Name: ground state depletion super-resolution imaging in mammalian cells
Uploaded: 2017-11-05T08:30:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Ground State Depletion Super-resolution Imaging in Mammalian Cells at JoVE.com

这项工作得到了从哈哈哈到 r.e.d.i (15SDG25560035) 的资助。作者想承认 Dr. 费尔南多桑塔纳使用他的徕卡 SR 3D 显微镜, 并 Dr. 约翰地狱为好心提供 FP1 抗体。

1. 洗涤玻璃片 <ol> <li> 在样品处理前的前一天, 清洁 #1. 5 玻璃片由 sonicating 在一个 1 M 解决方案的 KOH 为1小时。这将消除片上可能存在的任何荧光污染物. 
	<ol> <li> 用去水彻底冲洗片中的 KOH. </li>
		<li> 一旦在 KOH 中清洗, 将片储存在70% 乙醇中直到准备使用. </li>
	</ol> </li> </ol> 2。镀膜玻璃片 注意: 本节中的步骤应在细胞培养罩中进行, 以防止污染. 
<ol> <li> 使用镊子将单个片从70% 乙醇溶液中移除, 然后迅速火焰除去多余的。把片放进一个6井的盘子里。如果燃烧在文化敞篷不是一个选择, 考虑灭菌片在漂洗以后与去水并且/或者绝育在紫外光之下在文化敞篷为至少 30 min. </li>
	<li> 为了帮助细胞黏附, 在室温下用无菌的0.01% 聚 l-赖氨酸涂层片1小时. </li>
	<li> 吸聚 l-赖氨酸, 用无菌磷酸缓冲盐 (PBS) 冲洗片。空气干燥, 在冰箱里过夜. </li>
	<li> 第二天早上, 从冰箱中取出片, 并添加300和 #181; 层粘连蛋白, 浓度为50和 #181; 克/毫升, 在室温下至少30分钟。确保将层粘连蛋白直接放在片上. 
	注意: 这种层粘连蛋白的步骤对于 COS-7 细胞或 HEK293 细胞是不必要的, 但对于心室肌细胞是必需的。其他主要细胞, 如血管平滑肌细胞或神经元可能会更好地坚持胶原蛋白或纤维连接蛋白涂层片. </li>
</ol> 3。制备单元格 <ol> <li> 培养单元格 <ol> <li> 在10毫升 Dulbecco 和 #39 的 10 cm 培养基上生长 COS-7 细胞 (或优选的细胞系); 经改良的鹰培养基 (DMEM) 培养基, 辅以10% 胎牛血清 (FBS) 和1%青霉素/链霉素 (PS) 溶液。在37和 #176 的细胞培养孵化器中培养细胞; C 和 5% CO 2 . </li>
		<li> 当细胞达到 90% 70-100, 从 10 cm 盘中吸取 DMEM 培养基, 并加入5毫升0.05% 胰蛋白酶 EGTA 溶液。一旦细胞开始旋转和分离, 立即中和的胰蛋白酶与补充 DMEM。
		<ol> <li> 使用5毫升血清吸管从盘中取出细胞。将培养基移到盘底上数次, 以除去所有保持附着的细胞, 并在培养基中 homogenously 分布细胞. </li>
		</ol> </li> <li> 将细胞印在 35 mm 培养基上, 实现 70% 70-100 转染。用新鲜的 DMEM/FBS/PS 溶液使碟中的培养基体积达2毫升. </li>
		<li> 染 COS-7 细胞与所需的质粒 DNA 结构 ( 如 , sec61 和 #946;-mCherry 质粒), 使用适当的转染试剂, 并根据制造商和 #39; s 说明. 
		注意: 根据质粒和/或所使用的转染试剂, 蛋白质的表达可能需要24-48 小时. </li>
	</ol> </li> <li> 成年小鼠心室肌细胞 <ol> 使用成熟的共生灌注法分离成年小鼠心室肌细胞 <li> 6 。在中 199 (M199) 中, 再用 2.5% FBS 和 1% PS, 重新暂停产生的心肌细胞颗粒. </li>
