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>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agrobacterium tumefaciens 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 Agrobacterium tumefaciens. J Bacteriol 123:255-264.</td>
		</tr>
		<tr>
			<td>Agrobacterium tumefaciens C58&#916;tetRA::mini-Tn7T-GM-Ptac-ctrA &#916;ctrA</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 Agrobacterium tumefaciens. Appl Environ Microbiol. 82:5015-5025.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Media Components</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 &#176;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 KH2PO4 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>KH2PO4</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 (NH4)2SO4, 3.2 g/L MgSO4&#8226;7H2O, 0.2 g/L CaCl2&#8226;2H2O, and 0.024 g/L MnSO4&#8226;H2O to water. Autoclave.</td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Fisher Chemical</td>
			<td>A701</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>Fisher BioReagents</td>
			<td>BP213</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#8226;2H2O</td>
			<td>Fisher BioReagents</td>
			<td>BP510</td>
			<td></td>
		</tr>
		<tr>
			<td>MnSO4&#8226;H2O</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Optional Media Additives</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 &#181;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopy Materials</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 &#176;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Target-specific dyes</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 &#181;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 &#181;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 &#181;g/ml.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</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

15.6K 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 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

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

荧光显微镜下细胞电融合的可视化

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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 Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

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 ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. Therefore, it is considered a living antibiotic. To apply B. bacteriovorus as a living antibiotic, it is first necessary to understand the major stages of its complex life cycle, particularly its proliferation inside prey. So far, it has been challenging to monitor successive stages of the predatory life cycle in real-time. Presented here is a comprehensive protocol for real-time imaging of the complete life cycle of B. bacteriovorus, especially during its growth inside the host. For this purpose, a system consisting of an agarose pad is used in combination with cell-imaging dishes, in which the predatory cells can move freely beneath the agarose pad while immobilized prey cells are able to form bdelloplasts. The application of a strain producing a fluorescently tagged &#946;-subunit of DNA polymerase III further allows chromosome replication to be monitored during the reproduction phase of the B. bacteriovorus life cycle.

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Presented here is a protocol that describes monitoring of the complete life cycle of predatory bacterium Bdellovibrio bacteriovorus using time-lapse fluorescence microscopy in combination with an agarose pad and cell-imaging dishes.

使用延时荧光显微镜对细菌捕食者 Bdellovibrio 细菌的生命周期进行活细胞成像

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. ...

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of&#160;the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

We present a label-free live imaging protocol using transmitted light microscopy techniques to capture images, analyze and quantify growth kinetics of the filamentous fungus A. nidulans in both submerged cultures and solid media. This protocol can be used in conjunction with fluorescence microscopy.

使用实时显微镜和开源软件对 阿斯珀吉卢斯尼杜兰 生长速度的定量分析

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...