This research was supported by the AXonomIX research project financed by the Provincia Autonoma di Trento, Italy.

用于获取小鼠组织中的做法是由身体特伦托大学的动物保护（OPBA）（意大利）批准，协议没有。 04-2015，每art.31因为法令没有。二千〇一十四分之二十六。所有小鼠均保持在特伦托大学中心综合生物学（CIBIO），意大利的模式生物设施。 注意:为避免样品的任何RNA的降解，用准备DEPC处理过的水中所有的缓冲区最大限度地减少RNase污染。 1.全脑多核​​糖体的制备<ol><li>收集脑组织（15分钟） <ol><li>安乐死用CO 2窒息野生型小鼠品系C57BL / 6 5分钟36。小心从头骨36解剖大脑出来，所述组织置于1.5ml试管，并立即将其放置在液氮中。储存在-80℃直到使用。 </li></ol></li><li>裂解物的制备（30分钟） <ol><li>使用粉碎全脑组织液氮下用研钵和研杵。 </li><li>转移约25mg粉末的冷的离心管，并立即通过快速上下吹打25倍（以避免粉碎组织的解冻）添加0.8毫升裂解缓冲液（ 见表1）的和破坏细胞。 </li><li>离心管以12,000rpm xg离心1分钟，在4℃以沉淀细胞碎片。 </li><li>将上清转移到新的微量离心管中，并保持该管在冰上15分钟。 </li><li>离心管以12,000rpm xg离心5分钟，在4℃以沉淀细胞核和线粒体。 </li><li>将上清转移到新的离心管中。 </li><li>储存在-80℃的上清液为最多6个月或立即使用它。 </li></ol></li><li>蔗糖梯度制备和离心（2小时，30分钟） <ol><li>用无RNase水（焦碳酸二乙酯处理的水（用DEPC水）或商业）洗涤超速离心管广泛的ð3％H 2 O 2 / DEPC H 2 O解决方案。 </li><li>置于冰上的管中并在每个管（参见，蔗糖溶液表1）的底部添加5.5冷的50％的蔗糖溶液。小心逐滴添加15％的蔗糖溶液滴，力求贴近相间，以便保持一个尖锐相间，直到管被完全填充。当管被完全填充，用橡胶塞将其关闭，以避免气泡形成。 </li><li>在寒冷的房间轻轻水平放下管子，并保持在此位置2小时。在此时间后，慢慢伸直管放回竖直位置，并把它们在冰上。梯度现在准备好被使用。可替代地，使用常规的梯度前制备15-50％的蔗糖梯度。 </li><li>在寒冷的房间从渐变的顶部取出仔细1.0毫升和覆盖一滴蔗糖下降与胞浆裂解液（即 STE得到的上清液p 1.2）。 </li><li>小心地将管放入水平转头的水桶。使用超速离心机离心100分钟梯度180,000 xg离心在4℃下。 </li><li>离心分离后，离开管子自己水桶20分钟，4℃，让梯度稳定。 </li></ol></li><li>蔗糖梯度分馏（2小时） <ol><li>小心地从超速离心机转子删除一个超速离心管并将其安装密度梯度分馏系统的集热设备。收集1毫升级分监测吸光度在用UV / VIS检测器（参见图1上图 ）260纳米。置于冰上收集到的馏分。 </li><li>制备30-40微升感兴趣的级分的等分试样，在-80℃，直到使用它们存储之前保持他们在冰上。不使用行多于两个冻融循环等份或蔗糖级分（ 见图1中的下图 ）。 </li></ol> &lt;/ LI&gt; </ol> 2.样品制备原子力显微镜（3小时） <ol><li>使用磁带，剥去云母片。 </li><li>用DEPC水冲洗云母片3-4次，并把它变成一个小的培养皿中。然后用空气干燥的表面上。 </li><li>覆盖用200μl1mM的硫酸镍4的云母并在室温下孵育3分钟。 </li><li>除去镍溶液中，然后用空气干燥的表面上。通过将培养皿在冰上云母进行在4℃以后所有的步骤。 </li><li>在冰上解冻1.4.2获得等份，轻轻用在云母滴添加所有样品下降。使用100-200微升尖，传播云母的整个表面上的样品。在冰上孵育样品3分钟。 </li><li>覆盖一滴云母片下降用200μl冷缓冲区的AFM（ 见表1）和孵育在冰上1小时。 </li><li>成像液<ol><li>仔细缓冲区AFM捞出洗净云母片3-4时s的200微升冷缓冲区AFM以去除多余的蔗糖。然后洗云母片用冷水洗涤液的3倍（ 见表1），同时保留的解决方案的一些微升覆盖的云母表面。 </li><li>进入3点（图像采集）。 </li></ol></li><li>成像在空气中<ol><li>小心地取出缓冲区AFM和用200μl冷缓冲区AFM洗云母3-4次以除去过量的蔗糖。然后，洗云母用冷洗涤溶液3倍（ 见表1），并使用纸排出多余的水。 </li><li>保留样品的化学罩与培养皿部分开放的顶部下干燥。 2小时后，收于RT培养皿和存储。测量样品后2-3小时，因为它们是稳定多年。 </li></ol></li></ol> 3.图像采集（热稳定后，每幅影像15分钟） 注意:固定在云母多核糖体可在空气中或在液体中进行成像，使用AC模式。 <ol><li>附加云母使用双面胶带的样本保持器。 </li><li>插入以下制造商的指示在AFM中阶段的样品架。当显像液中，如果可能的话，尝试在温度低于25℃以保持样品，以增加在时间上多核糖体的稳定性。 </li><li>选择适用于交流成像的悬臂，它安装在刀片保持器下面的制造商的指示。在这里，使用悬臂用武力2-20牛顿/米的空中成像和液体成像约为0.1牛顿/米之间不变。 </li><li>调整在悬臂激光点和零的象限检测器的信号。 </li><li>选择一个适当的驱动频率并且具有10-20纳米的振幅驱动该悬臂。 </li><li>接近样品直至前端的表面接合。 </li><li>选择2×2微米的扫描区域，至少获得512×512像素的图像（像素宽度&lt;4海里），选择一个实时背景减除模式和20-25纳米的Z比例。 </li><li>检查ŧ他图像寻找在范围25-30纳米的空气和25时30纳米液体和宽度取得时特征10和15毫微米之间的高度圆形物体的存在。调整设定值和反馈参数，直到尖锐物体可视化。背景应显示良好的样品中的相对平坦的，具有一定的2-4纳米的高度的物体（参见图2A和B）。 </li><li>如果图像看起来不错（如点3.8所示），收购在不同的采样区域的几个（至少十）的2x2微米扫描。 </li><li> （可选）。如果有必要，获取选定多聚核糖体的高分辨率的图像（ 见图2C）。 </li><li>应用软件更正（平面减法和行由行校正算法）来纠正AFM图像去除任意倾斜和漂移的影响。 </li></ol> 4.数据分析（每幅影像30分钟） <ol><li> preferent:在ImageJ的（应用比例因子可选）导出图像ially使用无损压缩格式， 例如，在TIFF格式（ 见图3A）。 </li><li>使用ImageJ的宏工具集RiboPick.ijm（在ImageJ的宏/工具集子目录被复制）来计算在多核糖体的核糖体（参见图3B），并计算样品的统计特性（ 见图3C）。 <ol><li>启动加载使用&lt;读取图像&gt;工具，初始化程序的图像。 </li><li>挑选的标准ImageJ的&lt;点选择&gt;工具（Shift +左键点击允许核糖体的多选）核糖体中心。马克与&lt;马克核糖体&gt;工具选择的核糖体。核糖体坐标将出现在自定义文本窗口。 </li><li>添加更多的核糖体为相同的多核糖体重复在点4.2.2所示的过程。 </li><li>删除使用&lt;撤消最后挑&gt;工具（开始从最后添加的核糖体取出到第一个错误标记的核糖体212;在当前的多核糖体只）。 </li><li>当完成多聚核糖体，使用&lt;新建多聚核糖体&gt;工具。作为多核糖号添加作为覆盖到图像，文本窗口被更新。 </li><li>使用&lt;减结果，并靠近&gt;写核糖体坐标文件（用于图像的默认单位），并且总结了拾取核糖体和多核糖PNG图像。原始图像被关闭没有被保存。 </li></ol></li></ol>

<ol>
	<li>Buttgereit, F., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hierarchy+of+ATP-consuming+processes+in+mammalian+cells.">A hierarchy of ATP-consuming processes in mammalian cells.</a> Biochem J. 312, 163-167 (1995).</li><li>Russell, J. B., Cook, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetics+of+bacterial+growth:+balance+of+anabolic+and+catabolic+reactions.">Energetics of bacterial growth: balance of anabolic and catabolic reactions.</a> Microbiol Rev. 59 (1), 48-62 (1995).</li><li>Vogel, 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=Sequence+signatures+and+mRNA+concentration+can+explain+two-thirds+of+protein+abundance+variation+in+a+human+cell+line.">Sequence signatures and mRNA concentration can explain two-thirds of protein abundance variation in a human cell line.</a> Mol Syst Biol. 6, 400 (2010).</li><li>Schwanh&#228;usser, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473, 337-342 (2011).</li><li>Tebaldi, 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=Widespread+uncoupling+between+transcriptome+and+translatome+variations+after+a+stimulus+in+mammalian+cells.">Widespread uncoupling between transcriptome and translatome variations after a stimulus in mammalian cells.</a> BMC Genomics. 13, 220 (2012).</li><li>Kondrashov, N., 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=Ribosome-mediated+specificity+in+Hox+mRNA+translation+and+vertebrate+tissue+patterning.">Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning.</a> Cell. 145 (3), 383-397 (2011).</li><li>Topisirovic, I., Svitkin, Y. V., Sonenberg, N., Shatkin, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap+and+cap-binding+proteins+in+the+control+of+gene+expression.">Cap and cap-binding proteins in the control of gene expression.</a> Wiley Interdiscip Rev RNA. 2, 277-298 (2011).</li><li>Darnell, J. 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=FMRP+stalls+ribosomal+translocation+on+mRNAs+linked+to+synaptic+function+and+autism.">FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism.</a> Cell. 146 (2), 247-261 (2011).