我们感谢 Dr. 克里斯汀 Verhey (密歇根大学, 安阿伯) 和 Dr. 格雷戈里 Pazour (马萨诸塞大学医学院) 提供一些质粒。该项目得到了国家卫生研究院 (NIH GM097037、GM116204 和 GM122552 W.Y.) 的资助。

1. NIH-3T3 细胞制备速度显微镜从库存
<ol><li>在实验前1.5 周, 在37° c 解冻的情况下, 从冷冻库中恢复 NIH-3T3 细胞的新鲜培养, 并将细胞转化为25厘米2细胞培养烧瓶, 3 毫升 Dulbecco 的改良鹰培养基 (DMEM) 补充110毫克/毫升丙酮酸钠, 2 毫米谷氨酰胺, 10% 胎牛血清, 1% 青霉素/链霉素。</li>
	<li>在 5% CO2孵化器中孵育37° c 的细胞。</li>
	<li>分裂细胞在 80% 70-100, 大约每两天, 至少在实验日前三次, 以确保细胞周期的均匀性。处理细胞与0.25% 胰蛋白酶2分钟在37° c, 抽吸胰蛋白酶和替换它与2毫升媒介。将介质反复移出细胞簇, 去除所需的细胞数量, 使培养基总体积恢复到3毫升。 注意: NIH-3T3 以前是基因工程的, 以表达 NPHP-4, 一种蛋白质, 本地化到26, 融合在 C 总站到 mCherry。mCherry 是一种荧光, 可以在561纳米光照的照射下, 定量地定位其选择性屏障, 使原纤毛处于定向。</li>
	<li>实验前两天, 将细胞放入35毫米的玻璃底盘中, 60-70% 70-100, 1.5 毫升的培养基为1.1 步, 并将细胞返回孵化室。</li>
	<li>在实验的前一天, 化学染的细胞与所需的质粒。混合 500-1000 ng 所需的质粒 (见下面的注意) 在 1:2. 5 的比例与转染试剂在0.25 毫升的减少血清培养基中的30毫米玻璃底碟中的35分钟, 用0.25 毫升的质粒取代它转染试剂混合物加上额外的1.25 毫升的减少血清介质没有抗生素。减少血清培养基的目的是促进成功转染, 诱导原发性纤毛生长, 以及保持细胞存活足够长的时间进行实验。第二天将细胞返回孵化室进行实验。 注意: 当执行分子跟踪 IFT20, 一个质粒含有基因改造 IFT20 融合在其 C 总站到 GFP 是使用25。在执行分子跟踪 SSTR3, 一个质粒含有基因改造 SSTR3 融合在其 N 总站的受体肽 (AP) 域和 C 总站到 GFP 是使用22。除 SSTR3 结构外, 含有生物素连接比拉的质粒必须 co-expressed, 必须用10µM 生物素补充转染培养基。然后, 比拉将生物素附着在 ER 的新合成的 AP-SSTR3-GFP 分子的 AP 域上。Alexa647 共轭到三的四生物素结合位点上的亲和, 平均, 然后可以补充的媒体之前成像荧光标签的 AP-SSTR3-GFP 分子在外部表面的细胞22,27.该方法采用 GFP 和 AlexaFluor647;然而, 其他的荧光探针可以使用, 如果他们具有类似的高光稳定性和量子产量。</li>
	<li>如果使用外部标记的 SSTR3 结构, 在实验前将培养基从玻璃底盘中取出1小时, 用1毫升磷酸盐缓冲盐水 (PBS) 洗涤5次, 并添加1毫升的还原血清培养基, 辅以1µM Alexa647 共轭亲和.</li>
	<li>试验前不超过15分钟, 从玻璃底盘中取出培养基, 用1毫升 PBS 清洗转染和标记的细胞5次。</li>
	<li>放置1毫升的成像缓冲器 (20 毫米 HEPES, 110 毫米 KOAc, 5 毫米 NaOAc, 2 毫米 MgOAc, 1 毫米 EGTA, pH 7.3) 在玻璃底盘。 注意: 在成像缓冲区中, 单元格的生存时间不超过3小时。因此, 每道菜只进行2小时的实验。</li>
</ol>2. 速度显微镜
