Name: triggering cell stress and death using conventional uv laser confocal microscopy
Uploaded: 2017-02-03T12:00:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy at JoVE.com

This work was supported by the MND Research Institute of Australia and the Cure for MND Foundation [GIA 1638], the Snow Foundation, and Macquarie University [MQRDG 9201401462]. Imaging was carried out at the Microscopy Facility in the Department of Biomedical Sciences, Macquarie University. Other members of the MND research center are thanked for their contributions to the development of these methods and fish lines.

注:设计，实施和动物实验报告必须采取的现行准则15帐户。这些工作必须事先经当地动物福利机关批准（在本例中，麦考瑞大学的动物伦理委员会）。 1.准备斑马鱼进行安装和UV细胞消融<ol><li>生成斑马鱼（ 斑马鱼 ）表达荧光蛋白。 <ol><li>以表达在斑马鱼兴趣荧光蛋白质中，执行的质粒注射到斑马鱼蛋的单细胞阶段（如别处16中描述），或使用荧光的转基因株系。标记多种类型的细胞，通过杂交确定转基因系相关于感兴趣的问题创造化合物转基因斑马鱼线。放在晚上假底对配合坦克的每侧一雄一雌斑马鱼和光的发病删除分隔第二天早上（如其他地方17详述）。保持斑马鱼在28℃，并根据所建立的协议17,18处理它们。 </li><li>通过使劲穿过塑料滤茶含有胚胎水箱收集成功产卵后的胚胎。冲洗鸡蛋与系统中的水，并将其转移到卵水在培养皿中。 </li><li>检查它们在光学显微镜下，确定施肥。商店受精卵在培养皿中，并放置在孵化器在28°C 18。 </li></ol></li><li>可选:执行注射标记特定的细胞群。 注意:这是一种替代方法，允许表达和蛋白质的可视化，而无需提高稳定的转基因系。这种方法也是有利的，当目的蛋白质是有毒的，并禁止了稳定的反式的代基因线。 <ol><li>注入质粒构建到斑马鱼胚胎的单细胞阶段，如别处19，20，21中描述。 注意:此方法导致感兴趣的蛋白的镶嵌表达。感兴趣的蛋白质是从所选择的启动子驱动的由TOL2侧翼（ 例如，islet1 22 -3mnx1 23，24，满足25，或MPEG1 26）反向重复序列20。 </li></ol></li><li>鱼的年龄到所需的大小。 <ol><li>提高鱼3 - 5天受精后（DPF），并将其放置荧光化合物显微镜下。屏幕的动物进行适当的荧光表达，选择亮标记的鱼。分开适当的幼虫到另一个菜，鸡蛋水嵌入晚R ON（在28℃培养箱店）。 可选:胚胎可以放入在24小时后受精（HPF）0.2毫1-苯基-2- thioures（PTU）林格溶液来抑制色素沉着的形成。必须小心以PTU，因为它是有毒的，并且可以具有不良生理，遗传，或形态影响。 </li><li>为在早期发育阶段的研究（&lt;2旦），dechorionate胚胎使用尖锐的镊子手动。酶通过加入链霉蛋白酶（2毫克/毫升）的蛋水，并在28℃培养他们10分钟Dechorionate大量的胚胎。 </li><li>定期通过胚胎通过塑料巴斯德吸管缓解dechorionation。终止该进程时多数胚胎已经从他们的绒毛膜用鸡蛋水洗他们数次出现。 </li></ol></li><li>准备在琼脂糖斑马鱼嵌入解决方案。 <ol><li>加入4克/升MS222制备麻醉溶液（三卡因原液，pH 7.0）中滴加到含有蛋水的培养皿中。 50 mg / L的剂量是建议起点（ 图1A）。 </li><li>在鸡蛋水 - （1.5％0.8），并将其分装成1.5毫升微量离心管制备低熔点琼脂糖的库存。放置等分试样到预加热的热块（38 - 40℃），并让它平衡到设定的温度（〜30分钟; 图1B）。 </li><li>可选:对于长期成像（&gt; 4小时），准备35毫米的玻璃底培养皿内的小圆圈琼脂糖，并允许它来设置（ 补充图1）。 注:这个额外的步骤能有效地避免在较长的时间框架与斑马鱼全琼脂糖下降的任何移动。 <ol><li>要做到这一点，沿着玻璃底菜的内圆琼脂糖的地方〜300μL准备一个环形圈在中间一个小口，在其中放置鱼（步骤1.5.3; 补充图1） 。 </li></ol></li></ol></li><li>安装在琼脂糖显微镜的斑马鱼。 <ol><li>选择1 - 3预先筛选鱼消融和转移他们（用移液管）与麻醉液菜麻醉幼虫（步骤1.4.1; 图1C;约5分钟）。 注意:鱼时，他们表现出浅鳃盖运动和降低心脏率麻醉，不再显示触摸诱发逃避反应（TEER;未能游开后，轻轻一刷抚摸自己的尾巴）。保证适当的麻醉鱼的道德治疗，防止抽搐时转入琼脂糖或暴露在荧光灯。 </li><li>麻醉被确认后，用一个可调移液管（具有截止200μL的尖端设定为〜30微升）吸起来幼虫并让它沉到尖端的底部。