Name: application of high-speed super-resolution speed microscopy in live primary cilium
Uploaded: 2018-01-16T12:30:00.000+00:00
Duration: 7 min 53 s
Description: Watch this Scientific Journal Video about Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium at JoVE.com

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>

<ol>
	<li>Huang, B., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+fluorescence+microscopy.">Super-resolution fluorescence microscopy.</a> Annu Rev Biochem. 78, 993-1016 (2009).</li><li>Leung, B. O., Chou, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+super-resolution+fluorescence+microscopy+for+biology.">Review of super-resolution fluorescence microscopy for biology.</a> Appl Spectrosc. 65, 967-980 (2011).</li><li>Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+reveals+that+synaptotagmin+remains+clustered+after+synaptic+vesicle+exocytosis.">STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.</a> Nature. 440, 935-939 (2006).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313, 1642-1645 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat Meth. 3, 793-796 (2006).</li><li>Ma, J., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+distribution+of+transient+interactions+in+the+nuclear+pore+complex+obtained+from+single-molecule+snapshots.">Three-dimensional distribution of transient interactions in the nuclear pore complex obtained from single-molecule snapshots.</a> Proc Natl Acad Sci USA. 107, 7305-7310 (2010).</li><li>Ma, J., Goryaynov, A., Sarma, A., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-regulated+viscous+channel+in+the+nuclear+pore+complex.">Self-regulated viscous channel in the nuclear pore complex.</a> Proc Natl Acad Sci USA. 109, 7326-7331 (2012).</li><li>Ma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+three-dimensional+mapping+of+mRNA+export+through+the+nuclear+pore.">High-resolution three-dimensional mapping of mRNA export through the nuclear pore.</a> Nat Comm. 4, (2013).</li><li>Akey, C. W., Radermacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+Xenopus+nuclear+pore+complex+revealed+by+three-dimensional+cryo-electron+microscopy.">Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy.</a> J Cell Biol. 122, 1-19 (1993).</li><li>Akey, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+and+structure+of+the+nuclear+pore+complex+revealed+by+cryo-electron+microscopy.">Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy.</a> J Cell Biol. 109, 955-970 (1989).</li><li>Czarnecki, P. G., Shah, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ciliary+transition+zone:+from+morphology+and+molecules+to+medicine.">The ciliary transition zone: from morphology and molecules to medicine.</a> Trends Cell Biol. 22, 201-210 (2012).</li><li>Elf, J., Li, G. -. W., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+transcription+factor+dynamics+at+the+single-molecule+level+in+a+living+cell.">Probing transcription factor dynamics at the single-molecule level in a living cell.</a> Science. 316, 1191-1194 (2007).</li><li>Anzalone, A., Annibale, P., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+orbital+tracking+in+a+modified+two-photon+microscope:+an+application+to+the+tracking+of+intracellular+vesicles.">3D orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles.</a> J Vis Exp. , (2014).</li><li>Ritter, J. G., Veith, R., Veenendaal, A., Siebrasse, J. P., Kubitscheck, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+microscopy+for+single+molecule+tracking+in+living+tissue.">Light sheet microscopy for single molecule tracking in living tissue.</a> PloS one. 5, 11639 (2010).</li><li>Marshall, W. F., Nonaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia:+tuning+in+to+the+cell's+antenna.">Cilia: tuning in to the cell's antenna.</a> Curr Biol. 16, 604-614 (2006).</li><li>Scholey, J. M., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraflagellar+transport+and+cilium-based+signaling.">Intraflagellar transport and cilium-based signaling.</a> Cell. 125, 439-442 (2006).</li><li>Yang, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superresolution+pattern+recognition+reveals+the+architectural+map+of+the+ciliary+transition+zone.">Superresolution pattern recognition reveals the architectural map of the ciliary transition zone.</a> Sci Rep. 5, 14096 (2015).</li><li>Craige, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CEP290+tethers+flagellar+transition+zone+microtubules+to+the+membrane+and+regulates+flagellar+protein+content.">CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content.</a> J Cell Biol. 190, 927-940 (2010).</li><li>Kee, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+size-exclusion+permeability+barrier+and+nucleoporins+characterize+a+ciliary+pore+complex+that+regulates+transport+into+cilia.">A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia.</a> Nat Cell Biol. 14, 431-437 (2012).</li><li>Najafi, M., Maza, N. A., Calvert, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steric+volume+exclusion+sets+soluble+protein+concentrations+in+photoreceptor+sensory+cilia.">Steric volume exclusion sets soluble protein concentrations in photoreceptor sensory cilia.</a> Proc Natl Acad Sci USA. 109, 203-208 (2012).</li><li>Nachury, M. V., Seeley, E. S., Jin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+to+the+ciliary+membrane:+how+to+get+across+the+periciliary+diffusion+barrier.">Trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier.</a> Annu Rev Cell Dev Biol. 26, 59-87 (2010).</li><li>Ye, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+imaging+reveals+a+major+role+for+diffusion+in+the+exploration+of+ciliary+space+by+signaling+receptors.">Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors.</a> Elife. 2, 00654 (2013).</li><li>Ross, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Bardet-Biedl+syndrome+ciliary+proteins+perturbs+planar+cell+polarity+in+vertebrates.">Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates.</a> Nat Genetics. 37, 1135-1140 (2005).</li><li>Handel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+targeting+of+somatostatin+receptor+3+to+neuronal+cilia.">Selective targeting of somatostatin receptor 3 to neuronal cilia.</a> Neuroscience. 89, 909-926 (1999).</li><li>Follit, J. A., Tuft, R. A., Fogarty, K. E., Pazour, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intraflagellar+transport+protein+IFT20+is+associated+with+the+Golgi+complex+and+is+required+for+cilia+assembly.">The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly.</a> Mol Biol Cell. 17, 3781-3792 (2006).</li><li>Awata, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NPHP4+controls+ciliary+trafficking+of+membrane+proteins+and+large+soluble+proteins+at+the+transition+zone.">NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone.</a> J Cell Sci. 127, 4714-4727 (2014).</li><li>Howarth, M., Ting, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+proteins+in+live+mammalian+cells+with+biotin+ligase+and+monovalent+streptavidin.">Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.</a> Nat Protoc. 3, 534-545 (2008).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+super-resolution+imaging+by+stochastic+optical+reconstruction+microscopy.">Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.</a> Science. 319, 810-813 (2008).</li></ol>