注: 速度显微镜设置包括一个倒置荧光显微镜配备了 1.4 NA 100×油浸泡物镜目标, 35 兆瓦 633 nm 氦氖激光器, 50 兆瓦固态 488 nm 和 561 nm 激光器, 片上乘法增益电荷耦合器件摄像机和用于数据采集和处理的显微镜软件包 (图 1)。对于单个通道成像, GFP、mCherry 和 Alexa647 分别为 488 nm、561 nm 或 633 nm 激光器所激发。对于单分子跟踪, 单点光照用于跟踪单个荧光标记的分子。对于荧光成像, 将凹透镜放置在激光光照路径中, 以将光束扩展到均匀的光照场。荧光发射是收集相同的目标, 过滤的一个分色过滤器 (405/488/561/635) 和一个发射过滤器 (405/488/561/635), 并与上述 CCD 相机的运行在500赫兹为单分子跟踪或2赫兹荧光成像。
<ol><li>将玻璃底板粘贴到显微镜的舞台上, 并找到一个适当表达所需构造的单元。一旦找到合适的细胞, 将原纤毛底部的 NPHP4-mCherry 点与成像平面上与激光单点照明对应的位置对准。</li>
	<li>如果使用数字显微镜软件包 (参见材料表), 则在 "焦点控制" 窗口的 "相机" 选项卡中使用 "快照" 功能捕获 NPHP4-mCherry 和 IFT20-GFP 或 AP-SSTR3-GFP 的荧光图像。 注意: 这些图像将作为后续的单个分子位置的参考。</li>
	<li>一旦获得了参考图像, 局部减少了标记的单一分子的浓度。照片漂白的1兆瓦激光照明二十年代或直到荧光强度接近的背景荧光。 注: 当精确浓度可控时, 使用 0.1-1 nM 标记的单分子。</li>
	<li>为单分子跟踪准备, 减少激光照射功率〜0.15 兆瓦的单分子标记的 GFP 或〜0.5 兆瓦的分子标记的 Alexa647。</li>
	<li>一旦激光功率和成像参数设置, 最大增益和强化和 2 ms 帧率, 为单分子成像, 从事适当的照明激光和记录 non-photobleached, 标记的单一分子, 因为它们被运输通过点击 "焦点控制" 窗口 "相机" 标签中的 "流" 按钮, 通过 photobleached 区域。 注: 不超过2分钟的视频应该被抓获, 以尽量减少的影响, 睫状漂移到一个微不足道的水平。</li>
	<li>在捕获单分子视频后, 使用2D 高斯拟合算法处理视频, 如洛伊盖尔实验室的一瞥, 它精确地本地化了每个分子在一个包含感兴趣的区域的激发的质心。</li>
	<li>选择所有的单一分子位置的精确 < 10 nm 和纠正的纤毛中心的基础上分布的单分子位置装有2D 高斯函数。 注: 使用2D 到3D 变换算法, IFT20-GFP 和 AP-SSTR3 路线的3D 传输路线分别在睫状纤毛或睫状膜上清晰显示。</li>
</ol>3. 2D 到3D 转换
<ol><li>在纤毛中, 一旦收集了几千个定位的过渡分子 (信噪比 > 11), 选择纤毛的长轴作为 X 维度。制作位置的 Y 维度直方图, 并以 10 nm 增量获取 bin 总和。 注意: 2D 到3D 转换可以通过手工或任何软件或编程语言进行评估。作者成功地实现了在 Matlab 和 Python 2.7 中的转换。</li>
</ol>

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作者声明没有利益冲突。

该协议描述了速度显微镜在初级纤毛的应用, 这是一种高度依赖高效蛋白质传输的细胞信号器官。速度显微镜可以提供高分辨率 (< 10 nm) 的位置为荧光标记分子, 因为他们通过单点照明中心的中间。此前它已被应用于研究通过 NPC 的蛋白质贩运6,7,8。然而, 它可以扩展到研究通过任何 sub-diffraction 细胞腔的贩运。该技术比其他高分?...