通过释放液态与一滴转移幼虫进入预热琼脂糖（步骤1.4.2）幼虫放入琼脂糖（尽量减少蛋水进入琼脂糖的量; 图1D）。 </li><li>吸上来的鱼琼脂糖包围。很快免除入事先准备好的玻璃底35毫米的菜。 </li><li>使用解剖显微镜和标准油漆刷（长衬垫，大小1）至琼脂糖内的动物的位置上的一侧（头左侧），使主体和尾都是平（ 图1E）。如果有多个鱼工作，对准所有的鱼的菜，让他们使用的共聚焦显微镜以后容易找到。 注:快速进行定位和调整的这个程序（它可能需要一些练习，因为琼脂糖开始暴露在寒冷的温度后立即设置）。 </li><li>离开琼脂糖包埋鱼10 - 15分钟，直到琼脂糖设置牢固。小心补足35毫米的培养皿与含有三卡因〜2蛋毫升水（ 图1F）。 </li></ol></li></ol> 2.设置共聚焦显微镜和成像参数<ol><li>放置在培养皿上共聚焦显微镜阶段嵌入式幼虫并专注于动物脊髓背侧（使用亮场）。检查适当的倍率（40X）和荧光设置下的动物和可视化的感兴趣结构（ 例如，标记的神经元或胶质细胞运动的荧光强度），以确认所有的成像参数是根据需要为后续消融（ 图2）。我们经常使用的40倍物镜来执行我们的时间间隔学习。 </li><li>可选:要进行数小时执行时间推移研究中，最好是记录单个或几个时间点之前消融建立细胞及其环境的未受扰动的生理反应（ 例如，小胶质细胞运动以建立基线速度和运动）。 </li><li>确定结构的厚度为紫外线激光烧蚀兴趣URE。 <ol><li>使用的z驱动器，通过手动聚焦上下验证感兴趣结构（ 例如，细胞胞体）的顶部和底部。记下z平面将要烧蚀（ 例如，小区的中心）。 注:根据经验，这种方法是通过瞄准那名亮标记的脊髓神经元（高信噪比，使消融后易时移可视化; 例如， 图4）最有效和消融的中间胞体。细胞核荧光能的优势，以确保正确的定位和高效率的消融。 </li></ol></li></ol> 3.在斑马鱼脊髓进行单个细胞的有针对性的激光烧蚀注意:对于这个消融和可视化的方法中，使用共聚焦显微镜（莱卡SP5）。使用405纳米二极管细胞特异性DEST消融过程乱子是根据软件（徕卡应用套件，v2.7.3.9723）的详细。然而，配备了405-nm激光和FRAP（荧光漂白后恢复）或漂白模块任何常规共焦显微镜将允许同一小区的操作的性能，但可能具有稍微不同的设置，参数和名称。 <ol><li>通过单击下拉菜单在软件菜单（ 图3A，1和2）的顶端启动FRAP向导。观察具有不同步骤的新窗口，允许所述特定参数为激光烧蚀设置向上（ 图3B，3）。 </li><li>通过选择格式，扫描速度（ 图3B，4），和平均确定消融方法的图像参数（ 图3B中 ，5）。在400赫兹和线的扫描速度1,024×1024的图像格式4平均是最适用的。 注意:通常没有必要改变光谱检测（如激发或发射参数），因为他们已经在先前采集被确定。 <ol><li>如果z平面消融尚未选择（如步骤2.4中所述）中，按在"实时"按钮，并通过试样集中直到荧光结构或将要被消融的所需z平面是焦点。 </li></ol></li><li>一旦通用图像参数设定，访问"漂白"的步骤（ 图3C，6），以控制特定的消融元件。 注意:激光强度的组合（ 图3C，8），扫描速度，并已在步骤3.2（ 图3B，4和5），以及重复将在设置数被设定的平均化步骤3.5（ 图3E，12）中 ，将确定在ROI内的紫外激光的总停留时间，并因此，漂白效率。 <ol><li>通过激活它的漂白过程（ 图3C，8）接合405-nm激光。 注: - 80％在我们的实验装置与上述设定最成功用405-nm激光强度之间60来实现的。要知道，这种激光的输出功率是仪器专用，并会为每一个共焦设置有所不同。 </li><li>使用选项中的"放大"（ 图3C，7）通过减少扫描场，因此最大限度地停留时间在选定的投资回报率最大化漂白力度。另外，使用所选择的用于该方法的软件的"漂白点"选项。 </li></ol></li><li>通过在图像采集风使用任何的绘图工具选择用于烧蚀一个或多个ROI（ 图3D，10）流（ 图3D，9）。定位的轴突岗，例如具有大约4的圆形绘图工具 - 8微米。 注:消融区域是从一个单一的像素，以更大的面积，这取决于应用可调节的。 </li><li>建立ROI之后，选择"时间进程"按钮（ 图3E，11）和确认的周期的感兴趣区将被扫描/烧蚀（ 图3E，12）的数量。以根据需要在漂白过程之后刚刚之前，立即使整个图像的概况选择"预漂白"和"后漂白"帧。 </li><li>建立所有必要的消融参数，按"运行实验"（ 图3E，13）和监测消融后效率。 注:在我们的FRAP设置，单个图像会前，并与合适的激光E中的FRAP周期后拍摄xcitation（ 例如，488纳米的激发对于表达EGFP的细胞）。这些前后消融图片允许的选择消融参数是如何令人满意的投资回报率被漂白的效果如何是一个快速的判断。 </li><li>通过调整激光强度重复这个过程（ 图3C，8），扫描速度和平均化（ 图3B，4和5），并在壳体的重复（ 图3E，12）所选择的投资回报率仍然示出了完成后的高的荧光强度FRAP周期。 </li></ol> 4.执行后续处理，包括鱼"救市"或处置<ol><li>如果实验是终端，安乐死的动物用三卡因的过量。除去蛋水中，并用麻醉原液10分钟更换。为了确保安乐死，检查在显微镜下心跳停止。 </li><li>可选的:如果实验不是终端，从细镊子和刷子琼脂糖小心取出鱼。放置在新鲜鸡蛋水中鱼，并允许它在观察恢复15分钟。如果正常的游泳行为返回，回鱼的孵化器。 </li><li>根据该机构的批准的转基因生物废物流处理转基因动物。 </li></ol>