	</ol> </li> </ol> 4。电镀单元格 <ol> <li> 转染 COS-7 单元 <ol> <li> 从 35 mm 盘中抽出2毫升 DMEM/FBS/PS 解决方案, 并添加1毫升 Ca 2 + 免费 PBS。将该盘从盘中的2分钟内的收获细胞中, 先将钙的 2 + 免费 PBS 吸出, 再加入1毫升的0.05% 胰蛋白酶-EGTA 溶液。
		<ol> <li> 一旦细胞开始旋转和分离, 立即中和胰蛋白酶与1毫升补充 DMEM。在2毫升胰蛋白酶-EGTA-DMEM 悬浮液中, 轻轻地上下吸管多次, 以确保转染细胞均匀分布. </li>
		</ol> </li> <li> 在适当的密度下将 COS-7 细胞转染到多聚 l-赖氨酸涂层片上, 使单个电池可以可视化, 并以 DMEM/FBS/PS ( 如 、添加90和 #181 的大小来填充2毫升的碟形; L 细胞悬浮到片然后添加1910和 #181; L 补充 DMEM, 旋流盘均匀分布细胞). 
		注意: 细胞不需要计数, 但每道菜要添加的细胞体积会因细胞的70-100 而变化. </li>
		<li> 在一夜之间将镀膜细胞返回细胞培养箱, 以使细胞黏附并恢复. </li>
	</ol> </li> <li> 成年的小鼠心室肌细胞 <ol> 将层粘连蛋白和平板肌细胞直接吸到涂覆的片上, 以适当的密度使单个细胞可视化。这将取决于从隔离中获得的活细胞的密度. </li>
		<li> 平衡细胞悬浮在一个圆顶上的片和地方的细胞培养孵化器在37和 #176; C 和 5% CO 2 为〜45分钟, 以允许粘连. </li>
	</ol> </li> </ol> 5。细胞固定 <ol> <li> 从孵化箱中取出单元格并小心地吸入培养基. 
	<ol> <li> 用 PBS 冲洗粘附的单元格一次. </li>
		<li> 使用固定在单个应用程序和抗体上的定影液修复细胞. 
		注: 在选择固定剂时要牢记的一个重要标准是保存样品的细胞结构, 同时也要确保在抗原中没有构象的变化。为本议定书的目的固定与3% 甲醛/0.1% 戊二醛 (粉煤灰/GA) 和固定100% 甲醇讨论. </li>
	</ol> </li> <li> Sec61 和 #946 的转染 COS-7 细胞的灰化/ga 固定;-mCherry <ol> <li> 混合 1.9 ml 16% 和20和 #181; L 50% ga 并添加 PBS 以使最后的容量10毫升. </li>
		<li> 将1毫升新鲜的粉煤灰/GA 溶液添加到每道细胞中, 并在室温下固定10分钟. </li>
		<li> 在 PBS 中吸出煤灰/GA 并冲洗细胞3次. </li>
		<li> 用 PBS 清洗黏附的细胞, 每5次5分钟。将旋转的所有洗涤步骤设置为中等速度 ( 例如 , 每分钟50转). </li>
		<li> 接下来, 在去水中, 用1毫升新鲜制备的0.1% 质量/体积氢化钠减少游离醛基团. </li>
		<li> 冲洗细胞两次, 然后清洗3次, 每5分钟在 PBS, 以消除所有的痕迹硼氢化钠和酒精, 它产生时, 与自由醛反应. </li>
	</ol> </li> <li> 成年小鼠心室肌细胞的甲醇固定 <ol> 小心地在细胞中加入1毫升的 ice-cold 100% 甲醇。倾斜的6井板和吸管的甲醇对侧壁, 而不是直接到片。然后倾斜6井板回水平, 使甲醇均匀洗涤在细胞上。在-20 和 #176 固定5分钟; C. </li>
		<li> 用 PBS 吸出固定剂和冲洗细胞3次. </li>
		<li> 用 PBS 清洗黏附的细胞, 旋转5次, 每次5分钟. </li>
	</ol> </li> </ol> 6。阻止非特定绑定 <ol> <li> 阻塞解决方案中室温下的 1 h 块 (请参见 材料表 ). 