自 1873年, 由于来自目标12的光线衍射, 传统光学显微镜的分辨率被认为仅限于大约 200 nm。目前, 超分辨率光显微镜技术打破了这一限制, 并允许捕捉动态图像与 sub-diffraction (< 200 nm) 的决议。该技术一般分为两大类: 受激发射损耗 (STED) 显微镜的方法, 它产生 sub-diffraction 光照体积由于非线性光学响应的荧光在样本3;和光光显微镜 (手掌) 和随机光学重建显微镜 (风暴) 的超分辨率技术, 利用数学功能, 本地化质心的荧光, 然后重建这些质心要形成超分辨率图像4,5。目前, 由于相对简单的光学设置, 棕榈和风暴是广泛使用的只激活一个小子集的荧光在每个帧的一个长视频的生物准备。这使得更精确的定位由2D 高斯拟合的荧光点, 称为点扩散函数 (聚砜), 荧光标记的蛋白质在每个帧的视频。每个荧光标记的分子的2D 位置, 然后可以叠加在一个单一的成像平面上产生一个超分辨率的图像的生物准备1,2。虽然这些分子的本地化, 超分辨率的方法, 以显微镜肯定革命性的生物样品的成像是如何进行的, 仍然有挑战要克服。例如, 风暴和棕榈可以达到他们的最佳空间分辨率后, 固定的生物样品, 从而提出了静态表示的荧光标记蛋白, 这是一个类似的限制电子显微镜。此外, 为了实现在活细胞中每个荧光标记的蛋白质的高空间分辨率, 样品必须成像在非常长的帧, 不能捕捉蛋白质动力学。因此, 有必要克服这些主要的技术障碍。
为了获得一个高时空分辨率, 它非常适合在活细胞中检测快速移动的蛋白质或 rna, 我们在实验室中开发了超分辨速度显微镜 (图 1)6,7,8. 速度显微镜的几个主要技术进步使我们能够成功地追踪小分子、蛋白质、mRNA 和病毒通过本机核孔复合体 (npc) 的 nucleocytoplasmic 传输,6,7,8. 简要地, 以下速度显微镜的特点将用于跟踪快速移动的大分子通过微米旋转对称结构在活细胞, 例如 npc 和主要纤毛: (1) 倾斜或垂直照明, 使在焦平面的小衍射极限体积内的单分子激发 (图 1);(2) 倾斜的聚砜可以极大地避免焦荧光, 从而提高信噪比。(3) 光照中的 100-500 kW/cm2的光学密度允许数千个光子从单个荧光中收集, 并具有快速检测速度 (> 500 Hz)。(4) 快速检测速度还大大降低了分子空间定位错误 (< 10 nm) 在确定活细胞中移动荧光分子的空间轨迹时, 因为分子扩散是主要因素之一造成移动分子分子定位的缺陷。(5) 良好的2D 到3D 转换算法使我们能够为 NPC 或主要纤毛中的分子提供3D 空间概率密度地图。值得注意的是, 我们在笛卡尔和圆柱坐标系之间的转换过程被用来生成3D 空间概率密度图而不是3D 分子跟踪 (图 2)。此前, 电子显微镜数据显示, NPC9、10和主纤毛11都具有旋转对称结构。原则上, 随机扩散分子通过 NPC 或初级纤毛也应该有旋转对称分布。如图 2所示, 大量随机扩散的分子在气缸内将产生旋转对称分布, 在剖面图, 在 NPC, 进一步导致一个近似一致的空间在相邻的两个环之间的每个非常小的子区域中分布 (图 2E)。这种均匀分布导致圆柱系统中θ维数的空间分布是恒定的。然后, 3D 坐标 (r、x、θ) 可以简化为2D 坐标 (r、x、常量)。实际上, 我们在笛卡尔和圆柱系统之间的转换过程是从 2D (x, Y) 到 2D (R, x, 常数)。常数θ, 指<img alt="Equation 1" src="/files/ftp_upload/56475/56475eq1.jpg">的是空间密度p在图 2E中, 是通过使用等式A来计算的。
最终, 分子跟踪在生物研究中有着广泛的应用, 因此, 将开发大量的技术来填充特定的生物龛位12、13、14是很自然的。速度显微镜就是如此。以前, 当与3D 变换算法结合时, 这项技术被开发来解决通过 npc 传递分子的3D 传输路线, 一个 sub-diffraction-sized 和旋转对称的生物结构6。本文中, 原发纤毛也被证明是优秀的模型细胞器。主要纤毛是圆柱形的, 天线状的细胞器 (~ 125 nm 半径), 该项目从大多数哺乳动物的表面15,16,17。它们负责接收外部信号, 并传送通常与生长和新陈代谢相关的细胞内反应15,16。因此, 结构蛋白的通量、跨膜受体的再循环以及细胞内信使的传递是原发性纤毛的重要责任。在主要纤毛和细胞体之间的接合处是一种临界选择性屏障, 称为过渡区或正向, 所有这些蛋白传输必须发生11,18,19, 20。除了 intraflagellar 的浇注功能, 至少有两个传输过程, 即传输和被动扩散, 被认为是负责蛋白质的移动通过这个区域16,21,22. 从人类健康的角度来看, 失去初级纤毛和随后对下游信号的放松管制是许多癌症的特点。此外, 许多遗传性疾病, 如 Bardet Biedl 综合征和多囊肾病, 都与有缺陷的蛋白质转运有关23。sub-diffraction 极限大小和选择性蛋白转运的复杂过程, 使原纤毛成为这项技术的主要目标。在本方法中, 我们将展示一个睫状跨膜蛋白的跟踪, 生长抑素受体 3 (SSTR3)24, 在外部标有 Alexa 氟647和 IFT, IFT2025的成分, 用一个融合的 GFP 分子标记。

