We thank Dr. Roger Tsien for providing us with the miniSOG constructs. Dr. Xuejun Sun for help with the TEM. Lisa Lem, and Peter Shipple from the Cross Cancer Institute for supplying oxygen. Hilmar Strickfaden holds a postdoctoral fellowship by the Alberta Cancer foundation and was supported by the Bayrische Forschungsallianz. This work was supported by grants from the Canadian Institutes of Health Research and Alberta Cancer Foundation.

1.代miniSOG细胞系<ol><li>生长的U2OS（人骨肉瘤）细胞在含2ml即补充有10％胎牛血清（FBS），在37℃下在湿润的培养箱中5％的CO 2低葡萄糖Dulbecco改良的Eagle培养基（DMEM）的35mm培养皿气氛。 </li><li>转染的细胞时，它们是80％汇合通过脂质转染法，用纯化的转染质量（A269 / 280比率1.8-2.0）包含miniSOG和mCherry序列质粒构建根据转染试剂19的制造商的协议标签修复蛋白。该miniSOG表达质粒可以从钱学森实验室订购<a href="http://www.tsienlab.ucsd.edu/Samples.htm">：http://www.tsienlab.ucsd.edu/Samples.htm</a> </li><li>排序的细胞通过荧光活化细胞分选后两天转染表达重组蛋白与miniSOG和mCherry标记。收集细胞用，以避免被过度表达20个细胞中间荧光强度。 </li><li>培养上在10ml的DMEM培养基是补充有10％FBS和选择剂的G418（遗传霉素）的0.6微克/毫升10cm皿的阳性细胞。 </li><li>后可见菌落都出现在平板上，画面mCherry阳性菌落使用倒置荧光显微镜和绘制圆周围（上板的底部）具有永久性标记。使用低倍率的空气物镜（ 如 10倍），并选择使用无菌技术克隆通过仔细刮殖民地断，慢慢地吸吮他们进入无菌1000微升的枪头。 </li><li>扩展所选择的菌落，并利用在步骤1.1补充有10％二甲亚砜中描述的生长培养基冻结其中某些部分向下。通过他们成长在无菌18×18 平方毫米盖玻片，并将其暴露于测试细胞DRF形成辐射2戈瑞。经照射的细胞稳定表达miniSOG mCherry标记的修复蛋白必须显示双链断裂的典型焦图案特性时使用的过滤器设置用于成像mCherry用荧光显微镜显现。 </li></ol> 2.细胞培养<ol><li>培养U2OS细胞稳定表达miniSOG和mCherry在步骤1.1中概述的条件下标记修复蛋白。细胞生长至80％汇合在含2ml DMEM的35毫米直径的玻璃底菜。确保，重视盖玻片到塑料和所使用的塑料的胶水是耐水性和醇溶液和耐使用的树脂！ </li><li>之前进行的实验中，概述了大约1mm 2，其中包含的细胞通过使用荧光显微镜来识别感兴趣的区域，用无菌金刚石笔刮擦表达异位蛋白质的小区域。或者使用玻璃底菜牛逼帽子包含与内置于盖玻片预蚀刻的网格系统盖玻片。 注意：如果使用的是高品质的相关显微镜相结合ESI和荧光显微图片21，确保盖玻片厚度与油浸目标一致。它应具有的厚度号1.5（0.16-0.18毫米）。选择一个面积接近盘的中心区域进行审查的TEM，以避免与附近的边缘可能发生的树脂的聚合（见点4.10-4.13）。 </li></ol> 3. DNA损伤诱导<ol><li>照射细胞用γ辐射，可使用radiomimetic药物，或诱导由激光微照射的伤害上的共焦显微镜（例如，如在18,22或23中所述 ）。 </li><li>在这个例子中，使用405nm的溶胶照射细胞用γ照射的2和6戈瑞铯-137辐射源或激光器microirradiate共聚焦显微镜的致敏细胞20分钟，用0.​​5微克/毫升赫斯特之后的ID激光器。 </li></ol> 4.样品制备<ol><li>固定细胞用1ml的4％多聚甲醛小心在0.1M二甲胂酸钠缓冲液或者在0.1M磷酸盐缓冲液pH 7.4，在室温30分钟在黑暗中。 注意：多聚甲醛和砷酸钠是有毒的！戴上手套和处置有毒物质充分。 </li><li>用4ml的0.1M二甲胂酸钠缓冲液pH 7.4或磷酸盐缓冲液pH 7.4洗涤细胞两次。 </li><li>对待固定的细胞用2ml的50mM甘氨酸，5mM的氨基三唑和在0.1M二甲胂酸钠缓冲液或0.1M磷酸缓冲液（pH7.4）中的10mM氰化钾小心（检查pH值！）处理30分钟，以封闭未反应醛基和以抑制由血红素基团所产生的活性氧，从而可以提高的背景。 注意：氰化钾是剧毒！ W¯¯耳手套，工作引擎盖下和处置有毒物质充分。该反应对pH非常敏感，因此，重要的是将pH核实和准确。 </li><li>记录被在上一个倒置荧光显微镜步骤2.2在二维或三维中选择，如果相关显微术所需的区域。 </li><li>通过将10毫克的二氨基联苯胺盐酸盐片剂到2ml微量离心管中制备一1mg / ml的二氨基联苯胺盐酸小心溶液。添加980微升蒸馏水H 2 O的和20μl浓盐酸（11.65米）的并混合直至溶液为浅棕色并且是半透明的。稀释此溶液在0.1M磷酸盐或0.1M的二甲胂酸钠缓冲液中至1毫克/毫升的最终浓度，同时调整用氢氧化钠将pH值至pH 7.0和7.6之间（pH为7.4，推荐）。 注意：二氨基联苯胺是有毒的！戴上手套和处置有毒物质充分。 </li><li>对于光氧化河E放置用2ml的1毫克/毫升二氨基联苯胺盐酸盐在0.1M二甲胂酸钠缓冲液或磷酸盐缓冲液的缓冲液中。避光该溶液冷却下来在冰上4℃下 ，以提高氧的溶解度。通过该溶液鼓泡氧气饱和与氧的溶液（使用插入到溶液中并连接到一个氧气瓶的软管）。再次检查pH值，并在必要时进行调整。 注：它是用于光氧化反应的pH为7.0和pH 7.6之间至关重要。 </li><li>支座与固定的细胞的培养皿小心地配备了40倍的油浸物镜透镜倒置荧光显微镜，并将其移动到感兴趣的区域。通过使用滤镜立方体的GFP或CFP激发与蓝光miniSOG标签。 MiniSOG有一个最大激发在448 nm的肩膀，在473±10。 </li><li>即使在绿色荧光miniSOG公顷继续与照明š完全消失，直到聚合二氨基联苯胺的棕色光氧化产物出现在所传送的光信道。当氧化产物是可见的，在大多数的细胞，关闭荧光照明，停止光氧化。 注：光氧化最多可能需要取决于物镜，滤波器的传输波长和效率，以及照明源数分钟。 </li><li>后缀的细胞用1ml的2％戊二醛的0.1M二甲胂酸钠pH 7.4的缓冲液或磷酸盐缓冲液pH 7.4 30分钟。 </li><li>固定细胞与0.1％-0.5％的四氧化锇将膜20分钟在0.1M二甲胂酸钠缓冲液pH 7.4或0.1M磷酸盐缓冲液pH 7.4。 注：建议使用来稳定该膜所需的四氧化锇的可能的最低浓度。由于聚合的DAB析出物具有高密度的氮气，它可以直接用ESI，强烈的被检测锇染色是没有必要的，以确定所述miniSOG标记蛋白，可能是有害的ESI。四氧化锇很毒！在通风橱中操作和处置有毒废物充分。 </li><li>通过乙醇系列使用30％，50％，70％，90％，98％的100％乙醇的步骤（每次5分钟）脱水，所述细胞。接着，孵育细胞以1：1混合100％乙醇和丙烯酸树脂（LR白），并把该盘到振荡器上4小时孵育细胞至少在100另外4小时前，以促进渗透到细胞％丙烯酸树脂（LR白）。 </li><li>为了使树脂聚合，切断标记的2 ml离心管的盖用​​刀片和涂层与丙烯酸树脂加速器边缘。确保该加速器仅覆盖轮缘和不流入管内。随后，小心地充满管大约三分之二与使用巴氏吸管的LR白树脂。