	注: 各种溶液可用于阻断非特异性抗体结合, 包括3% 牛血清白蛋白 (BSA) 或与原抗体相同物种的免疫血清. </li>
</ol> 7。检测 <ol> <li> 主抗体孵化 <ol> 提取阻塞解决方案。请勿清洗或冲洗. </li>
		<li> 准备主要抗体溶液如下 (参见步骤 7.1.5): 将主要抗体稀释到10和 #181 的浓度; 抗体培养液中的 g/毫升 (或制造商和 #39; s 建议浓度) (请参阅 材料表).平衡这个小体积的片, 并仔细设置一个正方形的塑料石蜡薄膜在上面。这有助于传播小体积的抗体在片和防蒸发. </li>
		<li> 将6个井板放在湿度室中, 然后在冰箱中过夜. </li>
		<li> 在早上, 从湿度室中取出细胞, 小心地从片的表面去除石蜡薄膜, 并吸取主抗体溶液. </li>
		<li> 用 PBS 冲洗3次, 然后用洗涤块解决方案洗涤5次5分钟 (请参阅 旋转体上的材料表 ). 
		注: 本协议使用以下抗体, 抗 RFP 小鼠单克隆抗体的图像显示在 图 2 和兔多克隆 FP1 7 抗 Ca V 1.2 抗体 (来自 Prof. 的礼物在 图 3 中显示的图像。关于湿气室, 一个塑料饭盒与一个紧合的盖子, 内衬湿纸巾工作。PBS 可用于洗涤步骤, 但该协议提高了标签的特异性, 并通过使用稀释的堵塞溶液清洗步骤减少了背景. </li>
	</ol> </li> <li> 二次抗体孵化 <ol> <li> 吸入洗涤液并添加200和 #181; 共轭二次抗体溶液稀释 1:1, 000 抗体培养液。在片 (参见步骤 7.1.2) 的顶部放置一正方形石蜡膜. </li>
		<li> 在室温下在黑暗中孵育1小时. </li>
		<li> 吸入二次抗体, 用 PBS 冲洗3次. </li>
		<li> 用稀释的堵塞溶液冲洗细胞5次, 在摇杆上5分钟. </li>
		<li> 以 PBS 为5分钟清洗3次. 
		注: 本协议描述了使用 anti-mouse alexa 647 共轭继发抗体和兔 alexa 647 共轭继发抗体的 图 2 和 图 3 , 分别。对于单蛋白质标签, Alexa 647 是首选荧光由于其高光子计数 (提高定位精度) 和低占空比 (改善时间分辨率) 8 。许多其他荧光共轭的二次抗体选择, 如阿托或 Cy 染料, 是可利用的。请参阅 et al. 8 和 Chozinski et al. 9 用于综合评估几种荧光/染料对超分辨率成像的适用性. </li>
	</ol> </li> </ol> 8。后固定 (可选步骤) <ol> <li> 在 10 mL PBS 中稀释50和 #181; L 50% ga 以获得 0.25% ga 解决方案。在室温下, 在本解决方案中, 在10分钟后修复细胞. </li>
	<li> 用 PBS 洗涤3次, 在一个旋转的5分钟. </li>
</ol> 9。存储示例 <ol> <li> 将样品存储在冰箱 (4 和 #176; C) 中, 在铝箔衬里的容器中保存, 直到成像. </li>
	<li> 用于长期储存样品 (最多几个月), 在 PBS 中储存含有3毫米的叠氮化钠, 以防止细菌生长. </li>
</ol> 10。超分辨率成像 Photoswitching 缓冲准备 <ol> <li> 准备氧清除和 #39; GLOX 和 #39; 解决方法如下: <ol> <li> 在1.5 毫升离心管, 添加12.5 和 #181; L 过氧化氢酶 (34 毫克/毫升), 3.5 毫克葡萄糖氧化酶, 0.5 和 #181; l 1 米 (pH = 8) 到49.5 和 #181; l PBS. </li>
		<li> 漩涡简要地溶化组分入解答然后离心在 20800 x g 为3分钟在4和 #176; C. 
		注: 氧清除解决方案, 如 GLOX, 已被发现反对漂白。GLOX 应该放在冰箱里一周。每次使用时重复离心机步骤. </li>