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。该技术比其他高分辨率成像技术具有优势, 因为它能够分别在 < 10 纳米和 < 2 ms 的空间和时间分辨率下, 捕捉活细胞系统中单蛋白传输的动态。另一个好处是, 一个必须只荧光标签的兴趣蛋白和表达的结构通过转染, 一个共同的分子生物学方法研究蛋白质的本地化, 开始执行速度显微镜。我们必须记住, 用大功率激光器的单点照明是提供分离、高分辨率单分子位置的特性。然而, 这也限制了照明面积, 使技术不适合广泛的现场定位。幸运的是, 有许多广泛的领域超分辨率技术供研究者选择, 如果需要的话。另一个需要考虑的一点是, 速度显微镜只捕获移动单分子的位置。因此, 与 FRAP 的结合, 可以确定的总分子, 是流动的分数, 可能是一个有用的补充分析。
笛卡尔和圆柱坐标系之间的转换过程是生成一个虚拟的3D 概率密度图, 而不是基于3D 分子跟踪的3D 视图。详细地说, 电子显微镜数据表明, 原纤毛有一个旋转对称结构, 可以产生一个均匀的分子分布沿一定半径。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, 指的是空间密度p, 是通过使用等式A(<img alt="Equation 3" src="/files/ftp_upload/56475/56475eq3.jpg">图 2) 计算的。使用矩阵计算器计算向量, 它对应于横截面视图中相邻同心环之间的相对区域6、 <img alt="Equation 4" src="/files/ftp_upload/56475/56475eq4.jpg"> </img>7Aj位置 j 上的总交互站点直接从实验中测量;因此<img alt="Equation 3" src="/files/ftp_upload/56475/56475eq3.jpg">, pi (ri上的交互站点的空间概率密度) 可以通过求解的矩阵方程来计算.  
协议中的一个关键步骤是最小化参考图像采集和单分子视频之间的显微镜设置的任何扰动。如果发生任何运动, 就不可能满怀信心地将单个分子的位置映射到通过荧光成像获得的主要纤毛的超微结构。另一个重要的步骤是 photobleach 足够的光照区域, 以局部降低荧光标记的单分子的浓度, 但不太多, 人口是完全 photobleached 的。在 SSTR3 的情况下, 扩散系数比可溶性分子慢, un-photobleached SSTR3 分子的迁移到 photobleached 区的时间将长于两分钟, 而睫状漂移则超过了不可因素。如果适当水平的漂白是难以达到和高水平的背景荧光仍然存在, 一个角度照明激光可以用来减少荧光标记的单一分子, 兴奋以上的成像平面。这将减少背景, 提高每个捕获的单个分子的信噪比。
总之, 速度显微镜可以很容易地实现的传统显微镜设置, 增加了激光照明光源, 相关的镜子, 和高速 CCD 相机。其他类似技术的主要进展是倾斜激光, 大大降低了背景荧光和2维3维转换算法, 重建3D 空间概率密度图从2D 分子位置.从生物学的角度来看, 除了荧光共轭蛋白外, 不需要额外的标记。这些特性允许速度显微镜跟踪高时空 (5-10 nm, 0.4-2 ms) 的单个分子, 因为它们通过微米的生物腔或具有旋转对称结构的生物通道进行通信。再加上3D 概率密度变换算法, 标签蛋白质的3D 传输路线可以通过腔或通道来确定。该技术的应用已被用来区分3D 运输路线的蛋白质和 rna 通过 NPC, 在这里, 我们表明, 3D 运输的跨膜蛋白, SSTR3, 和胞浆蛋白, IFT20, 可以实现在主要纤毛。其他的超分辨率技术, 如 3 d-风暴, 已经获得了 x, y, z 位置的单一分子, 通过放置一个圆柱形透镜的光学路径, 造成不对称失真的分子, 根据其位置以上或以下焦点平面28。另一项进展可能尝试实现虚拟针孔技术, 以减少发射的宽度, 从而进一步提高分辨率。以上技术可以很容易地在速度显微镜上实现。总之, 速度显微镜提供了一个独特的方法, 同时确定运输动力学在分子水平和3D 地图运输路径超时空分辨率为间或 intra-organelle 分子贩运在某些情况下活细胞系统。

