我们感谢德国研究基金会 (DFG) 提供财政支持 (RO2414/5-1 给 KR)。

<ol>
	<li>Day, R. N., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+protein+palette:+tools+for+cellular+imaging.">The fluorescent protein palette: tools for cellular imaging.</a> Chem Soc Rev. 38 (10), 2887-2921 (2009).</li><li>Ishikawa-Ankerhold, H. C., Ankerhold, R., Drummen, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+fluorescence+microscopy+techniques--FRAP,+FLIP,+FLAP,+FRET+and+FLIM.">Advanced fluorescence microscopy techniques--FRAP, FLIP, FLAP, FRET and FLIM.</a> Molecules. 17 (4), 4047-4132 (2012).</li><li>Koestler, S. A., 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=Arp2/3+complex+is+essential+for+actin+network+treadmilling+as+well+as+for+targeting+of+capping+protein+and+cofilin.">Arp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin.</a> Mol Biol Cell. 24 (18), 2861-2875 (2013).</li><li>Steffen, A., 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=Rac+function+is+crucial+for+cell+migration+but+is+not+required+for+spreading+and+focal+adhesion+formation.">Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation.</a> J Cell Sci. 126, 4572-4588 (2013).</li><li>Lai, F. P., 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=Arp2/3+complex+interactions+and+actin+network+turnover+in+lamellipodia.">Arp2/3 complex interactions and actin network turnover in lamellipodia.</a> EMBO J. 27 (7), 982-992 (2008).</li><li>Dimchev, G., 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=Efficiency+of+lamellipodia+protrusion+is+determined+by+the+extent+of+cytosolic+actin+assembly.">Efficiency of lamellipodia protrusion is determined by the extent of cytosolic actin assembly.</a> Mol Biol Cell. 28 (10), 1311-1325 (2017).</li><li>Koppel, D. E., Axelrod, D., Schlessinger, J., Elson, E. L., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+fluorescence+marker+concentration+as+a+probe+of+mobility.">Dynamics of fluorescence marker concentration as a probe of mobility.</a> Biophys J. 16 (11), 1315-1329 (1976).</li><li>Patterson, G. H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photoactivatable+GFP+for+selective+photolabeling+of+proteins+and+cells.">A photoactivatable GFP for selective photolabeling of proteins and cells.</a> Science. 297 (5588), 1873-1877 (2002).</li><li>McKinney, S. A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+and+photostable+photoconvertible+fluorescent+protein.">A bright and photostable photoconvertible fluorescent protein.</a> Nat Methods. 6 (2), 131-133 (2009).</li><li>Gurskaya, N. G., 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=Engineering+of+a+monomeric+green-to-red+photoactivatable+fluorescent+protein+induced+by+blue+light.">Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light.</a> Nat Biotechnol. 24 (4), 461-465 (2006).</li><li>Lippincott-Schwartz, J., Patterson, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+fluorescent+proteins+for+diffraction-limited+and+super-resolution+imaging.">Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging.</a> Trends Cell Biol. 19 (11), 555-565 (2009).</li><li>Kremers, G. J., Piston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoconversion+of+purified+fluorescent+proteins+and+dual-probe+optical+highlighting+in+live+cells.">Photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells.</a> J Vis Exp. (40), (2010).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+slides+and+coverslips+for+microscopy.">Preparation of slides and coverslips for microscopy.</a> CSH Protoc. 2008, 4988 (2008).</li><li>Small, J. V., Rottner, K., Carlier, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Actin-based Motility. , (2010).</li><li>Kaverina, I., 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=Enforced+polarisation+and+locomotion+of+fibroblasts+lacking+microtubules.">Enforced polarisation and locomotion of fibroblasts lacking microtubules.</a> Curr Biol. 10 (12), 739-742 (2000).</li><li>Small, J., Rottner, K., Hahne, P., Anderson, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualising+the+actin+cytoskeleton.">Visualising the actin cytoskeleton.</a> Microsc Res Tech. 47 (1), 3-17 (1999).</li><li>Mikhailov, A. V., Gundersen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centripetal+transport+of+microtubules+in+motile+cells.">Centripetal transport of microtubules in motile cells.</a> Cell Motil Cytoskeleton. 32 (3), 173-186 (1995).</li><li>Rottner, K., Behrendt, B., Small, J. V., Wehland, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VASP+dynamics+during+lamellipodia+protrusion.">VASP dynamics during lamellipodia protrusion.</a> Nat Cell Biol. 1 (5), 321-322 (1999).</li><li>Svitkina, T. 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=Mechanism+of+filopodia+initiation+by+reorganization+of+a+dendritic+network.">Mechanism of filopodia initiation by reorganization of a dendritic network.</a> J Cell Biol. 160 (3), 409-421 (2003).</li><li>Small, J. V., Isenberg, G., Celis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+of+actin+at+the+leading+edge+of+cultured+cells.">Polarity of actin at the leading edge of cultured cells.</a> Nature. 272 (5654), 638-639 (1978).</li><li>Dang, I., 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=Inhibitory+signalling+to+the+Arp2/3+complex+steers+cell+migration.">Inhibitory signalling to the Arp2/3 complex steers cell migration.</a> Nature. 503 (7475), 281-284 (2013).</li><li>Anderson, K. I., Cross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+dynamics+during+keratocyte+motility.">Contact dynamics during keratocyte motility.</a> Curr Biol. 10 (5), 253-260 (2000).</li><li>Burnette, D. 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=A+role+for+actin+arcs+in+the+leading-edge+advance+of+migrating+cells.">A role for actin arcs in the leading-edge advance of migrating cells.</a> Nat Cell Biol. 13 (4), 371-381 (2011).</li><li>Humphries, A. C., 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=Clathrin+potentiates+vaccinia-induced+actin+polymerization+to+facilitate+viral+spread.">Clathrin potentiates vaccinia-induced actin polymerization to facilitate viral spread.</a> Cell Host Microbe. 12 (3), 346-359 (2012).</li><li>Riedl, 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=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5 (7), 605-607 (2008).</li><li>Kage, 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=FMNL+formins+boost+lamellipodial+force+generation.">FMNL formins boost lamellipodial force generation.</a> Nat Commun. 8, 14832 (2017).</li><li>Peckham, M., Miller, G., Wells, C., Zicha, D., Dunn, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+changes+to+the+mechanism+of+cell+locomotion+induced+by+overexpression+of+beta-actin.">Specific changes to the mechanism of cell locomotion induced by overexpression of beta-actin.</a> J Cell Sci. 114, 1367-1377 (2001).</li><li>Salmon, E. D., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+we+discovered+fluorescent+speckle+microscopy.">How we discovered fluorescent speckle microscopy.</a> Mol Biol Cell. 22 (21), 3940-3942 (2011).</li><li>Machan, R., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+applications+of+fluorescence+correlation+spectroscopy+in+live+systems.">Recent applications of fluorescence correlation spectroscopy in live systems.</a> FEBS Lett. 588 (19), 3571-3584 (2014).</li><li>Becker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging--techniques+and+applications.">Fluorescence lifetime imaging--techniques and applications.</a> J Microsc. 247 (2), 119-136 (2012).</li></ol>