请注意，该树脂不会进入接触机智H于轮辋的加速器！ </li><li>删除渗入细胞在培养皿的树脂和颠倒放置皿到垂直竖立的离心管，使得该管的宽度几乎完全填满了盘的玻璃覆盖的观察窗。 </li><li>等待1至2分钟的加速器以密封管和盖玻片，然后反转管，使得在管中的树脂现已覆盖细胞。 </li><li>放置与管盘放入烘箱，在60℃，并固化它12小时。 </li><li>当块被治愈，删除与从烘箱所附树脂填充的离心管盘和分离的离心管从盘。促进在液氮和热水通过冷冻 - 解冻循环的分离。 </li><li>丢弃玻璃底培养皿，并仔细剖开的离心管，用刀片以除去该块。 </li><li>标签永久性标记块。 </li><li>使用刀片为三M中的嵌段，使得只是包含先前标记与金刚石或钨笔的区域（或含有与感兴趣的细胞中的区域）中的1毫米2区保持。 </li><li>安装块在超微，具有修边刀修剪块切成约50nm的超薄切片，使用金刚石刀。 </li><li>拿起高传输300目网格的部分。这些网格有非常小的尘棒，从而覆盖更少细胞中所关注的区域。 </li><li>涂层，使用约0.2-0.4纳米碳的碳涂布机，以帮助TEM的电子束下稳定它们的网格的部分。 </li></ol> 5.电子显微镜<ol><li>加载网格成配备有能量过滤器的TEM。调整显微镜并根据制造商的说明，将能量过滤后，检查对切片中的细胞低倍率模式（通常传输），并比较它们与荧光数据在适当的时候（在步骤4.4获得）。 </li><li>一旦与有趣的功能（DNA损伤曲目或DNA修复灶）原子核被发现，切换到能过滤模式并记录厚度​​图。 注：此过程包括零丢失和未经过滤的图像的记录。厚度达0.3平均自由程（在入射光束的电子的30％得到由样品散射）是足够薄，以产生良好的元素映射。 </li><li>元素磷和氮的使用控制所使用的显微镜​​的能量过滤器的软件的记录比率的地图。记录在175电子伏特能量损失的磷地图后边缘图像以20电子伏特的预边缘图像，在120电子伏特能量损失的狭缝宽度，也以20电子伏特的狭缝宽度。在氮地图，记录后的边缘图像在447伏特与35伏特和预边缘图像的狭缝宽度在358电子伏特，也为35电子伏特的狭缝宽度。 注：由于成像元件的含量（phosphOrús酒店位和氮）是相对低的（约1％），选择“比率图像”（定性元素地图）比“3窗口方法”（定量元素地图），以产生具有更好的信噪比的图像。 </li></ol> 6.图像处理<ol><li>在图像处理软件可以处理所获取的图像的文件格式 （例如，数字显微）打开元素比地图。氮地图复制到红色通道和磷地图成RGB图像的绿色通道，然后叠加并对准地图。 </li><li>合成图像导出为标记的图像文件格式（TIFF）文件。在图像处理的软件，可以使用层（如，Photoshop中）打开图像。 </li><li>通过重新缩放的最大值和最小值在图像中的8位数据组（0到255）调整各信道的动态范围。 </li><li>减去从氮地图中的磷含量（使用“层”的窗口），以产生一个定性图，显示蛋白质的分布。 </li><li>转换在上一步分为“索引彩色”创建的核酸（磷）和蛋白质（氮）的地图和在该地图表示核酸分布和青色查找表CMYK颜色空间创建一个黄色的查找表以显示非核蛋白地图。颜色被选择为产生在两个元素地图之间的最大对比度。 </li><li>创建一个新的形象，呈现出进口磷和蛋白质的不同层的分布地图。使用以便在“屏幕”透明模式，看看这两个层相互叠加。 </li><li>打开在步骤5.2获得的零损失图像。此图像表示记录区，看起来像一个常规的TEM图像的图像，但只包含通过试样已通过而不与样品碰撞的电子。出口此图片为TIFF图像。 </li><li>在照片编辑器中打开导出的零损耗的图像，并将其复制到图像显示的地图元素的最上层。对齐使用“屏幕”透明模式下的图像。 </li><li>任选阈零损失使图像段源自所述miniSOG信号。添加得到的图像作为一个单独的层，如上所述。 </li></ol>