	</ol> </li> <li> 准备 photoswitching 诱导的硫醇解决方案, 如下所示: <ol> <li> 准备100毫米和 #946;-mercaptoethylamine (多边环境协定) 解决方案在 PBS 和修改 ph 值8或交替使用和 #946;-基 (#946; 我)。存储 aliquoted 的解决方案在冰箱 (-20 和 #176; C) 长达1周. </li>
	</ol> </li> <li> 在成像之前, 通过将硫醇和氧清除组件一起添加到冰上准备最终的交换缓冲器。500和 #181; 我 GLOX 添加5和 #181; l GLOX 到50和 #181; l (ph 值 = 8) 和445和 #181; l 盐水缓冲 (2.5 毫升 1 M 三 ph = 8, 29.22 毫克氯化钠 (10 毫米最后的集中), 5 g 葡萄糖和47.5 毫升水)。另外, 对于 GLOX #946; 我的解决方案添加5和 #181; 我 GLOX 到5和 #181; l 和 #946; 我和490和 #181; l 盐水缓冲. 
	<ol> <li> 将解决方案保留在冰上, 并根据需要在玻璃凹陷幻灯片上安装样品. 
		注: 含硫醇的溶液, 如和 #946; 我或多边环境诱导 photoswitching cyanine-based 染料, 如 alexa 647 10 或 xanthene-based 染料, 如 alexa 568。和 #946; 我的结果与 alexa 的 647, 而多边环境协定是首选的 alexa 568 或当2的蛋白质是用双重标记的方法, 同时利用 alexa 568 和647。由于酸化, 缓冲液在一段时间内会恶化。这将导致样品的平均光子计数的减少。为防止这种情况, 建议在2-3 小时后进行新鲜的 photoswitching 缓冲, 或者如果观察到平均光子计数的下降或诱导 photoswitching 的时间的显著延长。最近的一篇文章描述了一个新的图像缓冲牛 EA, 我们还没有测试, 但据报道, 显示稳定的 pH 值数天, 并表现优于 GLOX 多色成像应用 11 . </li>
	</ol> </li> </ol> 11。安装示例 
<ol> <li> 在抑郁症幻灯片上使用带有标记的单元格 (参见1-9 节) 安装片, 用100和 #181; L 成像缓冲区. </li>
	<li> 用硅胶胶水封住片的边缘, 以防止成像缓冲器的快速氧化。等待几分钟, 直到硅胶胶水已经完全治愈, 否则片将漂移时, 成像. 
	注: 如果与 #946 接触, 硅胶胶水不会硬化; 我。一定要擦干片的边缘, 以防止硅胶胶水接触成像缓冲器. </li>
</ol> 12。图像获取 <ol> <li> 请参见表 1, 以获取 图 2 和 图 3 中使用的图像参数列表。
	<ol> <li> 开始时, 根据所选的荧光选择适当的激光来照亮样品。本协议中使用的 SR-物料供应系统配备大功率激光器 (488 nm、1.4 kw/cm 2 ; 532 nm、2.1 kw/cm 2 ; 561 nm、2.1 kw/cm 2 642 nm、2.1 kw/cm 2 )。 图 2 使用了 642 nm 激光. </li>
	</ol> </li> <li> 使用高 NA 的油浸物镜。在这里使用的 SR-物料供应系统配备了 HC PL 160 x/1.43 NA 目标. </li>
	<li> 选择了适当的分色图像多维数据集。该协议中的 SR-物料供应系统配备了 488 hp t, 532 hp t, 568 hp t, 642 hp t 与发射带通滤波器505-605 毫微米, 550-650 毫微米和660-760 毫微米. </li>
	<li> 选择2D 获取模式。将相机设置为帧传输模式, 曝光时间为10毫秒 (100 Hz)。将 EM 增益设置为300。选择检测阈值. 