作者没有什么可透露的。

在这里, 我们讨论了本文所描述的技术中的关键步骤, 以及如何在不同的实验条件下优化它们的应用。
显微注射是一种方法, 可用于监测细胞的即时效应, 从引进外源蛋白, 抑制剂, 或药物。它可以特别有利于确定蛋白质的功能在困难的染细胞类型或在情况下, 长期表达不需要。必须指出的是, 某些细胞类型的存活取决于它们所播种的细胞外基质。大多数内皮, 上皮, 或成纤维细胞类, 甚至小的像鱼基质 (见当et 等人21和安德森和交叉22) 可以成功地注入。然而, 也有一些例外, 例如在层粘连蛋白上播种的 B16-F1 细胞, 它构成了一个优良的细胞迁移模型系统, 但与这种类型的基质注射不相容, 原因不明。对于 NIH3T3 成纤维细胞, 我们通常在纤维粘连蛋白基底上进行注射, 以及其他 photomanipulation 技术, 如酶 (即使有 photoactivation; 在此显示的 B16-F1 细胞) 可以同样良好地执行在这些成纤维细胞 (见例如,Köstler et 。3). 还必须考虑到, 不同的蛋白质, 根据其功能特性和实验目标, 可能需要不同的时间来引起变化, 从几秒钟到几小时不等。该技术的优点是, 在使用质粒转染时, 在单细胞水平比例如更精确地控制外源剂的用量/浓度。此外, 荧光标记的蛋白质是不必要的, 以保证其存在的细胞, 这可以增加灵活性, 如果同时多通道可视化其他荧光标签的蛋白质是必需的。显微注射对于分析特定蛋白质或蛋白质混合物对细胞形态或细胞骨架的动态变化的即时影响特别有用 (例如,. 21 Arp2/3 复合抑制剂 Arpin 对迁移的即时影响示例)。技术的缺点是它的侵袭性, 可能导致细胞损伤或影响细胞形态学。因此, 在执行 microinjections 时, 一个重要的考虑因素是监测细胞的生存能力。此处介绍的方法依赖于手动操作。在测试的条件, 以配合成功的注射, 如成纤维细胞生长在纤维连接层, 这里所描述的手动注射协议允许近100% 成功率;这是必要的, 当结合这种方法与复杂和耗时的后续实验, 包括视频显微镜或酶, 如以前发布的3。这并不排除偶尔, 个别细胞可能会遭受微注射事件, 这可以被安全地识别的突然变化, 细胞核和细胞质的对比, 其次是细胞边缘退缩。这样罕见的实验性案例被排除并且因而不考虑为进一步分析。
然而, 半自动方法也通常被使用, 例如使用快速 (< 300 ms) 机器控制的针降低与射入压力增量重合, 因此针只必须在每个细胞之上被安置在各自之前注射。半自动注射的成功率低于上面描述的人工方法, 这仅仅是因为它对速度进行了优化, 接着分析了成功地在这种治疗中幸存下来的多个细胞;因而它不依靠成功的射入单独细胞。因此, 相对于单细胞分析, 半自动注射更适合分析数个百细胞的注射效果,例如,通过视频显微镜在低放大率或在细胞固定和染色。无论采用何种详细的方法, 显微注射都不构成终点化验, 但可以结合多种技术, 包括酶或 photoactivation3。
当通过酶确定蛋白质周转率时, 必须优化激光强度, 具体取决于显微镜的设置和成像条件 (放大、目标、等, 以及细胞类型、结构和荧光蛋白等。漂白)。请注意, 在最佳激光功率下, 高效漂白与最小可能的光损伤相结合, 以避免在分析 (例如, lamellipodia 或丝状伪足) 中的结构收缩或完全收缩, 甚至在细胞层面上造成损害。理想的情况是, 至少70–80% 的漂白效率应该达到, 虽然完全漂白可能会受到极快的蛋白质周转的阻碍, 在这种情况下, 任何50% 以上的东西也可以接受。对给定结构和荧光染料的最佳漂白功率应进行实验测试, 从低激光功率开始, 然后逐渐增加。当然, 任何荧光染料可以根据定义被漂白与激光接近其峰值的励磁 (488 毫微米为常用的绿色染料, 如 FITC 或 EGFP)。然而, 波长较短的激光, 如近紫外激光器, 提供了更高的功率, 因此也可用于有效漂白常用染料。我们通常使用405纳米二极管激光器 (120 兆瓦) 漂白的 EGFP 和红色荧光染料 (如 mCherry), 虽然其效率略低的情况下 (不显示数据)。由于405纳米二极管也可以用于 photoactivation (见下文), 它赋予这个系统最大的灵活性。
对于 B16-F1 细胞结构和荧光蛋白 photobleached, 在65–100兆瓦之间应用了405纳米激光功率。在分析 photobleached 区域时, 重要的是要考虑给定的结构是否保留在其原始形状上。例如, 在分析 lamellipodia 尖端蛋白质的周转时, 应注意 lamellipodia 的曲率是否随着时间的推移而显著改变, 因为如果分析的区域/轮廓不完全包含整个结构在每个被测量的框架。此外, 应该指出, 嵌入到 lamellipodia 中的捆绑, 如 microspikes, 可能会导致荧光强度的偏差。如图 2b (在9的时间帧中为白色箭头) 所示, 一个类似 microspike 的结构位于测量的 photobleached 区域的旁边, 但在整个测量期间仍处于外部, 因此不会导致任何误差。对于蛋白质周转的分析, 在选择分析区域的位置和大小时, 重要的考虑因素是它们的荧光随时间变化不应受到细胞形态学或其它因素的改变的显著影响, 而不是很难避免购置漂白。例如, 对所分析结构提供大量定量贡献的结构, 在分析过程中不应脱离实测区域;此外, 不相关的, 荧光实体, 如水泡结构, 吸引蛋白质不应该进入兴趣领域的分析。为确定 lamellipodial 肌动蛋白聚合的速率, 应注意不要对缩回或梳理 (即,向上折叠) lamellipodia 进行分析, 因为这将强烈影响结果的准确性。此外, lamellipodial 地区的退缩可能会出现快速向后移位, 有可能导致高估 lamellipodial 肌动蛋白聚合速率。另外考虑的是细胞内正常化区域的距离 (作为获取漂白校正的参考位置) 从漂白的实际位置, 应该足够大, 以避免直接photobleached 地区的影响。