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			<td height="47" style="height:47px;width:123px;">25 cm2 tissue culture dish</td>
			<td style="width:204px;">Corning</td>
			<td style="width:101px;">VV-01936-00</td>
			<td style="width:191px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Penicillin/streptomycin</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">15140122</td>
			<td style="width:191px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Fetal bovine serum</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">10438018</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">DMEM</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">10566-016</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">OPTIMEM</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">31985062</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Trypsin</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">25300054</td>
			<td style="width:191px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Phosphate buffered saline</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:101px;">P3813-1PAK</td>
			<td style="width:191px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Transit LT1</td>
			<td style="width:204px;">Mirus</td>
			<td style="width:101px;">MIR 2300</td>
			<td style="width:191px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:123px;">35 mm glass bottom dish</td>
			<td style="width:204px;">MatTek</td>
			<td style="width:101px;">P35GCOL-0-14-C</td>
			<td style="width:191px;"></td>
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		<tr height="64">
			<td height="64" style="height:64px;width:123px;">AlexaFluor 647-conjugated streptavidin</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">S21374</td>
			<td style="width:191px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Biotin</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:101px;">B4501-100MG</td>
			<td style="width:191px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:123px;">633 nm He-Ne laser</td>
			<td style="width:204px;">Melles Griot</td>
			<td style="width:101px;">25-LHP-928-249</td>
			<td style="width:191px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:123px;">561 nm solid state laser</td>
			<td style="width:204px;">Coherent</td>
			<td style="width:101px;">OBIS 561-50 LS</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">488 nm solid state laser</td>
			<td style="width:204px;">Coherent</td>
			<td style="width:101px;">1185053</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">Inverted fluorescence microscope</td>
			<td style="width:204px;">Olympus</td>
			<td style="width:101px;">IX81</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:123px;">1.4-NA 100&#215; oil-immersion apochromatic objective</td>
			<td style="width:204px;">Olympus</td>
			<td style="width:101px;">UPLSAPO 100&#215;</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:123px;">On-chip multiplication gain charge-coupled-device camera</td>
			<td style="width:204px;">Roper Scientific</td>
			<td style="width:101px;">Cascade 128+</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">Dichroic filter</td>
			<td style="width:204px;">Semrock</td>
			<td style="width:101px;">Di01- R405/488/561/635-25x36</td>
			<td style="width:191px;"></td>
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		<tr height="64">
			<td height="64" style="height:64px;width:123px;">Emission filter</td>
			<td style="width:204px;">Semrock</td>
			<td style="width:101px;">NF01-405/488/561/635-25X5.0</td>
			<td style="width:191px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Slidebook 6.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td></td>
			<td style="width:191px;">digital microscopy software</td>
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application of high-speed super-resolution speed microscopy in live primary cilium