</li><li>Pircher, 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=An+mRNA-derived+noncoding+RNA+targets+and+regulates+the+ribosome.">An mRNA-derived noncoding RNA targets and regulates the ribosome.</a> Mol Cell. 54 (1), 147-155 (2014).</li><li>Simms, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+active+role+for+the+ribosome+in+determining+the+fate+of+oxidized+mRNA.">An active role for the ribosome in determining the fate of oxidized mRNA.</a> Cell Rep. 9 (4), 1256-1264 (2014).</li><li>Kraushar, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+defined+neocortical+translation+and+polysome+assembly+are+determined+by+the+RNA-binding+protein+Hu+antigen+R.">Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R.</a> Proc Natl Acad Sci U S A. 111 (36), E3815-E3824 (2014).</li><li>van Heesch, 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=Extensive+localization+of+long+noncoding+RNAs+to+the+cytosol+and+mono-+and+polyribosomal+complexes.">Extensive localization of long noncoding RNAs to the cytosol and mono- and polyribosomal complexes.</a> Genome Biol. 15 (1), R6 (2014).</li><li>Preissler, 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=Not4-dependent+translational+repression+is+important+for+cellular+protein+homeostasis+in+yeast.">Not4-dependent translational repression is important for cellular protein homeostasis in yeast.</a> EMBO J. 34 (14), 1905-1924 (2015).</li><li>Palade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+particulate+component+of+the+cytoplasm.">A small particulate component of the cytoplasm.</a> J Biophys Biochem Cytol. 1 (1), 59-68 (1955).</li><li>McQuillen, K., Roberts, R. B., Britten, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+nascent+protein+by+ribosomes+in+Escherichia+coli.">Synthesis of nascent protein by ribosomes in Escherichia coli.</a> Proc Natl Acad Sci U S A. 45 (9), 1437-1447 (1959).</li><li>Wettstein, F. O., Staehelin, T., Noll, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+aggregate+engaged+in+protein+synthesis:+characterization+of+the+ergosome.">Ribosomal aggregate engaged in protein synthesis: characterization of the ergosome.</a> Nature. 2 (197), 430-435 (1963).</li><li>Dimitriadis, E. K., 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=Tetrameric+organization+of+vertebrate+centromeric+nucleosomes.">Tetrameric organization of vertebrate centromeric nucleosomes.</a> Proc Natl Acad Sci U S A. 107 (47), 20317-20322 (2010).</li><li>Allen, M. 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=Atomic+force+microscope+measurements+of+nucleosome+cores+assembled+along+defined+DNA+sequences.">Atomic force microscope measurements of nucleosome cores assembled along defined DNA sequences.</a> Biochemistry. 32 (33), 8390-8396 (1993).