在建立 photoactivation 绿色荧光标记结构的最佳条件时, 应注意避免 photoactivation 中的瞬时漂白。在我们的工作中, 获得的最佳结果是激光功率低于通常使用的 EGFP 漂白的5-10 倍。对于光活化作用分子的图像获取, 应通过考虑要光活化作用和分析的区域和结构的大小以及光活化作用的潜在移动性来优化帧间的曝光时间和时间间隔。蛋白质到其他亚细胞位置。对于所有类型的荧光成像, 维持细胞的生存能力是至关重要的获得生理相关的结果。
原则上, 荧光蛋白 (如 mEos 或 Dronpa 变体) 的绿-红 photoconversion 是一种同样强大的方法, 跟随 lamellipodium 的亚细胞结构的动态和更替 (见 例如,Burnette et 。23). 后一种方法相对于 PA-GFP 的优势是在转换前后采用两种不同颜色的蛋白质动力学的可能性, 而不需要共同表达额外的红色荧光蛋白。然而, 在我们的初步实验中, 与 photoconverted 探针相比, 在 photoactivation 上实现的荧光信号的对比度变化和强度更大, 可能是由于绿色与红色的优越光谱特征。荧光探针 (未显示数据)。在任何情况下, 对肌动蛋白长丝周转的详细研究, 如 lamellipodia 或痘苗病毒诱导的肌动蛋白的尾巴迄今只发表了使用 PA-GFP 衍生物5,6,24。
在考虑哪一个细胞区域来分析以下 photoactivation 时, 应该考虑几个因素, 这是用这里所示的具体例子来讨论的 (在细胞质激活后, 在细胞边缘加入肌动蛋白单体), 但当然可以推断出各种类似的科学问题。首先, 当测量 cytosolically 光活化作用蛋白的 lamellipodial 合并率时, 例如, 在不同的实验条件下 (如Dimchev et 所示)。6), 胞浆区域的大小及其与 lamellipodial 边缘的距离应在实验组之间比较。同样重要的是, 当 photoactivating 胞浆区域时, 细胞厚度在靠近细胞核的位置更大。激活较厚的细胞区域可能导致更多的活化蛋白, 因为在细胞质中 homogenously 分布的蛋白质的分布。最后, 被激活的蛋白质的表达水平在个体细胞肯定是高度可变的。由于所有这些可变性的考虑, 重要的是要比较在细胞中其他地方的 cytosolically 活化蛋白相对于在特定区域激活后获得的总荧光的合并水平。
我们已经描述了如何使用显微注射作为一个工具, 以调查蛋白质对细胞形态学的影响, 并表明了这一点, 证明了有效诱导 lamellipodial 结构在 NIH3T3 成纤维细胞杏仁与小 GTPase Rac1。我们以前应用这一技术来干扰细胞中的 Arp2/3 功能杏仁与 C 终端 WCA 领域的疤痕/波3。杏仁细胞中的各种参数可以通过其他的化验方法来分析, 如酶或 photoactivation。我们描述了酶和 photoactivation 如何用于研究肌动蛋白单体的亚细胞动力学和流动性。我们的小组以前使用过酶5来调查定位到 lamellipodia 的蛋白质的更替情况, 如虚拟、Abi、cortactin、cofilin 和上限蛋白, 或用于阐明在存在中的病灶粘连中成分的更替情况。和没有 Rac 信号4。此外, 测量肌动蛋白聚合速率可以通过漂白 EGFP 标记的β肌动蛋白5来完成, 但存在替代方法。跟踪荧光不均匀性的实时细胞成像-兼容探针标记细胞肌动蛋白花丝,如Lifeact25, 也可以使用6, 26.这里的优点是可以避免β肌动蛋白的过度表达, 它能够增加细胞边缘突起和迁移, 从而可能干扰特定的化验或实验问题 (参见例如,凯奇et 。26;Peckham et 。27). 然而, Lifeact 探针的一个明显的缺点是它的快速的开/关动力学与肌动蛋白花丝结合, 因此, 在细胞中标记的肌动蛋白长丝结构的漂白只提供探针更替的信息,但不是肌动蛋白花丝的营业额, 它绑定25。以前使用的荧光不均匀性的跟踪6, 26 确实提供了一个实际的折衷, 类似于广泛使用的跟踪荧光斑点纳入丝状骨架结构 (请参见例如鲑鱼和酶28), 但可能不像 EGFP 标记的 F 肌动蛋白结构那样直接使用和精确。Photoactivation 已经被我们应用于估计单体肌动蛋白在凸 lamellipodia 中的比例, 以及它在细胞质的移动, 在实验性地调整胞浆 F 肌动蛋白水平6。该技术在检查从相对较大的区域 (如胞浆区域) 衍生的蛋白质的流动性和分布方面是有用的。然而, 研究从相对较小的光活化作用结构中提取的蛋白质的分布;例如,由于激活的荧光分子数量少, 信号微弱, 因而缺乏灵敏度, 因此生长锥可能具有挑战性。荧光 photoactivation 或 photoconversion 的潜在替代技术 (见上文) 可能包括逆酶, 它依赖于漂白整个细胞, 除了 ROI, 然后跟踪荧光分子的流动性远离这个地区。该技术不需要 overexpressing photoactivatable 版本的蛋白质, 但将始终涉及暴露在异常高剂量的激光功率, 可能导致不必要的副作用, 如光损伤。
显然, photoactivation 和酶不能区分蛋白质是否作为单体、脂肪酸、甚至小的低聚物移动, 以及它们是否与附加的结合性伙伴相结合。这类信息可以通过荧光相关光谱学技术 (29 ) 来获得, 或者是电影-烦恼30。尽管如此, 酶和 photoactivation 是直接评估细胞内局部和全球蛋白质动态的简单方法, 而不管感兴趣的蛋白质、亚细胞位置或研究的类型。