<ol>
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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=Changes+in+chromatin+structure+and+mobility+in+living+cells+at+sites+of+DNA+double-strand+breaks.">Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks.</a> The Journal of Cell Biology. 172, 823-834 (2006).</li><li>Goodson, H. V., Dzurisin, J. S., Wadsworth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+cell+lines+expressing+GFP-tubulin+and+photoactivatable-GFP-tubulin+and+characterization+of+clones.">Generation of stable cell lines expressing GFP-tubulin and photoactivatable-GFP-tubulin and characterization of clones.</a> Cold Spring Harbor protocols. 2010, (2010).</li><li>Zeyda, M., Borth, N., Kunert, R., Katinger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+sorting+conditions+for+the+selection+of+stable,+high-producing+mammalian+cell+lines.">Optimization of sorting conditions for the selection of stable, high-producing mammalian cell lines.</a> Biotechnology progress. 15, 953-957 (1999).</li><li>Dellaire, G., Nisman, R., Bazett-Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+light+and+electron+spectroscopic+imaging+of+chromatin+in+situ.">Correlative light and electron spectroscopic imaging of chromatin in situ.</a> Methods in enzymology. , 375-456 (2004).</li><li>Campbell, S., Ismail, I. H., Young, L. C., Poirier, G. G., Hendzel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+repressive+complex+2+contributes+to+DNA+double-strand+break+repair.">Polycomb repressive complex 2 contributes to DNA double-strand break repair.</a> Cell cycle. 12, 2675-2683 (2013).</li><li>Mortusewicz, O., 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=Recruitment+of+RNA+polymerase+II+cofactor+PC4+to+DNA+damage+sites.">Recruitment of RNA polymerase II cofactor PC4 to DNA damage sites.</a> J Cell Biol. 183, 769-776 (2008).</li><li>Bekker-Jensen, 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=Spatial+organization+of+the+mammalian+genome+surveillance+machinery+in+response+to+DNA+strand+breaks.">Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks.</a> The Journal of Cell Biology. 173, 195-206 (2006).</li><li>Ferrando-May, 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=Highlighting+the+DNA+damage+response+with+ultrashort+laser+pulses+in+the+near+infrared+and+kinetic+modeling.">Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling.</a> Frontiers in genetics. 4, 135 (2013).</li><li>Lukas, 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=53BP1+nuclear+bodies+form+around+DNA+lesions+generated+by+mitotic+transmission+of+chromosomes+under+replication+stress.">53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress.</a> Nature. 13, 243-253 (2011).</li><li>Harrigan, J. 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=Replication+stress+induces+53BP1-containing+OPT+domains+in+G1+cells.">Replication stress induces 53BP1-containing OPT domains in G1 cells.</a> The Journal of Cell Biology. 193, 97-108 (2011).</li><li>Aronova, M. 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=Reprint+of+'Three-dimensional+elemental+mapping+of+phosphorus+by+quantitative+electron+spectroscopic+tomography+(QuEST).">Reprint of 'Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST).</a> J. Struct. Biol. 161, 322-335 (2007).</li><li>Fussner, 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=Constitutive+heterochromatin+reorganization+during+somatic+cell+reprogramming.">Constitutive heterochromatin reorganization during somatic cell reprogramming.</a> The EMBO journal. 30, 1778-1789 (2011).</li><li>Kepkay, R., Attwood, K. M., Ziv, Y., Shiloh, Y., Dellaire, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KAP1+depletion+increases+PML+nuclear+body+number+in+concert+with+ultrastructural+changes+in+chromatin.">KAP1 depletion increases PML nuclear body number in concert with ultrastructural changes in chromatin.</a> Cell cycle. 10, 308-322 (2011).</li></ol>