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。

<table><tbody><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture dish</td><td>Corning</td><td>VV-01936-00</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>ThermoFisher</td><td>15140122</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ThermoFisher</td><td>10438018</td><td></td></tr><tr><td>DMEM</td><td>ThermoFisher</td><td>10566-016</td><td></td></tr><tr><td>OPTIMEM</td><td>ThermoFisher</td><td>31985062</td><td></td></tr><tr><td>Trypsin</td><td>ThermoFisher</td><td>25300054</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P3813-1PAK</td><td></td></tr><tr><td>Transit LT1</td><td>Mirus</td><td>MIR 2300</td><td></td></tr><tr><td>35 mm glass bottom dish</td><td>MatTek</td><td>P35GCOL-0-14-C</td><td></td></tr><tr><td>AlexaFluor 647-conjugated streptavidin</td><td>ThermoFisher</td><td>S21374</td><td></td></tr><tr><td>Biotin</td><td>Sigma-Aldrich</td><td>B4501-100MG</td><td></td></tr><tr><td>633 nm He-Ne laser</td><td>Melles Griot</td><td>25-LHP-928-249</td><td></td></tr><tr><td>561 nm solid state laser</td><td>Coherent</td><td>OBIS 561-50 LS</td><td></td></tr><tr><td>488 nm solid state laser</td><td>Coherent</td><td>1185053</td><td></td></tr><tr><td>Inverted fluorescence microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>1.4-NA 100&amp;times; oil-immersion apochromatic objective</td><td>Olympus</td><td>UPLSAPO 100&amp;times;</td><td></td></tr><tr><td>On-chip multiplication gain charge-coupled-device camera</td><td>Roper Scientific</td><td>Cascade 128+</td><td></td></tr><tr><td>Dichroic filter</td><td>Semrock</td><td>Di01- R405/488/561/635-25x36</td><td></td></tr><tr><td>Emission filter</td><td>Semrock</td><td>NF01-405/488/561/635-25X5.0</td><td></td></tr><tr><td>Slidebook 6.0</td><td>Intelligent Imaging Innovations</td><td></td><td>digital microscopy software</td></tr></tbody></table>