</li><li>Ban, N., Nissen, P., Hansen, J., Moore, P., Steitz, 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+complete+atomic+structure+of+the+large+ribosomal+subunit+at+2.4+&#197;+resolution.">The complete atomic structure of the large ribosomal subunit at 2.4 &#197; resolution.</a> Science. 289 (5481), 905-920 (2000).</li><li>Schluenzen, 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=Structure+of+functionally+activated+small+ribosomal+subunit+at+3.3+&#197;+resolution.">Structure of functionally activated small ribosomal subunit at 3.3 &#197; resolution.</a> Cell. 102 (5), 615-623 (2000).</li><li>Ben-Shem, 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+structure+of+the+eukaryotic+ribosome+at+3.0+&#197;+resolution.">The structure of the eukaryotic ribosome at 3.0 &#197; resolution.</a> Science. 334 (6062), 1524-1529 (2011).</li><li>Anger, A. 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=Structures+of+the+human+and+Drosophila+80S+ribosome.">Structures of the human and Drosophila 80S ribosome.</a> Nature. 497 (7447), 80-85 (2013).</li><li>Brandt, 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=The+native+3D+organization+of+bacterial+polysomes.">The native 3D organization of bacterial polysomes.</a> Cell. 136, 261-271 (2009).</li><li>Myasnikov, A. 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=The+molecular+structure+of+the+left-handed+supra-molecular+helix+of+eukaryotic+polyribosomes.">The molecular structure of the left-handed supra-molecular helix of eukaryotic polyribosomes.</a> Nat. Commun. 5, 5294 (2014).</li><li>Afonina, Z. A., Myasnikov, A. G., Shirokov, V. A., Klaholz, B. P., Spirin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+circular+polyribosomes+on+eukaryotic+mRNA+without+cap-structure+and+poly(A)-tail:+a+cryo+electron+tomography+study.">Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron tomography study.</a> Nucleic Acids Res. 42 (14), 9461-9469 (2014).</li><li>Afonina, Z. A., Myasnikov, A. G., Shirokov, V. A., Klaholz, B. P., Spirin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation+transitions+of+eukaryotic+polyribosomes+during+multi-round+translation.">Conformation transitions of eukaryotic polyribosomes during multi-round translation.</a> Nucleic Acids Res. 43 (1), 618-628 (2015).</li><li>Viero, 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=Three+distinct+ribosome+assemblies+modulated+by+translation+are+the+building+blocks+of+polysomes.">Three distinct ribosome assemblies modulated by translation are the building blocks of polysomes.</a> J Cell Biol. 208 (5), 581-596 (2015).</li><li>Brandt, F., Carlson, L. -. A., Hartl, F. U., Baumeister, W., Gr&#252;newald, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+three-dimensional+organization+of+polyribosomes+in+intact+human+cells.">The three-dimensional organization of polyribosomes in intact human cells.