在细胞和分子生物学的许多领域中, 监测蛋白质和其他分子的时空动力学已经成为一个必不可少的工具。先进的荧光显微技术包括荧光共振能量转移 (烦恼) 和焦虑荧光寿命成像 (烦恼-电影), 或酶, 荧光损失漂白 (翻转) 和 photoactivation 以及许多其他允许对于蛋白质-蛋白质相互作用的时空跟踪, 构象的变化, 以及在细胞1,2中确定不同蛋白质的扩散和定位的动力学。酶和 photoactivation 技术, 特别是, 广泛适用于检查调节肌动蛋白细胞骨架和细胞迁移。这些技术可以单独应用或与附加的微操作系统技术 (如显微注射 3) 结合使用, 并涉及荧光标记蛋白的表达.它们允许将蛋白质联想的动力学估计为参与细胞迁移的富含肌动蛋白的结构, 如丝状伪足或 lamellipodia、病灶粘连中蛋白质的更替4或分支肌动蛋白网络 5.它们还能确定 lamellipodial 肌动蛋白聚合速率, 单体肌动蛋白在细胞质内的分散度, 亚细胞肌动蛋白单体易位率在突出lamellipodia6和其他参数。
酶是一种可视化和量化在活细胞内蛋白质流动性的方法, 最初在二十世纪七十年代由阿克塞尔罗德7开发。一个在细胞内的感兴趣区域 (ROI), 用荧光标记的蛋白质填充, 瞬时暴露在高强度的激光中, 足以导致在给定的短时间内对该区域中的荧光分子进行漂白。在漂白过程中, 未漂白的、荧光标记的蛋白质, 会根据它们的时空动力学, 扩散和渗透到漂白的区域, 从而导致 photobleached 分子随着时间的推移而迁移。漂白地区荧光回收率的大小取决于各种因素, 包括给定分子的扩散率和传播速率, 以及在假定相关的漂白结构中的更替率。因此, 可溶性蛋白质将通过扩散迅速地在漂白的 ROI 中调节荧光的恢复, 而与结构紧密相关的蛋白质, 如焦粘连, 将有更长的周转时间, 因为它们的荧光恢复将依赖于蛋白质可溶性分数的扩散和结构相关分数的离解-联想动力学。荧光恢复通常是获得和量化, 直到初始水平的前漂白强度的荧光达到。但是, 如果初始荧光强度的一部分属于所谓的不动分数, 则无法通过扩散补充或以非常慢的速率补充, 而与包括移动的大多数分子相比, 这种情况不会发生。分数。为了确定蛋白质更替率, 酶曲线的产生, 代表了荧光恢复的程度随着时间的推移。从这些恢复曲线可以计算蛋白质回收率的平均半倍。通过创建平均酶数据的曲线拟合, 进而进行数学分析, 也可以推断流动分数的平均周转率是否构成一个分子的均匀种群的组合, 或者它是否由两个或更多的分子亚群在不同的速率翻转。除了通过定量方法估计蛋白质周转率外, 跟踪 lamellipodia 中 photobleached 区域的恢复也可以精确量化 lamellipodial 运动参数, 如逆行流、突起、和肌动蛋白聚合速率。因此, 酶构成了一种多功能工具, 用于评估活细胞结构中的各种参数。
Photoactivation 是一种用于跟踪来自指定细胞位置的蛋白质或分子的扩散和流动性的方法。例如, 该技术采用了一种野生型绿色荧光蛋白 (GFP) 的变体, 最初由帕特森和利平科特8开发, 这种变异方式使其荧光在接触到紫外线 (紫外线) 光 (大约400毫微米; 这里, 405 毫微米)。正如帕特森et al所描述的, 野生型 GFP 染色体作为中性酚类和阴离子 phenolates 的混合种群存在, 其产生的吸光度峰值约为 397 nm, 较小的是 475 nm。在紫外光照射下, 种群经历 photoconversion, 向阴离子形态转移。当兴奋 488 nm, photoconverted/光活化作用蛋白表现出3倍的荧光增加, 在实践中缺乏区分激活和非活化 GFP 由于高内在背景荧光。然而, 通过在 GFP 序列中引入单一氨基酸突变 (在位置 203), 降低了背景强度。由此产生的 T203H 突变体, 也称为 photoactivatable-gfp (PA-gfp) 的特点是显著降低了小峰的吸光度, 这在辐照与紫外线照射后增加近100倍, 随后激发 488 nm 光。因此, 过度表达的 PA-GFP 标记蛋白是一种广泛使用的方法, 它允许测定分子在细胞内的扩散和运动。我们以前应用了 PA-GFP 标记肌动蛋白, 以确定肌动蛋白单体分散的速率从胞浆区域, 不仅允许探索他们的流动性在细胞质, 而且他们的合并率到突出lamellipodial 肌动蛋白网络6。最近的文献还介绍了新颖的, 可转换的蛋白质, 原则上可以使用类似的方式, 但窝藏潜在的优势已经可见, 在照片转换之前。这组荧光蛋白的例子包括 Dendra2 和 mEos29,10,11,12。
在本文中, 我们解释了 microinjecting 细胞与蛋白质的方法。我们进一步解释了这项技术如何与酶结合, 通过漂白蛋白参与肌动蛋白细胞骨架调节和运动, 以及如何酶曲线和半时间恢复的移动分数可以获得。此外, 我们提供了一个例子, 酶技术如何可以用来确定肌动蛋白聚合速率的 lamellipodial 网络。我们还提供有关如何执行 photoactivation 实验的指示和提示, 可用于确定单体肌动蛋白的胞浆流动性和肌动蛋白在 lamellipodia 中的比例。当然, 这些技术不仅限于跟踪肌动蛋白骨架成分, 而且在可能需要适度的适应或优化的情况下, 可以广泛应用于其他细胞类型或研究不同的蛋白质、结构和参数。

