 氧要求和细胞结构。

<table><tbody><tr><td>&lt;strong&gt;Bacterial Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Agrobacterium tumefaciens&lt;/em&gt; C58</td><td>ATCC</td><td>33970</td><td>Watson B, Currier TC, Gordon MP, Chilton MD, Nester EW. 1975. Plasmid required for virulence of &lt;em&gt;Agrobacterium tumefaciens&lt;/em&gt;. J Bacteriol 123:255-264.</td></tr><tr><td>&lt;em&gt;Agrobacterium tumefaciens&lt;/em&gt; C58&amp;Delta;tetRA::mini-Tn7T-GM-Ptac-&lt;em&gt;ctrA &amp;Delta;ctrA&lt;/em&gt;</td><td></td><td></td><td>Figueroa-Cuilan W, Daniel JJ, Howell M, Sulaiman A, Brown PJB. 2016. Mini-Tn7 insertion in an artificial attTn7 site enables depeltion of the essentail master regulator CtrA in the phytopathogen &lt;em&gt;Agrobacterium tumefaciens&lt;/em&gt;. Appl Environ Microbiol. 82:5015-5025.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Media Components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ATGN Minimal Medium</td><td></td><td></td><td>To 1 L of sterilized water add 50 ml 20X Buffer, 50 ml 20X Salts, 12.5 ml 40% glucose. For plates, add 15 g Bacto Agar to 1 L of water and autoclave. Cool to 55 &amp;deg;C and add 50 ml 20X Buffer, 50 ml 20X Salts, 12.5 ml 40% glucose.</td></tr><tr><td>20X AT Buffer</td><td></td><td></td><td>Add 214 g/L KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; to water and adjust pH to 7.0 with sodium hydroxide. Autoclave.</td></tr><tr><td>NaOH</td><td>Fisher BioReagents</td><td>BP359</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Chemical</td><td>P288</td><td></td></tr><tr><td>20X AT Salts</td><td></td><td></td><td>Add 40 g/L (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;, 3.2 g/L MgSO4&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O, 0.2 g/L CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O, and 0.024 g/L MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;H&lt;sub&gt;2&lt;/sub&gt;O to water. Autoclave.</td></tr><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Chemical</td><td>A701</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher BioReagents</td><td>BP213</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher BioReagents</td><td>BP510</td><td></td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Chemical</td><td>M114</td><td></td></tr><tr><td>Glucose</td><td>Fisher Chemical</td><td>D16</td><td>Prepare 40% stock in water. Filter sterilize.</td></tr><tr><td>Bacto Agar</td><td>Fisher BioReagents</td><td>BP1423</td><td>Add 15 g to 1 L of water when preparing plates.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Optional Media Additives&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Kanamycin</td><td>GoldBio</td><td>K-120</td><td>Prepare as a 100 mg/ml stock solution in water and filter sterilize. Use at final concentration of 200 &amp;micro;g/ml.</td></tr><tr><td>IPTG</td><td>GoldBio</td><td>I2481C5</td><td>Prepare as a 1 M stock solution in water and filter sterilize. Use at final concentration of 1 mM as needed for induction.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscopy Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microscope Slides</td><td>Fisherbrand</td><td>12-550D</td><td>25 X 75 X 1.0 mm. Clean with Sparkle glass cleaner.</td></tr><tr><td>Microscope Cover Glass</td><td>Fisherbrand</td><td>12-541-B</td><td>22 X 22 mm. No. 1.5. Clean with Sparkle glass cleaner.</td></tr><tr><td>Sparkle Glass Cleaner</td><td>Home Depot</td><td>203261385</td><td>Ammonia and alcohol free.</td></tr><tr><td>Ultra Pure Agarose</td><td>Invitrogen</td><td>16500-100</td><td>Add to water, PBS, or media to a final concentration of 1 - 1.5%. Melt in microwave and place on 70 C</td></tr><tr><td>PBS</td><td>Fisher BioReagents</td><td>BP399500</td><td>10X solution to be diluted to 1X with sterile water.</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-999</td><td>Laboratory film used as gasket in agarose pad preparation.</td></tr><tr><td>VALAP</td><td></td><td></td><td>Add equal weights of lanolin, parafin wax, and petroleum jelly to a conical tube. Heat tube in 70 &amp;deg;C dry, bead or water bath to melt and mix. Apply VALAP while still molten.</td></tr><tr><td>Lanolin Butter</td><td>SAAQIN</td><td>SQ-LAB-R1</td><td></td></tr><tr><td>Petroleum Jelly</td><td>Target Corp.</td><td>06-17644</td><td></td></tr><tr><td>Paraffin Wax</td><td>Crafty Candles</td><td>263012</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Target-specific dyes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMSO</td><td>Fisher BioReagents</td><td>BP231-1</td><td>Use to dilute stock solutions of dyes as needed.</td></tr><tr><td>FDAAs (NADA, HADA,TADA)</td><td></td><td></td><td>FDAAs can be synthesized or acquired through agreement with Mike VanNieuwenhze (Indiana University). Prepare 100 mM stock solution in DMSO. Use at a final concentration of 5 mM.</td></tr><tr><td>DAPI</td><td>ThermoFisher Scientific</td><td>62247</td><td>Prepare 1 mg/ml stock solution in DMSO. Use at final concentration of 1 &amp;micro;g/ml.</td></tr><tr><td>SYTOX Orange Nucleic Acid Stain</td><td>Invitrogen</td><td>S11368</td><td>Stock concentration is 5 mM in DMSO. Use at final concentration of 5 &amp;micro;M.</td></tr><tr><td>FM4-64</td><td>Invitrogen</td><td>T3166</td><td>Prepare 8 mg/ml stock solution in DMSO. Use at final concentration of 8 &amp;micro;g/ml.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dry bath</td><td>Sheldon Manufacturing, Inc.</td><td>52120-200</td><td></td></tr><tr><td>Metallic thermal beads</td><td>Lab Armor</td><td>42370-002</td><td></td></tr><tr><td>Epifluorescence microscope equipped with an EMCDD camera</td><td></td><td></td><td>Nikon Eclipse TiE equipped with a QImaging Rolera em-c2 1K electron-multiplying charge-coupled-device (EMCCD) camera is used in this work.</td></tr></tbody></table>