The authors have nothing to disclose.

ESI可作为极好的工具用于检查染色质和核糖核蛋白的不同状态在细胞核中，因为它是能够专门映射是富含磷的区域。它深刻增强细节，可以用电子显微镜来获得并且不依赖于非特异性对比方法的量。 ESI的一个缺点是，它需要比在相同的加速电压常规的TEM较薄部分。这可以通过在与连续切片或使用层析方法28来克服。 虽然大量的信息可以通过查看磷和氮的分布来获得?...

尽管近年来在1光学显微镜显著的进步，细胞生物学家仍在遭受分辨率的差距。这限制涉及大分子复合物之间的协调相互作用中基本的细胞过程中的结构-功能关系的了解（例如，在染色质重塑，DNA修复，转录的RNA和DNA复制）。虽然透射电子显微镜（TEM）■提供所要求的分辨率，已经挑战来定义这些过程无法的结构因为标注的特定蛋白质，同时还能够以确定的可视化结构的生化成分。在没有内部的膜，以帮助区分核结构中，核一直特别具有挑战性。电子光谱成像（ESI）通过允许DNA，RNA和PROT的同时检测和分化解决某些限制EIN基核结构2-5。 电子光谱成像： 为了映射以高灵敏度和分辨率在电子显微镜下元素分布，可以用一个图像光谱仪，其选择已通过与元素的在样本 6内壳层电子相互作用非弹性散射电子。因为能量的单元特定的量被丢失原子在试样的电离的结果，这些电子可以被分离并使用被附接到电子显微镜的分光计可视化。因此，已经相互作用与试样的电子的频谱分析揭示关于样品7的元素组成的定性和定量的信息。电子通过检体传递时不损失能量被发现在电子能量损失的“零损耗峰”谱。这些电子的丰度是相关的试样的质量，密度和厚度，并包括通过检体通过，而不碰撞与样品或通过试样通道中失去能量的电子。这个信息可以是为特定的元素本的原子数，在试样8的绝对定量是有用的。 因为生物样品包括主要光元件很差偏转电子在TEM的入射光束，染色方法使用重金属盐具有以生成所述样品中的对比度得以应用。特异性大多数这些造影剂和无法的可视化多个污点其中特异性是可能的缺乏在细胞核的研究已限制常规电子显微镜的值。 ESI有超过常规的TEM显著的优点，特别是对细胞核的结构的研究。有可能利用DNA和含有RNA的大分子复合的富磷性质区分蛋白质复合核蛋白复合物和解决基于核酸它们的密度不同核蛋白复合物。剩余的生物材料可以根据它的丰度氮气进行成像。不同的解剖结构中的映射只是这两种元素的分布和相对丰度的分析为我们提供了许多关于核信息。例如，它是容易识别染色质，并在地图上的核糖体代表磷丰度。所述interchromatin空间，核孔复合物，和核机构，另一方面，可以容易地在该氮地图图像中检测到。 迷你单态氧发生器系统（miniSOG） 尽管ESI代表一种强大的技术原位染色质结构来研究，因为它需要的优势在磷和氮之间的元素组成的特征比率，元素组成通常不能使用的蛋白质复合物的不同群体之间进行区分。抗体标记的小的金颗粒在纳米范围内已被广泛地用于绘制单个分子的位置。由于金颗粒通常连接到一个二级抗体，它将出现的周围由第一抗体检测的表位大约20nm的圆周内。在后包埋样品，抗体只能检测是在部分的表面露出的表位。虽然这是可能的，以证明抗原的存在，并将其涉及到细胞的特定解剖结构，所获得的信息是不完整的，因为大部分的表位是由树脂遮蔽。预嵌入技术，其使用类似的协议与用于荧光显微镜，在整个允许访问抗原样品的整个深度，但所需要的使抗体穿透细胞通常需要去除脂膜，并删除无法固定醛组分通透步骤。此外，优选的醛固定液超微结构保存，戊二醛，通常破坏的表位，因此，多聚甲醛，通常需要。这是在蛋白质 - 蛋白质交联效果较差。抗体的被标记有金纳米颗粒的另一缺点是，金是创建一个强烈的对比，可以掩盖样品，它具有弱对比度性的结构细节非常电子致密材料。 绿色荧光蛋白（GFP）作为表达的蛋白质标签的出现改变了使用荧光显微镜的解答在细胞生物学的问题。标记的蛋白质与小荧光域允许其在体内的分布制图</e米&gt;而不需要透过程，可以改变结构。为了翻译用蛋白质标记映射蛋白质分布在细胞中，以电子显微镜的优雅原则，需要一个系统，该系统能够产生一个信号接近感兴趣的结构，并产生以TEM对比度。聚合的二氨基联苯胺]是经常用于组织学检测抗体结合的污点。此染色是使用辣根过氧化物酶（HRP）缀合的第二抗体通常沉积。尽管反应产生一个可靠的结果，HRP是不是在细胞9的胞质溶胶催化活性。 HRP的反应产物也可以扩散远离一代的网站，这样它们的分辨率比纳米金法10恶化。为了绕开这些问题，FLASH / ReAsH系统的开发11。它由重组融合蛋白，其拥有四半胱氨酸基序。这个主题可以结合一个biarsenic荧光。当激发时，结合的闪光或ReAsH是能够产生反应性高的单重态氧，因此photoconvert二氨基联苯胺（DAB）插入在标签的蛋白质的位点紧接该沉淀的聚合物。民建联聚合物可染色用四氧化锇，这是电子致密，因而可以用于映射重组融合蛋白在TEM的分布。 在2011年，舒等人 10提交的miniSOG系统，它由来自拟南芥的黄素蛋白是荧光，并且能够在与448纳米的蓝色光激发而产生很多单线态氧自由基衍生的小106个氨基酸融合标记的。这些单线态氧自由基可用于光氧化二氨基联苯胺，以形成上和标签蛋白，这是比免疫标记的抗体11相当接近的表面附近的聚合物。虽然闪存/ ReAsH系统需要使氟铬和二氨基联苯胺成执行光转化前的细胞中，miniSOG系统只需要二氨基联苯胺和轻，此外是大约两倍于聚合二氨基联苯胺作为有效。在这里，miniSOG采用与ESI的组合，以图的DNA修复灶的超微结构。 DNA修复灶（DRF） 未修复DNA双链断裂构成严重威胁到细胞，因为它们可以导致易位和遗传信息的丢失。反过来，这可导致衰老，癌症和细胞死亡。参与DNA双链断裂修复，许多蛋白积聚在病灶周围的DNA双链组装打破12-14。虽然它们的功能是未知的，它们所代表的部位在包含DNA双链断裂，并且是DNA双链断裂修复的部位的核心。 DNA修复FOCI（DRF）的特点是荧光显微镜和它们作为生物标志物用于DNA损伤12,15。它们大量相对于双链断裂的大小和被认为是相对均匀的，直到最近的超高分辨率显微镜研究揭示在每个聚焦16个子条块分子的一些证据。为了了解这些位点的组织，有必要以可视化的所有潜在的生物结构的相对于彼此。这不能用荧光显微镜下进行，但有可能通过电子显微镜17,18。这里，描述了一种方法，结合了电子光谱成像与miniSOG方法，来说明这种组合方法的潜力，以探索的DNA双链断裂修复的超微结构。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>35 mm glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Gridded 35 mm glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-2-14-CGRD&#160;</td>
			<td>not made for immersion objectives</td>
		</tr>
		<tr>
			<td>LR White: acryl resin</td>
			<td>emsdiasum</td>
			<td>14381</td>
			<td></td>
		</tr>
		<tr>
			<td>LR White accelerator</td>
			<td>emsdiasum</td>
			<td>14385</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#8242;-Diaminobenzidine tetrahydrochloride 10 mg tablets</td>
			<td>Sigma</td>
			<td>D5905</td>
			<td></td>
		</tr>
		<tr>
			<td>Slim Bar Grids 300 Mesh</td>
			<td>SPI</td>
			<td>1161123</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten-Point Lab Pen</td>
			<td>emsdiasum</td>
			<td>41148</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide</td>