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			<td height="21" style="height:21px;width:365px;">B16-F1 mouse skin melanoma cells&#160;</td>
			<td style="width:575px;">American Type Culture Collection, Manassas, VA</td>
			<td style="width:167px;">CRL-6323</td>
			<td style="width:511px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:365px;">NIH-3T3 cells</td>
			<td>American Type Culture Collection, Manassas, VA</td>
			<td>CRL-1658</td>
			<td></td>
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			<td height="21" style="height:21px;">DMEM 4.5g/L glucose&#160;</td>
			<td>Life Technologies, Thermno Fisher Scientific, Germany</td>
			<td style="width:167px;">41965-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ham&#8217;s F-12 medium</td>
			<td>Sigma-Aldrich</td>
			<td style="width:167px;">N8641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum&#160; (FCS)</td>
			<td>PAA Laboratories, Linz, Austria</td>
			<td style="width:167px;">A15-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>F7524</td>
			<td>Lot054M3396</td>
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		<tr height="21">
			<td height="21" style="height:21px;">MEM Non essential amino acids&#160;</td>
			<td>Gibco, ThermoFisher Scientific, Germany</td>
			<td style="width:167px;">11140035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glumatine 200mM (100x)</td>
			<td>Life Technolgies</td>
			<td style="width:167px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen-Strep 5000 U/mL</td>
			<td>Life technologies</td>
			<td style="width:167px;">15070063</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate (100 mM)</td>
			<td>Gibco, ThermoFisher Scientific, Germany</td>
			<td style="width:167px;">11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L-2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin coating buffer&#160;</td>
			<td style="width:575px;"></td>
			<td style="width:167px;"></td>
			<td>Self-made: 50mM Tris ph7.4, 150mM NaCl</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fibronectin from human plasma&#160;</td>
			<td>Roche Diagnostics, Mannheim, Germany</td>
			<td style="width:167px;">11 051 407 001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Jetpei</td>
			<td>Polyplus Transfection, Illkirch, France</td>
			<td style="width:167px;">101-10N</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:365px;">JetPei buffer</td>
			<td>Polyplus Transfection, Illkirch, France</td>
			<td style="width:167px;">702-50</td>
			<td>150mM NaCl</td>
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		<tr height="21">
			<td height="21" style="height:21px;">PA-GFP-actin plasmid DNA&#160;</td>
			<td style="width:575px;"></td>
			<td style="width:167px;"></td>
			<td>described in Koestler et al.2008</td>
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		<tr height="21">
			<td height="21" style="height:21px;">pEGFP-actin plasmid DNA</td>
			<td>Clontech, Mountain View, CA, USA</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Rac1 protein for microinjection</td>
			<td></td>
			<td style="width:167px;"></td>
			<td>Purified as GST-tagged version, and cleaved from GST prior to injection</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:365px;">Microinjection buffer</td>
			<td></td>
			<td style="width:167px;"></td>
			<td>Self-made: 100mM NaCl, 50mM Tris-HCl ph7.5, 5mM MgCl2, 1mM DTT</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:365px;">Dextran, Texas Red, 70,000 MW, Lysine Fixable</td>
			<td>Molecular Probes, Thermno Fisher Scientific, Germany</td>
			<td style="width:167px;">D1864</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope circular cover glasses 15mm, No.1&#160;</td>
			<td>Karl Hecht, Aisstent, Sondheim, Germany</td>
			<td style="width:167px;">1001/15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf Femtotips Microloader Tips</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td style="width:167px;">5242 956 003</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:365px;">Eppendorf Femtotip Microinjection Capillary Tips</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td style="width:167px;">930000035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone Grease&#160;</td>
			<td>ACC Silicones, Bridgewater, England</td>
			<td style="width:167px;">SGM494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminium Open Diamond Bath Imaging Chamber</td>
			<td>Warner instruments</td>
			<td style="width:167px;">RC-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automatic temperature controller</td>
			<td>Warner Instruments</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Microscope: Axio Observer&#160;</td>
			<td>Carl Zeiss, Jena, Germany</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolSnap-HQ2 camera&#160;</td>
			<td>Photometrics, Tucson, AZ</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lambda DG4 light source&#160;</td>
			<td>Sutter Instrucment, Novato, CA</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser source&#160;</td>
			<td>Visitron Systems</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Eppendorf FemtoJet microinjector&#160;</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td style="width:167px;"></td>
			<td>With built-in compressor for pressure supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Nikon Narishige Micromanipulator system</td>
			<td style="width:575px;">Nikon Instruments, Japan</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Visiview software v2.1.4</td>
			<td>Visitron Systems, Puchheim, Germany</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metamorph software v7.8.10</td>
			<td>Molecular Devices, Sunnyvale, CA</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Sigma Plot v.12</td>
			<td style="width:575px;">Systat Software Inc.</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