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。宽场成像的 mCherry 标签是用来本地化的, 而 GFP 是用来确保一个细胞表达了所需的 SSTR3 结构。此外, 使用 633 nm 照明的 Alexa647 标签的宽场成像是必要的, 以确认该细胞已正确表达了比拉质粒, 并吸收了足够的生物素从媒体。正确认的特征是具有与背景荧光 (图 3B) 不同的可见纤毛。有效的标记 AP-SSTR3-GFP 结构与亲和共轭 Alexa647 只能发生在这些条件下。一旦确定了纤毛的选择性标记的 Alexa647 已获得, 漂白和单点照明的 633 nm 激光将允许单一分子的可视化。在数据分析之前, 将单分子视频叠加在广域光照中是一个有用的验证步骤 (图 3C)。例如, 如果激光没有正确对准, 没有一个单一的分子会出现与 co-localize。另一个 pre-data 分析验证步骤, 以确保足够的信噪比是检查的强度在单分子视频的长度 (图 3E)。这种分析可以在 ImageJ 中使用 "图形 z 轴剖面" 函数快速执行。图 3F中的峰值对应于具有5.0 的信噪比的单分子的外观。这张图也能确保足够的漂白发生, 就像单分子的分离频率所证明的那样。图 3G演示 < 0.5 的低信噪比。一种解释是, 激光不是直接对准正向的。另一个原因可能是激光照射功率过低。这两种解释导致低激发, 因此, 低排放的荧光在中度。经过这些检查后, 2D 高斯拟合的单一分子将产生他们的轨迹, 在 (图 3H)。叠加许多轨道将产生的运输路线的标记蛋白的兴趣在对 (图 3I)。由于2D 高斯拟合对应于检测器上的像素值, 因此 x、y 单分子数据可以与主纤毛的宽场图像合并, 以进一步说明感兴趣的蛋白质的本地化 (图 3J).
IFT20 是 IFT 复合体的一个组成部分, 它携带货物沿初级纤毛25。Arl13b-mCherry, 一种广泛使用的睫状标记蛋白被用来标记原发性纤毛19。广域 epi 荧光显微镜用于图像的主要纤毛表达 Arl13b-mCherry 和 IFT20-GFP, 然后切换到速度显微镜跟踪单个 IFT20-GFP 蛋白在原发性纤毛后 pre-photobleaching 的 GFP荧光下降到分子水平在照明领域的速度显微镜 (图 4A-4C)。
最终, 可以从实时单元的主纤毛 (图 4D) 中获得具有系统定位精度 < 16 nm 的数百个分子 IFT20-GFP 位置。根据我们的模拟, 250 个半径为 95 nm 的单分子位置足以生成可靠的3D 传输路由 (图 5)。IFT20-GFP 传输路线的最终确定基于从十纤毛收集的数以千计的分子 IFT20-GFP 位置 (图 4E)。由于原纤毛具有旋转对称结构, 2D 到3D 变换算法应用于2D 投影数据。有趣的是, IFT20 的3D 空间概率密度图表明, 只有一个高密度的区域具有 ~ 60-nm 宽度的峰值在 ~ 95 nm 沿原纤毛半径 (图 4E)。此高密度区域可能与纤毛微管 co-localizes, 与 IFT 路由的已知位置 (图 4F) 一致。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56475/56475fig1.jpg"> 图 1.简化的速度显微镜示意图.垂直 (路径 1) 或倾斜 (路径 2) 点扩展函数用于照亮焦平面中的单个荧光分子。"d" 指的是垂直照明激光光束通过目标的中心和角度照明的距离, 这是通过将照明激光聚焦在目标的边缘来实现的。由光学斩波器调节的两个或两个以上的激光器具有开关励磁模式, 用于跟踪通过 NPC 或主纤毛的单分子。关闭时间至少十倍以上的漂白时间的荧光标签上的目标分子。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56475/56475fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56475/56475fig2.jpg"> 图 2.2D 到3D 转换过程的图形演示.示意图演示了2D 到3D 的分子转换算法, 例如, 如果它们在中心流明 (a-F) 或一个管的外围区域 (g-L) 内扩散。(a) 在管内随机扩散分子的理想3D 空间位置可以在圆柱坐标系 (R、X、θ) 中进行协调。