<ol>
	<li>Becker, T. S., Rinkwitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+genomics+model+for+human+neurological+and+polygenic+disorders.">Zebrafish as a genomics model for human neurological and polygenic disorders.</a> Dev Neurobiol. 72 (3), 415-428 (2012).</li><li>Wilt, B. 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=Advances+in+light+microscopy+for+neuroscience.">Advances in light microscopy for neuroscience.</a> Annu Rev Neurosci. 32, 435-506 (2009).</li><li>Philips, T., Robberecht, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation+in+amyotrophic+lateral+sclerosis:+role+of+glial+activation+in+motor+neuron+disease.">Neuroinflammation in amyotrophic lateral sclerosis: role of glial activation in motor neuron disease.</a> Lancet Neurol. 10 (3), 253-263 (2011).</li><li>Robberecht, W., Philips, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+scene+of+amyotrophic+lateral+sclerosis.">The changing scene of amyotrophic lateral sclerosis.</a> Nat Rev Neurosci. 14 (4), 248-264 (2013).</li><li>White, D. T., Mumm, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nitroreductase+system+of+inducible+targeted+ablation+facilitates+cell-specific+regenerative+studies+in+zebrafish.">The nitroreductase system of inducible targeted ablation facilitates cell-specific regenerative studies in zebrafish.</a> Methods. 62 (3), 232-240 (2013).</li><li>Jagadeeswaran, P., Paris, R., Rao, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+thrombosis+in+zebrafish+larvae:+a+novel+genetic+screening+method+for+thrombosis.">Laser-induced thrombosis in zebrafish larvae: a novel genetic screening method for thrombosis.</a> Methods Mol Med. 129, 187-195 (2006).</li><li>Johnson, C. S., Holzemer, N. F., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+of+the+zebrafish+pronephros+to+study+renal+epithelial+regeneration.">Laser ablation of the zebrafish pronephros to study renal epithelial regeneration.</a> J Vis Exp. (54), e2845 (2011).</li><li>Matrone, 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=Laser-targeted+ablation+of+the+zebrafish+embryonic+ventricle:+a+novel+model+of+cardiac+injury+and+repair.">Laser-targeted ablation of the zebrafish embryonic ventricle: a novel model of cardiac injury and repair.</a> Int J Cardiol. 168 (4), 3913-3919 (2013).</li><li>Sieger, D., Moritz, C., Ziegenhals, T., Prykhozhij, S., Peri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+Ca2++waves+transmit+brain-damage+signals+to+microglia.">Long-range Ca2+ waves transmit brain-damage signals to microglia.</a> Dev Cell. 22 (6), 1138-1148 (2012).</li><li>Lewis, G. M., Kucenas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+nerve+transection+and+time-lapse+imaging+of+glial+cell+behaviors+in+live+zebrafish.">Motor nerve transection and time-lapse imaging of glial cell behaviors in live zebrafish.</a> J Vis Exp. (76), (2013).</li><li>Rosenberg, A. F., Wolman, M. A., Franzini-Armstrong, C., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+nerve-macrophage+interactions+following+peripheral+nerve+injury.">In vivo nerve-macrophage interactions following peripheral nerve injury.</a> J Neurosci. 32 (11), 3898-3909 (2012).</li><li>Villegas, R., 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=Dynamics+of+degeneration+and+regeneration+in+developing+zebrafish+peripheral+axons+reveals+a+requirement+for+extrinsic+cell+types.">Dynamics of degeneration and regeneration in developing zebrafish peripheral axons reveals a requirement for extrinsic cell types.</a> Neural Dev. 7, 19 (2012).</li><li>O'Brien, G. S., 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=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. (24), (2009).</li><li>Soustelle, L., Aigouy, B., Asensio, M. L., Giangrande, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV+laser+mediated+cell+selective+destruction+by+confocal+microscopy.">UV laser mediated cell selective destruction by confocal microscopy.</a> Neural Dev. 3, 11 (2008).</li><li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+the+ARRIVE+guidelines+for+reporting+animal+research.">Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.</a> PLoS Biol. 8 (6), 1000412 (2010).</li><li>Avdesh, 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> J Vis Exp. (69), e4196 (2012).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio).Vol.4th ed. , (2000).</li><li>Clark, K. J., Urban, M. D., Skuster, K. J., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+zebrafish+using+transposable+elements.">Transgenic zebrafish using transposable elements.</a> Methods Cell Biol. 104, 137-149 (2011).</li><li>Kwan, K. 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=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev Dyn. 236 (11), 3088-3099 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), (2009).</li><li>Higashijima, S., Hotta, Y., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+cranial+motor+neurons+in+live+transgenic+zebrafish+expressing+green+fluorescent+protein+under+the+control+of+the+islet-1+promoter/enhancer.">Visualization of cranial motor neurons in live transgenic zebrafish expressing green fluorescent protein under the control of the islet-1 promoter/enhancer.</a> J Neurosci. 20 (1), 206-218 (2000).</li><li>Arkhipova, V., 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=Characterization+and+regulation+of+the+hb9/mnx1+beta-cell+progenitor+specific+enhancer+in+zebrafish.">Characterization and regulation of the hb9/mnx1 beta-cell progenitor specific enhancer in zebrafish.