micromanipulation techniques allowing analysis of morphogenetic dynamics and turnover of cytoskeletal regulators

研究蛋白质的时空动力学可以揭示它们在不同语境中的功能重要性。本文讨论了漂白 (酶) 和 photoactivation 技术后荧光恢复方法在亚细胞位置蛋白质时空动力学研究中的应用。我们还展示了这些技术如何能够直接确定与肌动蛋白骨架调节和细胞运动有关的各种参数。此外, 细胞的显微注射还被描述为一种替代疗法 (可能之前或补充上述 photomanipulation 技术), 以触发移位蛋白对细胞的瞬时影响形态和功能。微操作系统, 如蛋白质注射或局部应用血浆膜通透性药物或骨架抑制剂, 可以作为一个强有力的工具, 以记录特定治疗的直接后果的细胞行为在单细胞和亚细胞水平。这是在这里的例子, 立即诱导 lamellipodial 细胞边缘突出的注射重组 Rac1 蛋白, 建立了一个季度前。此外, 我们提供了一个协议, 以确定增强的绿色荧光蛋白 (EGFP)-虚拟的营业额, 肌动蛋白长丝聚合酶显著积累在 lamellipodial 提示 B16-F1 细胞, 使用酶和包括相关数据分析和曲线拟合。我们还提出了评估 lamellipodial 肌动蛋白网络聚合速率的指导原则, 如表达 EGFP 标记β肌动蛋白的细胞所示。最后, 给出了如何调查细胞胞浆内肌动蛋白单体流动率的指示, 其次是肌动蛋白在快速灯丝组装部位的 lamellipodia, 如突出的小贴士, 使用 photoactivation方法。这些协议都不限于肌动蛋白细胞骨架的成分或调节器, 但可以很容易地扩展到类似的方式探索蛋白质在不同亚细胞结构或功能中的时空动力学和功能上下文。