			<td>emsdiasum</td>
			<td>19100</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Coater 208carbon</td>
			<td>Cressington</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra microtome Leica EM UC6</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop CS5</td>
			<td>Adobe</td>
			<td></td>
			<td>might even work with older versions</td>
		</tr>
		<tr>
			<td>Digital Micrograph V.2.30</td>
			<td>Gatan</td>
			<td></td>
			<td>might even work with older versions</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma</td>
			<td>H-1399</td>
			<td></td>
		</tr>
		<tr>
			<td>Effectene</td>
			<td>Quiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MiniSOG-Constructs</td>
			<td>Tsien-Lab</td>
			<td></td>
			<td><a href="mailto:tsienlab@yahoo.com">tsienlab@yahoo.com</a></td>
		</tr>
		<tr>
			<td>MDC1 miniSOG mCherry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>53BP1 miniSOG mCherry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rad52 miniSOG mCherry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cesium 137 radiation source &#34;MARK 1&#34;</td>
			<td>&#160;(J.L. Shepherd &#38; Associated)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ/FiJi</td>
			<td>open source</td>
			<td></td>
			<td>http://fiji.sc/Fiji</td>
		</tr>
		<tr>
			<td>2 ml Eppendorf tubes</td>
			<td>Fisherbrand</td>
			<td>05-408-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond Knife ultra 35&#176;</td>
			<td>Diatome</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trimming Knife ultratrim</td>
			<td>Diatome</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cacodylate trihydrate</td>
			<td>emsdiasum</td>
			<td>12300</td>
			<td>Caution Toxic!</td>
		</tr>
		<tr>
			<td>Glutaraldehyde EM Grade 8%</td>
			<td>emsdiasum</td>
			<td>16020</td>
			<td>Caution Toxic!</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>emsdiasum</td>
			<td>21180</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>emsdiasum</td>
			<td>21190</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>emsdiasum</td>
			<td>19202</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide 4% solution</td>
			<td>emsdiasum</td>
			<td>19150</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Micoscope Axiovert 200M</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisherbrand</td>
			<td>A142-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide Solution 10M</td>
			<td>Fluka</td>
			<td>72068</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Medigas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-Amino-1,2,4-triazole</td>
			<td>Sigma</td>
			<td>A8056</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium cyanide</td>
			<td>Sigma</td>
			<td>207810</td>
			<td>Caution Toxic!</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>emsdiasum</td>
			<td>15058</td>
			<td>Caution Toxic!</td>
		</tr>
		<tr>
			<td>Razor blade Single Edge Carbon Steel</td>
			<td>emsdiasum</td>
			<td>71960</td>
			<td>Caution Sharp!</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D 5546</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>lifetechnologies</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>lifetechnologies</td>
			<td>11811-023</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope 200 kV</td>
			<td>JEOL</td>
			<td>2100</td>
			<td></td>
		</tr>
		<tr>
			<td>GIF Tridiem 863 Energy filter</td>
			<td>Gatan</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of minisog tagged dna repair proteins in combination with electron spectroscopic imaging (esi)