</a> Mol. Cell. 39, 560-569 (2010).</li><li>Wu, X., Liu, W. Y., Xu, L., Li, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+of+ribosomes+and+initiation+complexes+from+rat+liver+as+revealed+by+atomic+force+microscopy.">Topography of ribosomes and initiation complexes from rat liver as revealed by atomic force microscopy.</a> Biol Chem. 378 (5), 363-372 (1997).</li><li>Yoshida, T., Wakiyama, M., Yazaki, K., Miura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+electron+and+atomic+force+microscopic+observation+of+polysomes+on+carbon-coated+grids+prepared+by+surface+spreading.">Transmission electron and atomic force microscopic observation of polysomes on carbon-coated grids prepared by surface spreading.</a> J. Electron Microsc (Tokyo). 46 (6), 503-506 (1997).</li><li>Mikamo, E. 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=Native+polysomes+of+Saccharomyces+cerevisiae+in+liquid+solution+observed+by+atomic+force+microscopy.">Native polysomes of Saccharomyces cerevisiae in liquid solution observed by atomic force microscopy.</a> J. Struct. Biol. 151, 106-110 (2005).</li><li>Mikamo-Satoh, E. 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=Profiling+of+gene-dependent+translational+progress+in+cellfree+protein+synthesis+by+real-space+imaging.">Profiling of gene-dependent translational progress in cellfree protein synthesis by real-space imaging.</a> Anal. Biochem. 394, 275-280 (2009).</li><li>Bernab&#242;, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+translational+control+in+non-model+stressed+organisms+by+polysomal+profiling.">Studying translational control in non-model stressed organisms by polysomal profiling.</a> J. Insect Physiol. 76, 30-35 (2015).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IH+Image+to+ImageJ:+25+years+of+image+analysis.">IH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Lauria, 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=RiboAbacus:+a+model+trained+on+polyribosome+images+predicts+ribosome+density+and+translational+efficiency+from+mammalian+transcriptomes.">RiboAbacus: a model trained on polyribosome images predicts ribosome density and translational efficiency from mammalian transcriptomes.</a> Nucleic Acids Res. , (2015).</li><li>Al Omran, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+the+Ependymal+Cilia+in+the+Lateral+Ventricles+of+the+Mouse+Brain.">Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain.</a> J Vis Exp. (100), e52853 (2015).</li><li>Warner, J. R., Rich, A., Hall, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscope+studies+of+ribosomal+clusters+synthesizing+hemoglobin.">Electron microscope studies of ribosomal clusters synthesizing hemoglobin.</a> Science. 138, 1399-1403 (1962).</li><li>Yazaki, K., Yoshida, T., Wakiyama, M., Miura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysomes+of+eukaryotic+cells+observed+by+electron+microscopy.">Polysomes of eukaryotic cells observed by electron microscopy.</a> J. Electron Microsc (Tokyo). 49, 663-668 (2000).</li></ol>