</a> Dev Biol. 365 (1), 290-302 (2012).</li><li>Flanagan-Steet, H., Fox, M. A., Meyer, D., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+synapses+can+form+in+vivo+by+incorporation+of+initially+aneural+postsynaptic+specializations.">Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations.</a> Development. 132 (20), 4471-4481 (2005).</li><li>Hall, T. 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=The+zebrafish+candyfloss+mutant+implicates+extracellular+matrix+adhesion+failure+in+laminin+alpha2-deficient+congenital+muscular+dystrophy.">The zebrafish candyfloss mutant implicates extracellular matrix adhesion failure in laminin alpha2-deficient congenital muscular dystrophy.</a> Proc Natl Acad Sci U S A. 104 (17), 7092-7097 (2007).</li><li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, 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=mpeg1+promoter+transgenes+direct+macrophage-lineage+expression+in+zebrafish.">mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.</a> Blood. 117 (4), 49-56 (2011).</li><li>Morsch, 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=In+vivo+characterization+of+microglial+engulfment+of+dying+neurons+in+the+zebrafish+spinal+cord.">In vivo characterization of microglial engulfment of dying neurons in the zebrafish spinal cord.</a> Front. Cell. Neurosci. , (2015).</li><li>Grunwald, D. J., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Headwaters+of+the+zebrafish+--+emergence+of+a+new+model+vertebrate.">Headwaters of the zebrafish -- emergence of a new model vertebrate.</a> Nat Rev Genet. 3 (9), 717-724 (2002).</li><li>Lele, Z., Krone, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+system+in+developmental,+toxicological+and+transgenic+research.">The zebrafish as a model system in developmental, toxicological and transgenic research.</a> Biotechnol Adv. 14 (1), 57-72 (1996).</li><li>Veldman, M. B., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+developmental+model+organism+for+pediatric+research.">Zebrafish as a developmental model organism for pediatric research.</a> Pediatr Res. 64 (5), 470-476 (2008).</li><li>Kabashi, 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=Gain+and+loss+of+function+of+ALS-related+mutations+of+TARDBP+(TDP-43)+cause+motor+deficits+in+vivo.">Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo.</a> Hum Mol Genet. 19 (4), 671-683 (2010).</li><li>Laird, A. S., 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=Progranulin+is+neurotrophic+in+vivo+and+protects+against+a+mutant+TDP-43+induced+axonopathy.">Progranulin is neurotrophic in vivo and protects against a mutant TDP-43 induced axonopathy.</a> PLoS One. 5 (10), 13368 (2010).</li><li>Lemmens, R., 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=Overexpression+of+mutant+superoxide+dismutase+1+causes+a+motor+axonopathy+in+the+zebrafish.">Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish.</a> Hum Mol Genet. 16 (19), 2359-2365 (2007).</li><li>Ramesh, 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+genetic+model+of+amyotrophic+lateral+sclerosis+in+zebrafish+displays+phenotypic+hallmarks+of+motoneuron+disease.">A genetic model of amyotrophic lateral sclerosis in zebrafish displays phenotypic hallmarks of motoneuron disease.</a> Dis Model Mech. 3 (9-10), 652-662 (2010).</li><li>Hanneman, E., Trevarrow, B., Metcalfe, W. K., Kimmel, C. B., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+pattern+of+development+of+the+hindbrain+and+spinal+cord+of+the+zebrafish+embryo.">Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo.</a> Development. 103 (1), 49-58 (1988).</li><li>Lewis, K. E., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cells+to+circuits:+development+of+the+zebrafish+spinal+cord.">From cells to circuits: development of the zebrafish spinal cord.</a> Prog Neurobiol. 69 (6), 419-449 (2003).</li><li>Ilieva, H., Polymenidou, M., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-cell+autonomous+toxicity+in+neurodegenerative+disorders:+ALS+and+beyond.">Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond.</a> J Cell Biol. 187 (6), 761-772 (2009).</li><li>Philips, T., Rothstein, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+in+amyotrophic+lateral+sclerosis.">Glial cells in amyotrophic lateral sclerosis.</a> Exp Neurol. 262, 111-120 (2014).</li><li>Mazaheri, 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=Distinct+roles+for+BAI1+and+TIM-4+in+the+engulfment+of+dying+neurons+by+microglia.">Distinct roles for BAI1 and TIM-4 in the engulfment of dying neurons by microglia.</a> Nat Commun. 5, 4046 (2014).</li><li>van Ham, T. J., Kokel, D., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cells+are+cleared+by+directional+migration+and+elmo1-+dependent+macrophage+engulfment.">Apoptotic cells are cleared by directional migration and elmo1- dependent macrophage engulfment.</a> Curr Biol. 22 (9), 830-836 (2012).</li><li>Radford, R. 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=The+established+and+emerging+roles+of+astrocytes+and+microglia+in+amyotrophic+lateral+sclerosis+and+frontotemporal+dementia.">The established and emerging roles of astrocytes and microglia in amyotrophic lateral sclerosis and frontotemporal dementia.</a> Front Cell Neurosci. 9, 414 (2015).</li></ol>