	注意: 低阈值产生高背景的图像。高阈值可能会过滤掉感兴趣的信号。确定基于负控制的阈值, 如 non-transfected 细胞的成像片或只用二次抗体孵育的细胞. </li>
	<li> 启用自动事件控制, 并将每个图像的事件设置为 8. </li>
	<li> 在 "物料供应处购置" 选项卡中输入像素大小 (以 10 nm 或 20 nm 像素大小开始)。确保 #34; 直方图模式和 #34; 在图像获取之前, 在高分辨率图像计算选项的 "物料供应" 工具选项卡中选择. </li>
	<li> 对细胞膜上的蛋白质进行成像, 选择 TIRF 模式并修改入射角以确定穿透深度. </li>
	<li> 将电子发送到黑暗状态 (称为泵浦), 将激光强度设置为 100%. </li>
	<li> 在帧相关性为0.20 时, 选择单分子检测, 同时抽取和设置自动获取. </li>
	<li> 用于获取, 将激光强度设置为 50%. </li>
	<li> 将获取帧的数目设置为 6万. 
	注意: 调查人员可能会发现, 一个样本所需的帧数多于或少于此。修改帧数根据观察荧光漂白和停止承购, 当没有进一步事件是可检测的时. </li>
	<li> 开始购置. 
	注意: 在收购开始时, 所有染料分子都处于荧光状态, 然后切换到黑暗状态。当帧相关性达到0.20 时, 图像将开始被获取。当每帧检测到少于8事件时, 405 激光将打开, 鼓励荧光从黑暗状态过渡到基态。当为 n = y 动物的数据集获取图像时, 如 TIRF 穿透深度、检测阈值和获取的帧数等参数应保持不变. </li>
</ol> 13。图像分析 <ol> <li> 用于量化蛋白质簇大小, 使用和 #39; 分析粒子和 #39; ImageJ 的特征. 
	注: 可以使用随机光学重建 microscopy-based 相对定位分析 (风暴-RLA) 或类似的 13 进行进一步分析。
	<ol> <li> 使用 ImageJ 执行群集分析 ( 图 3 ), 如下所示: </li> <li> 打开要分析的图像文件。这应该是一个 10 nm 直方图单分子本地化地图生成的成像软件上面描述 (第12节). </li>
		<li> 调整亮度和对比度以可视化图像: 单击图像菜单, 选择调整, 然后亮度和对比度。单击 "自动" 选项. </li>
		<li> 将图像类型更改为8位: 选择 "图像" 菜单, 然后选择 "类型", 然后选择8位. </li>
		<li> 设置图像的比例: 打开 "分析" 菜单, 单击 "设置缩放", 然后输入以下参数: 距离 = 1, 已知距离 = 10, 1 像素 = 10 nm. </li>
		<li> 若要选择要获取的度量值, 请打开 "分析" 菜单, 选择 "设置度量值", 然后选中 "区域" 选项旁边的复选框. </li>
		<li> 调整图像的阈值。打开 "图像" 菜单, 选择 "调整", 然后选择 "阈值", 选择 "过/下"。将最大值移动到 255, 并将最小值移动到 1, 然后单击 "应用". </li>
		<li> 要分析粒子, 打开 "分析" 菜单, 选择 "分析粒子"。放置复选标记以选择像素单位、显示结果和汇总。将大小设置为 4-无穷大 (因为分辨率为 ~ 20 nm), 请选择 "裸轮廓" 选项, 然后单击 "确定"。将创建一个摘要文件, 其中将包含分析详细信息, 如图像的平均簇大小和簇数. 
		注意: 在对特定簇中的蛋白质数量做出结论时应谨慎, 因为局部点的数量与蛋白质的数量没有线性相关。本地化与抗体相结合的染料, 产生连锁错误, 将荧光标签从表位中取代 #62; 10 nm 在任何方向。此外, 如果使用多克隆抗体, 不止一个抗体可以结合到一个单一的抗原和混淆的问题更进一步, 商业二级抗体共轭, 平均, 3-6 分子染料, 所以一个荧光并不总是等于一种蛋白质。通过将染料直接共轭到主抗体 (消除次) 或使用 nanobody-based 标记方案 14 , 可以减少连杆错误. </li>
	</ol>
	</li>
</ol>