在速度上, 只有3D 密度图最终是从2D 空间位置计算出来的。然而, 这是一个理想化的例子, 以说明3D 密度图是等价的, 无论2D 或3D 的空间位置是已知的。(B) 在 a 的3D 分子位置被投射到一个2D 平面在笛卡尔协调系统 (X, Y, Z) 通过显微镜成像。(C) 一个非常薄的切片 (x) 从气缸沿 x 维度切割。(D) C 所示的切片中的3D 空间位置可以在一个狭窄的2D 区域内预测。(E) 在 C 中显示的薄切片中所有位置的剖面视图。这些位置可以分为同心环之间的分区域。考虑到圆柱体中的 high-number 随机分布的分子和切割非常薄的切片, 相邻两个环的每个子区域 (Si) 中的位置 (pi) 的空间密度将是旋转对称和均匀的。这些位置可以沿 Y 维度进一步投影到1D。如果沿 Y 维度的位置聚集在带有 j 列的直方图中。每列 (Aj) 中的总位置数等于, 可以在 (F) 中进行实验测量. <img alt="Equation 2" src="/files/ftp_upload/56475/56475eq2.jpg"> </img>(G L)类似于上述, 转换过程是提出的分子扩散在一个外围区域的管。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56475/56475fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56475/56475fig3.jpg"> 图 3. 3D. 通过速度显微镜绘制活原纤毛 SSTR3 的运输路线的空间位置.(A) 速度显微镜的图形表示法, 用于跟踪 GFP 标记的 SSTR3, 通过 NPHP4-mCherry 标记的。(B) 荧光图像的 NPHP4-mCherry (红色), AP-SSTR3-GFP (绿色), Alexa647 用于外部标签 AP-SSTR3-GFP (白色), 和最终合并图像。缩放栏: 2 µm. (C) 跟踪一个单一的 Alexa 647 标记的 AP-SSTR3-GFP (白色), 通过点照明领域的四帧 (2 ms/帧)。标尺: 2 µm. (D) 从 (C) 的单分子轨迹 (白色) 叠加在 NPHP4-mCherry (红色) 和 AP-SSTR3-GFP (绿色) 的荧光图像上。比例栏: 2 µm. (E) 白色虚线正方形突出了单点照明的区域, 以中心的位置。标尺: 2 µm. (F) 光子计数 vs 帧数图为一个高信噪比的单分子视频, 由除以背景荧光的单个分子的最大荧光来确定。(G) 光子计数 vs 帧数图为一个低信噪比的单分子视频。(H) 从 (E) 和 (D) 到每个帧的轨迹由2D 高斯拟合, 并叠加在准确的图形表示, 也本地化为2D 高斯拟合。2D 高斯拟合过程符合一个高斯函数的 X 和 Y 维度的强度剖面的一个领域的兴趣 (君) 包括单一分子的聚砜. (I) 2D 超分辨率空间分布的220个人 Alexa 氟647标签从单个主纤毛收集的 AP-SSTR3-GFP 位置。(J) 超分辨率单分子位置 (I) 叠加在 NPHP4-mCherry (红色) 和 AP-SSTR3-GFP (绿色) 的荧光图像上。比例栏: 2 µm. (K) 通过2D 到3D 变换算法, 得到了 AP-SSTR3-GFP 在原纤毛上沿 R 维数的空间概率密度分布。基于高斯函数拟合, Alexa 氟647标记的 AP-SSTR3-GFP 主要位于 131±3 nm 半径的纤毛膜上, 宽度在 24±1 nm 的半最大 (FWHM)。(L) 横断面视图的空间概率密度分布 (绿云) 的 Alexa 氟647标记 AP-SSTR3-GFP 的主要纤毛。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56475/56475fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56475/56475fig4.jpg"> 图 4. 3D. 在速度显微镜下绘制的活原纤毛内 IFT20 的运输路线的空间位置.(A-C)Arl13b-mCherry (a) 和 IFT20-GFP (B) co-expressed 在 NIH3T3 细胞和合并 (C) 中的代表图像。