The authors declare that they have no competing financial interests.

激光烧蚀途径激光辅助消融技术使细胞的个人或小团体的精确瞄准。结合高分辨率的显微镜和动物模型的遗传操作，如斑马鱼这种技术可以让研究人员系统地研究单个细胞的命运和受伤后的相互作用。 这里所描述的紫外线（405纳米）的激光烧蚀协议概括了单个细胞可以如何强调或杀死选择性（以剂量依赖性的方式），而相邻的神经元，神经胶质细胞，和轴突留下无恙。我们已成功地应用在细胞培养实验这一做法，并在这里描述的斑马鱼脊髓详细的方法。我们通过其它细胞（ 图5，A和网络内选择性地强调个别神经元显示在斑马鱼脊髓这种方法的实施<strong&gt; B），或立即无条件恢复杀死单个神经元（ 图5，C和D）。 先前，专业激光系统，例如脉冲氮激光或双光子激光系统，被要求以诱导组织损伤和运动神经横断10，11，12，13。这些激光系统已成功地使用，以引起细胞损伤，如血栓形成中的动脉和静脉6，急性肾损伤7，心肌损伤8，并研究钙波和脑损伤9后小胶质细胞的反应。此外，苏斯戴尔及其同事使用一种传统的共聚焦装置（351纳米和364纳米紫外激光）来诱导果蝇 14 &lt;损害上皮细胞和神经胶质细胞/ SUP&gt;。 斑马鱼模型的相关性为了解ALS（和其他人类疾病） 斑马鱼是一种广泛使用的模式生物，尤其是对发育研究28，29，30。虽然他们有一定的局限性，其对人类疾病模型，给致病分子机制的认识潜力是巨大的。斑马鱼模型已经很好建立为MND的研究，并已导致重要的分子的见解31，32，33，34。转基因斑马鱼线可以快速产生（4 - 5个月），并允许一个特定的细胞类型，功能，使它们一个有价值的除了ALS的当前动物模型的选择性跟踪。斑马鱼的胚胎/幼虫光学透明，并提供独特的experi精神的优点，允许长期实时成像在大脑或脊髓，不能在啮齿动物模型中容易地实现（或人类）的单细胞水平。当与分子技术，如单细胞消融相结合，这提供了用于在体内研究精确的分子机制的唯一实验平台。 运动神经元可以有选择有针对性的使用紫外激光烧蚀斑马鱼的脊髓神经元开始在出生后10小时内制定并后大约48小时，35，36确立。这种快速发展使得在短的时间框架，并与高通量这些神经元的可视化。运动神经元提供大脑和肌肉之间的重要联系，并在ALS中运动皮层（上位运动神经元），脑干和脊髓（下运动神经元）会受到影响。这些神经元的损失不可避免地导致万亩SCLE萎缩和无力。在斑马鱼的脊髓运动神经元可以通过不同的凸起和由像-3MNX1运动神经元特异性启动子的使用来确定。针对这种投射神经元的胞体显露沿着轴突投射的顺行退化随着时间的推移（ 图4和视频1）。脊髓运动神经元的单细胞分辨率成像还证实激光烧蚀（参见图4和补充视频3参考27）后，磷脂酰丝氨酸易位和随之而来的Annexin V的标签。虽然我们在紫外激光消融方法后死亡的神经元报Annexin V的激活，我们不能确定它是在这个进程加快引发死亡的级联反应，神经退行性疾病或正常细胞的动态平衡过程中发生的神经元死亡完全匹配。 虽然这种消融方法是高度可再现和具体的，不同的嵌入策略也可能影响紫外烧蚀的效率。根据我们的经验，这是最成功的，以我们在嵌入式我们的鱼琼脂糖层最小化。包埋介质与蛋水的附加层可以减少最终由小区接收的紫外线功率由于衰减而发生的散射效应的较厚的层沿着光束路径。 在未来，不同的转基因鱼线的交叉将使对直接和短期的可视化（高达12小时）其他受影响的细胞，如神经胶质细胞的反应，以激光诱导的细胞破坏。例如，星形胶质细胞和非细胞自主毒性在神经变性疾病如ALS一直在研究聚光灯和散发性和家族性ALS 37,38的致病性都在很大程度上牵连。然而，机制底层胶质毒性和选择性对运动神经元仍不清楚。我们和其他人最近采取了这种方法的优点，研究通过小胶质细胞的神经元死去的吞噬和可视残存神经元的27，39，40的间隙。 高分辨率显微术和用于神经炎症标记物相结合的消融技术将允许研究在未来扩展单细胞功能和互连细胞系统的理解。 在体内设置这些过程的描述是不仅在发育的设置，而且在神经变性疾病，包括MND，其中细胞相互作用可能被削弱3，41的模型至关重要。