极限到光学分辨率和鉴定特定蛋白质种群透射电镜的挑战已经在细胞生物学障碍。许多现象无法通过体外分析在简化的系统进行说明，并且需要额外的结构信息在原位 ，特别是在1毫微米和0.1微米之间的范围内，以便被完全理解。这里，电子光谱成像，透射电子显微技术，允许蛋白质和核酸的分布同时映射，和表达标签，miniSOG，被组合以研究DNA双链断裂修复病灶的结构和组织。

ESI 与常规的TEM图像比较所述核（ 图1）的ESI 图像 （例如， 图7-1）中揭示了解剖结构，可以很容易分辨的显着增加。染色质脊显示为黄色，这是很容易识别在小鼠细胞中的chromocenters。核仁可以容易地从染色质区分通过圆形结构和不同的颜色，由于预组装的核糖体已经含有高量的蛋白质，其是富含氮，除了核糖核酸，其中富?...

Watch this Scientific Journal Video about Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI at JoVE.com

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

在联合电子能谱成像miniSOG标记的DNA修复蛋白可视化（ESI）

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

visualization-minisog-tagged-dna-repair-proteins-combination-with

Research

JoVE Journal

Biology

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

10.0K 次观看.  University of Alberta. Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

视频: Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

科研

生物学

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

免疫荧光DNA修复蛋白相互作用的可视化

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

基于光二极管的光学成像，用于记录非转基因无脊椎动物的单神经元分辨率的网络动态

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...