<ol>
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Probing the stoichiometry of membrane proteins by single-molecule localization microscopy.</a> Sci Rep. 5, 14072 (2015).</li></ol>

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最近的技术爆炸使得成像超出了衍射极限, 为哺乳动物细胞信号在空间和时间上的复杂性提供了新的窗口。这些技术包括风暴、STED、棕榈、物料供应、SIM 和它们的变体 (例如、dSTORM、FPALM)。这些技术背后的科学家的聪明才智使我们能够绕过物理学定律对光的衍射所施加的限制。尽管取得了这一巨大成就, 但每种技术都做出了某种妥协, 以达到超分辨率, 因此, 有其局限性。细胞生物学家和 biop...

细胞信号反应转换内部和外部环境, 以启动细胞的反应。它们调节人类生理学的各个方面, 作为激素和神经递质释放、心跳、视力、受精和认知功能的基础。这些信号级联的中断可能会有严重的后果, 包括癌症, 帕金森病和阿尔茨海默氏症等病理生理条件。几十年来, 生物学和医学研究人员成功地使用了荧光蛋白、探针和生物传感器, 并以荧光显微镜作为主要工具, 了解这些细胞的精确时空组织信号.
光学技术的优势, 如荧光, 共焦, 或全内反射荧光 (TIRF) 显微镜是他们的灵敏度, 速度和与活细胞成像的兼容性, 而主要的限制是他们的衍射有限的分辨率, 意味着结构或蛋白质复合物小于 200-250 nm 不能解决。随着确定性超分辨率的理论和实际发展 (例如, 受激辐射损耗显微镜 (STED1), 结构化照明显微镜 (SIM2) 或随机超分辨率 (例如, 光定位显微镜 (PALM3), 或基态损耗 (物料供应量4,5), 在荧光显微镜中的横向和轴向分辨率已经超出了衍射屏障, 以数十纳米。因此, 调查人员现在有无与伦比的能力, 以可视化和理解如何蛋白质动力学和组织转化为功能的近分子水平。
基态损耗显微镜后, 个别分子返回 (GSDIM), 或简单的政府物料供应, 因为它是已知的, 绕过衍射限制, 减少同时发射荧光4,5的数量。高能激光用于激发荧光标签的样品, 用光子轰击电子, 并增加他们将经受 "自旋翻转" 的几率, 并从激发态4进入三重或 "暗态"。这实际上耗尽了基态, 因此得名 "基态损耗"。在三重状态, 荧光不发射光子和样品出现变暗。然而, 这些荧光随机返回到基态, 并可以通过几个光子发射 excited-to-ground 状态转换, 然后返回到三重状态。在任何给定的时间荧光发射, 从单个荧光发出的光子爆发在空间上和世俗上不同于邻近的荧光。光子的爆发可以与高斯函数相匹配, 计算的质心与荧光的位置相对应, 它的定位精度取决于透镜的数值孔径 (NA), 光的波长用于激发和关键的是, 每荧光发出的光子数。物料供应处的一个限制是, 由于只有一个荧光的子集在任何时候积极发出, 数千张图像必须收集超过几分钟, 以建立一个完整的本地化地图。较长的采集时间与高激光功率要求相结合, 意味着物料供应处更适合于固定而非活体样品。
本文介绍了用于膜和内质网 (ER) 驻留蛋白的超分辨率显微成像固定样品的制备 (如需消耗品和试剂清单, 请参阅材料表)。如何方便地对此协议进行调整, 以量化心肌细胞肌中 L 型电压门控 ca2 +通道 (cav1.2) 的大小和聚类, 或用于可视化 ER 的细胞分布,被证明。了解这些单元格组件的分布和组织对于了解许多 Ca2个相关的信号级联的启动、翻译和最终功能至关重要。例如, cav1.2 通道对于激发-收缩耦合至关重要, 而受体介导的 ca2 +则可能是哺乳动物细胞中最普遍的信号级联。