荧光显微镜长期以来被用于研究在斑马鱼的CNS转基因，特别是其对发展1效应的影响。高分辨率显微术已经允许参与脑发育，肌肉生成，以及许多其他发育事件2的细胞过程的详细映射。研究单个细胞的死亡一直是更具挑战性，主要是由于在标准成像过程中选择性诱导细胞死亡的技术困难。然而，单细胞分辨率成像和高度针对性消融技术的组合允许的应力和损伤直接细胞应答的调查，以及随之而来的细胞 - 细胞相互作用的。理解这些过程是关键的，特别是用于神经变性疾病如运动神经元病（MND），其中神经元的神经胶质细胞的相互作用已经显示出向进展的疾病3。 MND，或肌萎缩性侧索硬化症（ALS），是一种破坏性的神经退行性疾病，影响在脑干，运动皮层，和脊髓运动神经元。这些神经元的丧失导致肌肉松弛，病人在3死- - 5年确诊4。在脊髓链接到肌肉纤维的运动神经元和在促进肌肉收缩中起重要作用。这些神经元的这种交流或死亡的失败逐渐减弱肌肉和影响患者的吞咽，行走，说话，呼吸的能力。可视化运动神经元和短期后果的死亡活的动物提供了极好的机会更好地了解参与正常细胞稳态和疾病的动态过程。 斑马鱼已成为一个有吸引力的模型系统来研究神经退行性疾病1。这个是由于该模型生物体提供，如外部施肥，短发育时间，对神经系统的光接入和易于转基因的优点。此外，为了容易地生成化合物转基因斑马鱼的能力允许对于不同的细胞类型的多个标记策略。遗传消融方法杀死特定的细胞类型可以相当广泛的干扰，但缺乏针对单个细胞5的精细控制。激光辅助的技术，在另一方面，提供优良的时间和空间的控制和已被用于不同的动物模型。尽管大多数方法使用专门的设备，如脉冲激光器6，7，8，9，10，11，12或双光子调校13，其他研究小组最近已采取在常规共焦显微镜14 UV激光的优点。 此处所描述的技术结合高分辨率共焦显微镜用UV激光介导的方法，使细胞应激或死亡中选择的运动神经元的剂量依赖性的方式。它依赖于使用通常安装405纳米的激光，已在细胞培养和活体动物试验成功，并允许细胞相互作用，如神经元死亡后，小胶质细胞间隙的详细特征。