图 1g, h显示 NIH3T3 成纤维细胞的相对比图像, 前和10分钟后显微注射 Rac1, 这是一个小的细胞家族 GTPase 能够诱导 lamellipodia 形成通过与波复合体的相互作用。在微注射 (图 1g) 之前, 该单元格首先被可视化, 以确认其生存能力和形态学,例如,缺少 lamellipodia。在注射后10分钟后, 细胞已经明显改变了它的形态学, 这是预期从这种治疗, 并表示成功注射 (图 1h)。
为了简单和清晰起见, 我们接下来为细胞中的酶和 photoactivation 分析提供了模范的结果, 而这些研究还没有杏仁。
在图 2EGFP 中显示了在 lamellipodium 提示处标记的虚拟的营业额的分析.请注意, 虚拟另外目标到新生和焦点粘连, 小和拉长点在细胞内部18,19。lamellipodial 区域的荧光强度在尖端有明确的虚拟积累被漂白和测量每一个时间框架, 通过遵循 ROI 的轮廓之前, 期间, 漂白后, lamellipodium 突出向前。由于漂白的 EGFP-虚拟蛋白在这些部位被非漂白分子回收, 观察到荧光的逐渐恢复 (图 2b)。在图 2c中可以看到以这种方式获得的酶恢复曲线, 并将其规范化为前漂白强度 (表示为 1)。漂白效率可以变化, 约20% 的价值之前漂白在这个例子, 从价值确定从 t0 (第一帧后漂白)。荧光的增加到达一个高原在例子显示在大约80% 荧光在漂白之前。在静态结构中, 在实验的时间过程中, 如焦粘度, 恢复后达到的前漂白强度和高原荧光的差异定义为不动分数 (如果,图 2中的红色箭头)c、e), 而在漂白和完全恢复时间之间恢复的荧光量被定义为移动分数 (图 2c、e中的绿色双头箭头)。请注意, 在动态变化的结构, 如在这里分析的 lamellipodium 尖端, 其范围可能不仅代表不动分子, 但也从降低的凸出速度, 因为 EGFP 虚拟强度是众所周知的依赖于此参数18。要计算恢复的半时间, 在斯格码绘图 (图 2d) 上创建了一个拟合曲线。在这种情况下, 从解决等式 2中提取的 "b" 参数值等于 0.0754, 当应用到对数函数 (等式 4) 时, 将导致估计的恢复时间为9.19 秒 (图 2d, 远右面板), 在这个特定的单元格中相对较快, 这与以前发布的平均5相比。必须指出的是, 在同一人群中, 恢复半衰期有时会有很大差异。因此, 为了获得具有代表性的结果, 我们建议将此参数确定为至少15-20 个单元格的平均值。为了说明方差的程度, 生成了每个时间点平均从15个单元 EGFP 虚拟恢复的算术方法 (图 2e), 并以类似的方式创建和显示平均曲线匹配 (图 2f)。
lamellipodial 肌动蛋白网络的聚合速率包括正向网络凸出和逆行流的总和。酶可用于测量肌动蛋白聚合速率的转染细胞 (在这种情况下 B16-F1) 与 EGFP 标记β肌动蛋白和漂白一个突出的 lamellipodial 区域 (图2 g).为分析 lamellipodial 肌动蛋白网络聚合, EGFP 标记的β肌动蛋白漂白后的荧光恢复是随着时间的推移进行评估的。由于肌动蛋白单体的聚合进展在 lamellipodial 肌动蛋白花丝的有刺的末端 (所有指向前面20), 网络经常移位后方和进步批转, 速度可以容易地通过漂白上的荧光极化恢复获得。当漂白带到达 lamellipodium 后部与薄板的过渡区后, lamellipodium 的荧光恢复就完成了, 其特征是水平排列的纤维束密度较低。比 lamellipodium 中观察到的要慢得多。如图 2g所示, 荧光恢复可以被可视为水平线向边缘的直线, 向后流向薄板, 这允许测量凸出和逆行流的距离 (单独表示在图 2g的最右侧面板中, 分别为橙色和红色双头箭头。
我们还应用 photoactivation 在 B16-F1 细胞转染的 PA-GFP-肌动蛋白, 以跟踪肌动蛋白单体在细胞质的流动性和其在突出 lamellipodia 的比例。如图 3a、b所示, 一个胞浆区域通过暴露于 405 nm 激光光活化作用, 而在 gfp 通道上每1.5 秒获取图像, 以可视化 gfp 标记的光活化作用肌动蛋白的分布。光活化作用 GFP-肌动蛋白在图 3 b 中的胞浆区域中可见扩散.光活化作用胞浆区的荧光强度下降率表现为 t0 (photoactivation 后第一帧) 初始强度的百分比;图 3c). 光活化作用肌动蛋白也集成在 lamellipodia 的提示, 其中新的肌动蛋白单体添加到增长的刺状的两端拉长肌动蛋白花丝在突出。为了估计 lamellipodial 的速率, 我们测量了一个二维轮廓/区域约5µm 宽度和1µm 高度的荧光强度;该地区在 lamellipodium 的尖端不断被重新定位。肌动蛋白在 t0 (图 3d) 中代表了光活化作用胞浆区域的荧光强度百分比。随着肌动蛋白纤维的伸长, 新的肌动蛋白单体在 lamellipodial 前被纳入。这些肌动蛋白单体的一小部分是随机衍生的胞浆池, 其中单体被光活化作用。这导致 lamellipodia 在 photoactivation 后的第一二十年代, 荧光的快速增加。随着新的单体被添加到 lamellipodial 前面, 以前加入的肌动蛋白单体流动与花丝向薄板逆行流动。随着时间的推移, ROI 是完全充满了荧光单体和一个高原在荧光达到 (图 3d)。然后观察到, 当光活化作用单体在整个细胞中扩散后, 荧光的逐渐下降, 非光活化作用肌动蛋白单体越来越多地被重新添加到 lamellipodial 的前方。荧光的减少将发现一个新的高原, 当平衡在光活化作用和非光活化作用单体之间的整个细胞被到达 (没有显示的数据) 时, 将很快到达。
肌动蛋白单体在整个细胞质的流动性是通过测量在光活化作用区域远端位置相同大小的区域的荧光强度而得出的 (举例说明在图 3 a 上的颜色编码区域标记为R1-R5)。如图 3e所示, 这些区域中的荧光强度逐渐减少, cytosolically 光活化作用区域, 因为光活化作用肌动蛋白单体的分数变得越来越稀释非活化 (即,非荧光) 单体。此外, 荧光的峰值是在以后达到的: 测量的区域从光活化作用区域的距离越远, 肌动蛋白单体扩散到这些区域所需的时间就越长。通过对到达荧光高原的半时间进行量化, 可以得出一个代表肌动蛋白单体渗入每个区域的程度的代表性值。该区域越远, 光活化作用肌动蛋白扩散到它所需的时间越长, 因此要达到的荧光平台需要更多的时候, 最终导致更高的t 1/2 值(图 3 e).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57643/57643fig1.jpg"> 图 1: 成像室组件和显微注射过程.(a) 成像室组件。(b) 硅脂在塑料封口机的开口周围小心涂抹。(c) 盖玻片位于成像腔开口中心的单元侧。(d) 通过将塑料封口机放置在盖玻片的顶部并拧紧侧面夹具来建立安全密封。(e) 显微镜介质被 pipetted 到腔槽中。(f) 成像室位于显微镜阶段, 热检测器和电极连接到加热单元预设置为37°c, 并且允许细胞在显微镜开始前至少适应30分钟。在这个例子中, 显微镜阶段也配备了机器人执行 microinjections, 和注射针被浸入介质覆盖细胞层在成像室。(g) NIH3T3 成纤维细胞在显微注射前通过相衬显微术进行可视化。红十字在核周车厢表明未来微注射的地点, 对应于高细胞质区域由于接近大核。(h) 10 分钟后, 微注射与 Rac1, 细胞的反应由 lamellipodia 的显著形成周围的整个细胞周围 (由绿色箭头指示)。<a href="/files/ftp_upload/57643/57643fig1large.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/57643/57643fig2.jpg"> 图 2:酶允许确定蛋白质周转率或 lamellipodial 肌动蛋白聚合.(a) 代表在 lamellipodial 区域漂白前表示 EGFP 虚拟的 B16-F1 细胞的典型例子。不同颜色的轮廓/形状被标记, 以表明哪些区域被考虑为荧光强度测量随着时间的推移。注意标记有感叹号的红色轮廓, 它标签的胞浆区域位于包含多个囊泡和细胞表面褶边的区域内。在选择荧光参考区域时应避免这样的动态区域, 因为它们的特点是荧光的短期波动很强, 可能导致不准确的结果。(b) EGFP 虚拟在漂白前后表达细胞的 Lamellipodial 区域。在紫色标记的区域内漂白后的荧光信号恢复是随着时间的推移而可视化的。箭头表示 microspike 的尖端, 虚拟可能由于肌动蛋白花丝聚合的高密度而富集19。(c) 酶恢复曲线的一个示例, 它从量化 photobleached lamellipodium (紫色轮廓) 的荧光强度到 b. 右侧红色和绿色线条分别表示不动和移动分数。(d) c (左面板) 中的酶恢复曲线的适当性, 以及用于派生恢复半时间 (右面板) 的计算方法的示例。(e) 以15个细胞的荧光恢复曲线为平均值的酶恢复曲线的一个例子, 用 SEM 条表示样本种群内的变异性程度。(f) 从平均酶恢复曲线适合15个单元格 (左面板) 的曲线拟合, 以及用于推导恢复半时间 (右面板) 的计算方法示例。(g) 显示 EGFP 标记β肌动蛋白的 B16-F1 细胞在 lamellipodial 区域漂白之前和之后 lamellipodium 的时间推移板, 随后在 lamellipodium 随着时间的推移荧光恢复。在最右边的面板上, 为凸出和逆行距离测量的值 (分别为橙色和红色)。在图像面板下进行计算, 揭示了 lamellipodial 肌动蛋白网络的聚合速率与凸出和逆行距离的总和。<a href="/files/ftp_upload/57643/57643fig2large.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/57643/57643fig3.jpg"> 图 3:Photoactivation 在整个细胞内进行单体跟踪的 PA-GFP 肌动蛋白.(a) 一个代表 B16-F1 细胞的典型例子, 表达 pa-GFP 肌动蛋白, 在触发 photoactivation 之前, 在胞浆区域 (pa) 表示。不同颜色的轮廓标记, 以表明哪些区域被考虑为荧光强度测量随着时间的推移。(b) 说明 photoactivation 后的 PA-GFP 肌动蛋白的时间分布。注意在光活化作用、胞浆区 (红圈) 中荧光的逐渐减少, 因为光活化作用肌动蛋白扩散远离它。由于其扩散到前端和组装到网络, 光活化作用肌动蛋白单体逐渐增强, 在 lamellipodia (青色地区) 和整个细胞质 (不同的颜色编码区域) 的距离和时间依赖的方式。(c) 代表, 光活化作用胞浆区内荧光的时间下降 (b 中的红色轮廓)。(d) lamellipodial 区域荧光强度的时间变化 (b 中青色轮廓)。(e) 曲线代表了胞浆区域荧光强度的时间变化 (在 b 中的颜色编码), 因为定位于 photoactivation 区域的可变距离。注意到达荧光高原的一半时间 (在右侧的图例中表示) 随着给定区域与 photoactivation 区域的距离增加, 可能与肌动蛋白单体扩散所需的增加时间相关。各自区域。<a href="/files/ftp_upload/57643/57643fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators at JoVE.com