圆圈 (青色) 表示在原纤毛上的速度显微镜单点照明的位置生长在细胞的一侧 (白色虚线), 其边缘由 IFT20-GFP 荧光在细胞体和明亮的场光照 (不显示)。标尺:10 µm. (D) 从单一初级纤毛收集的286个 IFT20-GFP 地点的超分辨率空间分布。(E) 通过2D 到3D 变换算法, 得到了 IFT20-GFP 在主纤毛内沿 R 维数的空间概率密度分布。基于高斯函数拟合, IFT20-GFP 主要定位在 95±1 nm 半径为 56±5 nm 半最大 (FWHM) 的全宽度。(F) IFT20-GFP 在原纤毛中的空间概率密度分布 (绿云) 的剖面图, 在 h。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56475/56475fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56475/56475fig5.jpg"> 图 5.模拟用于估计2D 分子位置的最小数目, 以生成2D 到3D 变换算法的可靠3D 空间概率密度映射.(a) 100 计算产生的分子地点随机抽样从径向密度正态分布的中心分布在 95 nm (对应于我们的实验确定的 IFT20 的主要运输路线)。16 nm 的定位精度是模拟每个分子的位置通过抽样从一个正态分布的σ = 16 nm。如图 2C所示, x 维度中的非常薄切片 (x) 用于转换算法, 因此在计算生成的2D 分子位置中不显示 x 维度。(B) Y 维项目数据和 3D r 维密度之间的转换为五不同的模拟数据集生成了 3D r 维密度直方图 (每一个都有100点)。上面的数字是峰值位置±拟合错误。(C D)基于250分子位置的仿真结果。(E-F)基于500分子位置的仿真结果。(G I)平均3D 密度直方图分别从 100, 250 和500点模拟。误差线表示直方图 bin 高度的可变性, 而峰值上方的数字是平均峰值位置±标准偏差。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56475/56475fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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 San Sebastian

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

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

科研

发育生物学

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

脉络丛上皮细胞的纤毛运动的观察<em&gt;离体</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

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 ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

生物学

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 ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

通过免疫荧光简单检测原发性西莉亚

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

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.

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

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

秀丽隐杆线虫种系内突触复合体的超分辨率显微镜

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...