<table><tbody><tr><td>Agarose low melting</td><td>Fisher-Scientific</td><td></td><td>Dissolve in egg water with tricaine (0.02%) at 0.8 - 1.5%</td></tr><tr><td>Tricaine</td><td>Argent Labs</td><td>MS-222</td><td>0.02%</td></tr><tr><td>Harvard standard wall Borosilicate with filament Capillary Glass</td><td>World Precision Instruments, Inc.</td><td>GC100F-10 (short)&lt;br/&gt; GC100F-15 (long)</td><td>Pull to a resistance of 2 - 7 M&amp;Omega;</td></tr><tr><td>Egg water&amp;nbsp;</td><td></td><td></td><td>0.6 g Instant Ocean sea salt, 4 mL of 1 mg/mL methylene blue in 10 L deionized water</td></tr><tr><td>1-phenyl-2-thiourea (PTU)</td><td>Sigma</td><td>P7629</td><td></td></tr><tr><td>Pronase</td><td>Sigma</td><td>10165921001</td><td></td></tr><tr><td>Heat block</td><td>Select BioProducts</td><td>Digital block heater (SBD110_)</td><td></td></tr><tr><td>Microinjection apparatus</td><td>World Precision Instruments, Inc.</td><td></td><td>Microinjection was performed by hand under a steromicroscope (Leica) with a loaded glass needle and a Picospritzer II (General Valve corporation)</td></tr><tr><td>Stereoscope</td><td>Leica</td><td></td><td>Bright field illumination microscopy was performed on a Leica M165FC stereo dissection microscope (Leica)</td></tr><tr><td>Microscope</td><td>Leica&amp;nbsp;</td><td>SP5</td><td></td></tr><tr><td>35 mm glass bottom dish</td><td>MatTek Corporation</td><td>P35G-0-20-C</td><td></td></tr></tbody></table>

triggering cell stress and death using conventional uv laser confocal microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