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

我们描述了微和 photomanipulation 技术, 如酶和 photoactivation 如何能够确定运动参数和蛋白质在迁移细胞内的时空动力学。实验读数包括亚细胞动力学和运动调节器的更替或底层肌动蛋白骨架。

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

micromanipulation-techniques-allowing-analysis-morphogenetic-dynamics

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.0K 次观看.  Technische Universität Braunschweig. 我们描述了微和 photomanipulation 技术, 如酶和 photoactivation 如何能够确定运动参数和蛋白质在迁移细胞内的时空动力学。实验读数包括亚细胞动力学和运动调节器的更替或底层肌动蛋白骨架。

视频: Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis和显微操作的微循环活体荧光成像

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

啮齿动物附睾精子磷酸分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic &#8216;Touch DNA&#8217; Evidence

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a &#8216;blind-swabbing&#8217; approach will co-sample cellular material from the different individuals, even if the individuals&#8217; cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim&#8217;s DNA may be found in significant excess thus masking any potential perpetrator&#8217;s DNA.
In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, &#8216;smart analysis&#8217; method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 &#181;l microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., &#8220;clumps&#8221;) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

Here we describe an optimized and efficient removal strategy for the collection of bio-particles present in &#8216;touch DNA&#8217; samples, together with an enhanced amplification protocol involving a one-step 5 &#181;l micro-volume lysis/STR amplification, to permit the recovery of short tandem repeat (STR) profiles of the bio-particle donor(s).

单人的生物粒子从法医"触摸DNA&#39;证据回收简体显微增强遗传分析

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

高分辨率成像和芽殖酵母个人星体微管动力学分析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

小鱼肾注射液循环系统中微粒的简易有效管理与可视化

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

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