<table><tbody><tr><td>KOH</td><td>Thermo Scientific</td><td>P250-500</td><td>To clean coverglass</td></tr><tr><td>#1.5 coverglass 18 x 18 mm</td><td>Marienfeld Superior</td><td>0107032)</td><td>To grow/process/image cells</td></tr><tr><td>10x PBS</td><td>Thermo Scientific</td><td>BP3994</td><td>Dilute to 1x with de-ionized water</td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P4832</td><td>Aids with cell adhesion to cover glass</td></tr><tr><td>laminin</td><td>Sigma</td><td>114956-81-9</td><td>Aids with cell adhesion to cover glass</td></tr><tr><td>Medium 199</td><td>Thermo Scientific</td><td>11150-059</td><td>Ventricular myocyte culture media</td></tr><tr><td>DMEM 11995</td><td>Gibco</td><td>11995</td><td>Cell culture media</td></tr><tr><td>Fetal bovine Serum (FBS)</td><td>Thermo Scientific</td><td>10437028</td><td>Media supplement</td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma</td><td>P4333</td><td>Media supplement</td></tr><tr><td>0.05% trypsin-EDTA</td><td>Corning</td><td>25-052-CL</td><td>Cell culture solution</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-019</td><td>Transient transfection reagent</td></tr><tr><td>Ca2+-free PBS</td><td>Gibco</td><td>1419-144</td><td>Cell culture</td></tr><tr><td>100 % Methanol</td><td>Thermo Fisher Scientific</td><td>A414-4</td><td>Cell Fixation</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Cell Fixation</td></tr><tr><td>Glutaraldehyde</td><td>Sigma Aldrich</td><td>SLBR6504V</td><td>Cell Fixation</td></tr><tr><td>SEAblock</td><td>Thermo Scientific</td><td>37527</td><td>BSA or other blocking solution alternatives exist</td></tr><tr><td>Triton-X 100</td><td>Sigma</td><td>T8787</td><td>Detergent to permeabilize cells</td></tr><tr><td>Rabbit anti-CaV1.2 (FP1)</td><td>Gift</td><td>N/A</td><td>Commercial anti-CaV1.2 antibodies exist such as Alomone Labs Rb anti-CaV1.2 (ACC-003)</td></tr><tr><td>Mouse monoclonal anti-RFP</td><td>Rockland Inc.</td><td>200-301-379</td><td>Primary antibody</td></tr><tr><td>Alexa Fluor 647 donket anti-rabbit IgG (H+L)</td><td>Invitrogen (Thermo Scientific)</td><td>A31573</td><td>Secondary antibody</td></tr><tr><td>Alexa Fluor 568 goat anti-mouse IgG (H+L)</td><td>Invitrogen (Thermo Scientific)</td><td>A11031</td><td>Secondary antibody</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>Prevents microbial growth for long term storage of samples</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C40</td><td>Photoswitching buffer ingredient</td></tr><tr><td>Glucose oxidase</td><td>Sigma</td><td>G2133</td><td>Photoswitching buffer ingredient</td></tr><tr><td>Tris</td><td>Sigma</td><td>T6066</td><td>Photoswitching buffer ingredient</td></tr><tr><td>beta-Mercaptoethylamin hydrochloride</td><td>Fisher</td><td>BP2664100</td><td>Photoswitching buffer ingredient</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>63689</td><td>Photoswitching buffer ingredient</td></tr><tr><td>NaCl</td><td>Fisher</td><td>S271-3</td><td>Photoswitching buffer ingredient</td></tr><tr><td>Dextrose</td><td>Fisher</td><td>D14-212</td><td>Photoswitching buffer ingredient</td></tr><tr><td>Glass Depression slides</td><td>Neolab</td><td>1 &amp;ndash; 6293</td><td>To mount samples</td></tr><tr><td>Twinsil</td><td>Picodent</td><td>13001000</td><td>To seal coverglass</td></tr><tr><td>sec61&amp;beta;-mCherry plasmid</td><td>Addgene</td><td>49155</td><td></td></tr><tr><td>Leica SR GSD 3D microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td></td></tr><tr><td>Washing block solution</td><td></td><td></td><td>20 % SEAblock in PBS</td></tr><tr><td>Primary antibody incubation solution</td><td></td><td></td><td>0.5 % Triton-X100, 20 % SEAblock, in PBS</td></tr><tr><td>Secondary antibody incubation solution</td><td></td><td></td><td>1:1000 in PBS</td></tr></tbody></table>