主要纤毛是在许多真核细胞的表面上的 microtubule-based 突起, 它包含了在细胞运动和信号中具有重要作用的独特的蛋白质补充。由于纤毛无法合成自己的蛋白质, 近200独特的睫状蛋白需要在胞和原发纤毛之间进行贩运。然而, 这仍然是一个技术上的挑战, 以地图三维 (3D) 的运输途径的这些蛋白质在活原发纤毛的位置, 由于目前现有的技术的局限性。为了克服这一挑战, 最近我们开发并使用了一个高速虚拟3D 超分辨率显微术, 称为单点边缘激发 sub-diffraction (速度) 显微镜, 以确定3D 空间位置的传输路径胞浆和细胞膜蛋白都是活细胞的主要纤毛。在本文中, 我们将演示的详细设置速度显微镜, 细胞表达荧光蛋白标记的睫状蛋白, real-time 分子跟踪个体蛋白在活纤毛和成就3D 空间概率密度图谱的传输路线的睫状蛋白。

本节演示了从在原纤毛的 SSTR3 上执行速度显微镜获得的数据, 以研究由 15 nm 外部链接器连接到 Alexa647 (图 3a) 的传输路径。它为验证3D 变换算法的双重目的服务。Alexa647 应只标记主纤毛的外部表面, 因此, 3D 运输路线应在该位置显示高密度的运输路线。NIH-3T3 稳定表达 NPHP3-mCherry, 必须转染 AP-SSTR3-GFP 和孵化根据上述协议与亲和共轭 Alexa647。...

Watch this Scientific Journal Video about Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium at JoVE.com

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

最近, 我们映射了三维 (3D) 的运输路线的空间位置的各种蛋白质转运内原纤毛的活细胞。本文详细介绍了新应用于活原纤毛的3D 超分辨率荧光成像方法的实验装置、生物样品的处理过程和数据分析。

高速超分辨速度显微镜在活原纤毛中的应用

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

application-high-speed-super-resolution-speed-microscopy-live-primary

Universidad de Cartagena UDEC

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Biology

Unit 1

Cells and their Components

SCIENTISTS IN ACTION

8.2K 次观看.  Temple University. 最近, 我们映射了三维 (3D) 的运输路线的空间位置的各种蛋白质转运内原纤毛的活细胞。本文详细介绍了新应用于活原纤毛的3D 超分辨率荧光成像方法的实验装置、生物样品的处理过程和数据分析。

视频: Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

基于液滴在微流体装置的被动抽水高速输送系统

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

科研

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 ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

试验样品为优化风暴超高分辨率显微镜

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

中G3BP应力颗粒动力学在Live原代细胞可视化

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

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 ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

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 ...

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 ...