live cell fluorescence microscopy to observe essential processes during microbial cell growth

核心细胞过程, 如 DNA 复制和分离, 蛋白质合成, 细胞壁生物合成和细胞分裂依赖于蛋白质的功能, 这是必不可少的细菌生存。一系列的靶向染料可以作为探针, 以更好地理解这些过程。用亲油性染料染色可以观察膜结构、脂微的可视化以及膜泡的检测。使用荧光 d-氨基酸 (FDAAs) 来探测肽生物合成的地点, 可以显示细胞壁生物或细胞生长模式的潜在缺陷。最后, 核酸染色可以显示 DNA 复制或染色体分离可能存在的缺陷。青菁 DNA 染色标签活细胞和适用于定时显微镜, 使 real-time 观察的核形态学在细胞生长。细胞标记协议可应用于蛋白质耗竭突变体, 以识别膜结构、细胞壁生物或染色体分离的缺陷。此外, 延时显微镜可以用来监测形态学变化, 作为一个基本的蛋白质被删除, 并可以提供额外的洞察力的蛋白质功能。例如, 基本细胞分裂蛋白的耗尽导致丝或分支, 而细胞生长蛋白的耗竭可能导致细胞变得更短或更圆。在这里, 细胞生长的协议, 目标特定的标签, 和延时显微镜提供的细菌植物病原体农杆菌介导。同时, 目标特定的染料和延时显微镜能够在A.最后, 所提供的协议可以随时修改, 以探测其他细菌的基本过程。

野生的目标特定标记A. 农杆菌单元格 为了说明细胞形态不受洗涤步骤或处理的影响, 1% 甲基亚砜 (这是用来稀释荧光染料), 细胞直接成像从文化 (图 2A, 远左面板), 洗后的细胞离心的描述在 1.2 (图 2A, 左面板), 或后孵化细胞与1% 亚砜10分钟和洗涤细胞 (图 2A, 右?...

Watch this Scientific Journal Video about Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth at JoVE.com

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

了解细菌中基本过程的功能是很有挑战性的。荧光显微镜与靶向特定染料可以提供关键的洞察力的微生物细胞生长和细胞周期进展。在这里,农杆菌介导被用作一种模型细菌, 用来强调活细胞成像的方法, 用于描述基本过程。

活体细胞荧光显微镜观察微生物细胞生长过程中的基本过程

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

live-cell-fluorescence-microscopy-to-observe-essential-processes

Research

JoVE Journal

Biology

15.7K 次观看.  University of Missouri. 了解细菌中基本过程的功能是很有挑战性的。荧光显微镜与靶向特定染料可以提供关键的洞察力的微生物细胞生长和细胞周期进展。在这里,农杆菌介导被用作一种模型细菌, 用来强调活细胞成像的方法, 用于描述基本过程。

视频: Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

科研

免疫与感染

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes underlying central cell cycle events. However, imaging of these molecules in slow-growing bacteria represents a challenge due to photobleaching of fluorophores and phototoxicity during image acquisition. Here, we describe a simple protocol to circumvent these limitations in the case of Myxococcus xanthus (which has a generation time of 4 - 6 h). To this end, M. xanthus cells are grown on a thick nutrient-containing agar pad in a temperature-controlled humid environment. Under these conditions, we determine the doubling time of individual cells by following the growth of single cells. Moreover, key cellular processes such as chromosome segregation and cell division can be imaged by fluorescence live-cell imaging of cells containing relevant fluorescently labeled marker proteins such as ParB-YFP, FtsZ-GFP, and mCherry-PomX over multiple cell cycles. Subsequently, the acquired images are processed to generate montages and/or movies.

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Bacterial cells are spatially highly organized. To follow this organization over time in slow growing Myxococcus xanthus cells, a set-up for fluorescence live-cell imaging with high spatiotemporal resolution over several generations was developed. Using this method, spatiotemporal dynamics of important proteins for chromosome segregation and cell division could be determined.

生长缓慢的社会细菌Myxococcus xanthus的全营养细胞周期的荧光活细胞成像

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

生物化学

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

生物学

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

活细胞荧光显微镜研究细菌的亚细胞蛋白定位和细胞形态变化

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

单细胞物种间细菌相互作用的动力学可视化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动:Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

通过电穿孔内在和生物分子荧光观察生活中的微生物

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...