ground state depletion super-resolution imaging in mammalian cells

荧光显微镜和细胞生物学的进展是密切相关的, 增强的能力, 可视化细胞事件往往导致戏剧性的飞跃, 我们的理解细胞的功能。超分辨率显微镜的发展和可用性大大扩展了光学分辨率的极限, 从 ~ 250-20 nm。生物学家不再局限于在衍射有限区域内描述分子间的相互作用, 相反, 现在有可能对单个分子的动态相互作用进行可视化定位。在这里, 我们概述了一个协议的可视化和量化的细胞蛋白的地面状态损耗显微镜的固定细胞成像。我们提供的例子来自两种不同的膜蛋白, 一个元素的内质网 translocon, sec61β, 和一个等离子膜局部电压门控 L 型 ca2 +通道 (caV1.2)。讨论了具体的显微镜参数、固定方法、光开关缓冲配方、图像处理的缺陷和挑战。

如导言中所记载的, 有许多不同的超分辨率显微成像模式。该协议, 重点在物料供应的超分辨率成像。有代表性的图像和本地化映射显示在图 2和图 3中。
图 2显示了转染 ER 蛋白的 COS-7 细胞, mCherry-Sec61β, 并使用上述方法进行处理。图 2A<str...

Watch this Scientific Journal Video about Ground State Depletion Super-resolution Imaging in Mammalian Cells at JoVE.com

Ground State Depletion Super-resolution Imaging in Mammalian Cells

本文概述了一种用于检测哺乳动物细胞中的一种或多种等离子和/或胞内膜蛋白的方法。在这里, 我们讨论的好处和考虑使用这种方法的可视化和量化的细胞蛋白。

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

ground-state-depletion-super-resolution-imaging-in-mammalian-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

7.1K 次观看.  University of California School of Medicine, Davis. 本文概述了一种用于检测哺乳动物细胞中的一种或多种等离子和/或胞内膜蛋白的方法。在这里, 我们讨论的好处和考虑使用这种方法的可视化和量化的细胞蛋白。

视频: Ground State Depletion Super-resolution Imaging in Mammalian Cells

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

用于活细胞超分辨率显微镜和单分子跟踪的传统 BODIPY 结合物

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

生物化学

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

免疫与感染

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

神经元的密集核心囊泡的超分辨率成像

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

神经科学

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

发育生物学

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

超分辨率成像细菌科机械

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

超分辨率成像研究原发神经元中蛋白质和突触标记的共同定位

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

在Cytokineticž圈的活细菌超分辨率成像使用快速三维结构照明显微镜（F3D-SIM卡）

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. 
However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). 
To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.