这里所描述的方法允许运动神经元中使用商业共焦显微镜的FRAP模块斑马鱼脊髓消融。表达在神经细胞绿色荧光蛋白特异性启动子，如控制下的转基因斑马鱼线- 3mnx1，islet1，或满足 ，分别使用。 GFP的由运动神经元的启动子驱动（如-3mnx1或满足 ）的表达使细胞体，主轴突，和外周分支延伸到肌肉（ 图4和视频1）的高分辨率可视化。 在3至5天龄鱼的脊髓神经细胞已经成功地烧蚀，具有60的总停留时间 - 第80号在上述步骤的〜70％的激光功率和一般设置3.成功消融当荧光消融后立即消失实现永不恢复（ 图5，C和D）。在与其他的激光线（如488纳米激光线）消融的尝试并没有导致永久性衰落，和荧光短的时间框架内恢复。重要的是，这种技术显示出在UV-烧蚀神经元的凋亡细胞死亡的特征，如膜联蛋白V的存在，常染色体变性一致的形态变化，并且烧蚀神经元27的轴突起泡。 这种方法的特异性使用photoconvertible荧光团枫（暴露于UV光之后切换从绿色到红色的发射），其中一个已定位的神经元被转化（ 图5，A和B）未经蜂窝迹象的实验证实销毁超过几个小时。较高的激光功率的使用，而不是联To在附近的细胞（〜20微米），以消融部位（ 图5，C和D）的目标神经元（不经光或荧光的再现）和光转换（无死亡）的灭绝。 此激光诱导消融技术的一个重要的优点是该方法的剂量依赖性。定位到不同强度的细胞，微调多层可通过调整激光功率（ 图3C，8），扫描速度和行平均（ 图3B，4和5），ROI的大小待消融（ 图3D，10），以及重复（ 图3E，12）。值得注意的是，这种方法也可用于细胞应激应用于单个细胞而不是诱导细胞死亡。例如，微调已经相当有价值一个神经元的死亡中，以评估细胞过程。长轴突投射是被烧蚀低UV激光强度的运动神经元透露特有的"起泡"（形成和细胞囊泡碎片），它开始在目标胞体和沿轴突随着时间的推移不断（40 - 90分钟; 图4 ; 3D 视频中呈现1本消融的电影）。因此，调制不同激光烧蚀参数和因此引起的细胞应激的水平和死亡的时间过程允许研究的实验灵活性高的水平。 <img alt="图1" src="/files/ftp_upload/54983/54983fig1.jpg" /> 图1:实时成像斑马鱼的嵌入。 （AF）嵌入实时成像步骤:（A）三卡因加入到鸡蛋水，麻醉的斑马鱼<html>的50毫克/升た起始剂量率。 （B）的低熔点琼脂糖（0.8 - 1.5％）的混合物，并加热至38 - 40℃。 （C）使用移液管，在筛选和选择的斑马鱼被转移到用三卡因溶液菜。成功镇静后（浅鳃盖运动，降低心脏速率，缺乏触诱发反应的），一条鱼被转移到预热琼脂糖（D）。最大限度地减少被转移到琼脂糖以防止随后的稀释蛋水的量。 （五）转让琼脂糖一滴-包含斑马鱼到玻璃底35毫米的菜（30〜50微升）。解剖显微镜下进行这一点，并用毛刷轻轻对准斑马鱼的首选方向。等待10 - 15分钟，直到琼脂糖被设置，并添加三卡因溶液〜2毫升的菜（F）。ANK"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54983/54983fig2.jpg" /> 图2:神经元的可视化和小胶质细胞在3-DPF斑马鱼的脊髓。在3天的转基因斑马鱼的脊髓小胶质细胞和神经元的可视化表达（A）GFP阳性神经元（islet1:GFP）和（B）mCherry阳性小胶质细胞（MPEG1:GAL4，UAS:mCherry）。 （C）与亮场图像一起神经元和胶质信道的复合图像。 （C）中的原理插入描绘了鱼的方向，并概述所提出的区域。比例尺= 30微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54983/54983fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3:在紫外激光烧蚀的方法的步骤（如在协议所概述的，步骤3）。步骤控制在共聚焦软件（徕卡应用套件）的FRAP软件模块。 （一）启动FRAP模块进行紫外激光烧蚀的工具。 （B）中设置的z平面像格式，速度和平均烧蚀等FRAP设置，这将决定激光的停留时间。激光强度和"放大"选项，以最大限度地提高效率漂白（三）控制。 （D）的感兴趣区域（ROI）中的一个或多个区域的选择，将被烧蚀。 （E）设置漂白的时间过程确定漂白周期和在感兴趣区的总激光停留时间。 <a href="http://ecsource.jove.com/files/ftp_upload/54983/54983fig3large.jpg" target="_blank">请点击她的</a><html> E要查看此图的放大版本。 <img alt="图4" src="/files/ftp_upload/54983/54983fig4.jpg" /> 图4:紫外烧蚀神经元的顺行变性。紫外切除脊髓神经元的神经退行性疾病的时间推移成像。 （AF）单脊髓神经元的紫外线照射（ 满足:GAL4，UAS:EGFP; A;圆）导致神经元萎缩，随着时间的推移围捕（AC）的身体，其次是轴突碎片（CF;箭头） 。在轴突变性开始在SOMA（消融部位），进展顺向轴突远端直到最后，在索玛荧光消失，整个轴突呈"起泡"（DF）。比例尺= 20微米。该消融的3D渲染时间推移电影显示在视频1。ES / ftp_upload / 54983 / 54983fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/54983/54983fig5.jpg" /> 图5:单细胞UV照射的使用在运动神经元一photoconvertible荧光团（枫）的效果的确认。通过photoconvertible荧光枫的一个神经元激活的单细胞的紫外线照射的验证。 （AD）的标有枫神经元的UV照射。 （A）一个单一的神经元（圆）与10秒30％的激光功率的光转化只在个别目标神经元（B）导致的枫光转化（从绿色到红色）。注意，转化细胞存活几个小时，并显示出恶化的无视觉迹象，如起泡或向上舍入。单神经元消融 （C<html> ;圆）具有较高的激光功率（95％对10秒）导致了该神经元（D立即消失）和枫的后续光转化在约20微米的半径范围内的一小部分周围神经元。比例尺= 20微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54983/54983fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="视频1" src="/files/ftp_upload/54983/54983vid1.jpg" /> 视频1:在图4所示的紫外线烧蚀神经元的三维表面再现（了Imaris）。 如图4所示神经元的延时视频是使用可视化软件（了Imaris，位平面）呈现的表面。它突出烧蚀体细胞的收缩过程，随后轴突碎片顺行朝向单元的远端。euron_ablation-3D_rendered.mov"目标="_空白"&gt;请点击这里下载该文件。 补充图1:可选琼脂糖塑像为长时间成像。 为了避免长期采集过程中的鱼类和琼脂糖的运动准备环形沿着玻璃底35mm培养皿（A）的中间边缘琼脂糖形圈。为让〜10分钟琼脂糖集并转移鱼入内圆琼脂糖（B）的下降。尽量减少琼脂糖的量为嵌入（定向后捞出刷过量琼脂糖到外部）。 <a href="http://ecsource.jove.com/files/ftp_upload/54983/Supplementary_Figure_1.jpg" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy at JoVE.com

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

触发细胞应激和死亡使用传统的紫外线激光共聚焦显微镜

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

triggering-cell-stress-death-using-conventional-uv-laser-confocal

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.4K 次观看.  Macquarie University. Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

视频: Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

科研

癌症研究

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

遗传学

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

利用分子荧光互补的方法照亮 Caspase 活化的途径

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

生物化学

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

生物工程

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

生物学

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

飞秒激光的光刺激激活目标细胞中的细胞外信号调节激酶(ERK)信号或线粒体事件

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

激光微辐射研究S阶段DNA招募

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...