The authors have no competing financial interests in this paper and nothing to disclose.

由于DNA的结构被证明是至关重要来形容转录和染色质组织提高了我们的基因表达的转录调控认识的过程，它分析多聚核糖体的组织和结构，提高真实的理解是必不可少的翻译及其调控。 所描述的协议中，轻轻地固定在一个平面上的多核糖体的最佳密度，适于原子力显微镜成像被获得。使用具有方便制备的样品这些条件下，每小时近50多核糖体可以被收购，准备作进一步的分析和表征。此外，存储在RT干燥的样品已经被证明是适合于后一年以上成像。 为了成功地获得我们的协议核糖体的图像，还有一些关键的步骤:使用已立即正确dissectio后快速冷冻组织n和储存在-80℃下，以尽量减少RNA降解;使用裂解缓冲液含有环己酰亚胺和RNase抑制剂，以避免由mRNA核糖体解离;云母样品沉积过程中进行冰上孵育所有;并准备对空气原子力显微镜成像的样本时，要广泛地洗上与放线菌酮存在缓冲区和RNase自由水云母样品。最后一点是特别重要的。如果图像似乎包含光滑的表面大约在合适的高度，但100-1000纳米宽，这是一个不好洗样品的明确指示（通常核糖体/多聚核糖体仍然可以观察到淡淡的，嵌入的对象）。解决此问题的方法，是将重复洗涤过程，但是这通常不减轻对已经干燥样品的问题。在这种情况下，这是值得与新的等分试样再次开始。 由于原子力显微镜的分辨率，这种方法用于研究核糖无法解决STRUC在多聚核糖体核糖体接口王兴仁细节。事实上，AFM是冷冻电镜，代表有效和有力的技术，获取和分析数千个多核糖体，并更好地了解核糖整体核糖体组织互补和简单的方法。重要的是，该协议具有识别与RNA 27兼容丝的独特优势。这个非常相同的方法可用于从任何生物组织或细胞系获得的任何多核糖体样品。更重要的是它也可以成功地使用，以获得使用体外翻译系统的特定转录信息，最近由劳里亚和同事35证实。这种方法将铺平道路，几个实验，以获得多聚核糖体形成或不同的细胞或组织条件下，在多聚核糖体组织的变化的动力学的理解。

蛋白质的合成是在细胞1,2-最耗能的过程。因此，并不奇怪，蛋白质丰度主要控制在翻译，而不是转录水平3,4,5。多核糖体是转换的mRNA信息为功能蛋白质读数基波高分子组分。多聚核糖体是迄今公认的大分子复合物，几个平移控制收敛6-13。尽管数百对核糖体结构14-16研究，详细的分子见解翻译的动态和多聚核糖体的拓扑结构遇到了兴趣有限。因此，原生多聚核糖核蛋白复杂的组织结构及其在翻译的潜在影响仍然是相当模糊的问题。多聚核糖体可能隐藏还是未知数有序和职能部门，潜在的镜像什么核小体和后生CONTROLS已经代表了转录领域。事实上，这样一个有趣的假设的调查需要更多的研究和新的技术途径。在此行中，结构的技术和原子力显微镜可以卓有成效协作解开的新机制，用于控制基因表达的，类似于什么是为核小体17,18来实现的。 由于核糖体14-16的发现，它的结构已被广泛表征在原核生物19,20，酵母21以及最近在人22，在蛋白质合成的基础上提供的机制的分子描述。多核糖体进行初始确认在内质网的膜，从而形成典型的2D几何机构14。正如前面提到的，多聚核糖体装配一直没有固定利率，作为核糖体结构的对象。在过去，多聚核糖体已基本上由TR研究ansmission EM为基础的技术。只是在最近，冷冻电镜技术，使纯化多聚核糖体的三维重建体外翻译系统23-26，人类细胞裂解液27或28的细胞。这些技术提供了有关在多核糖体23，26-28和在小麦胚芽多核糖体24相邻的核糖体的接触表面进行初步分子描述的核糖体的核糖体组织更精致的信息。因此，冷冻电镜断层扫描允许与分子细节核糖体的核糖体相互作用的公开内容，但它是由大量的后处理的负担，并重建分析需要大量的计算资源进行数据处理。此外，为了得到核糖体的核糖体相互作用的分子细节，核糖体的高度解决地图是必需的，并且这种参考核糖体仅可用于几种。重要的是，冷冻电镜断层扫描是无法检测无RNA。 Theref矿石，需要新的技术来全面了解机器翻译的组织。 旁冷冻电镜，原子力显微镜也已在低等真核生物29-33和人类27用作用于多核糖体的直接成像的有用工具。相比EM，AFM不需要样品固定或标签。此外，测量结果可以在接近生理条件，并具有明确确定两个核糖体和裸RNA链27上的唯一的可能性来进行。能够相对快速地执行单个多核糖体的成像，在纳米分辨率的小的后处理工作获得成千上万的图像相比，广泛和重后处理和重建分析通过冷冻电镜显微术所需的。因此，AFM数据处理和分析，也不需要昂贵的工作站和高计算能力。因此，该技术收集多核糖体的形状信息，形态特征（如高度，长度和宽度），核糖体密度的无RNA的存在和每多核糖体27的核糖体比冷冻电镜更高的吞吐量的数量。在这样的方式，AFM代表了一个强大的和互补的方式来EM技术来描绘核糖27。 在这里，我们提出了一个完整的管道从净化到AFM应用到图像和分析小鼠大脑多聚核糖体的数据分析。该协议的重点是净化问题上用于原子力显微镜成像云母基材多聚核糖体的精确沉积。除了 ​​可与通过AFM社区使用的通用软件容易地进行常规颗粒分析，一个ImageJ的34插件 ​​，称为RiboPick，提出用于计数每多核糖体27，35的核糖体的数目。

<table>
	<tbody>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>01810</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DNAseI</td>
			<td>Thermo Scientific</td>
			<td>89836</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>RiboLock RNAse Inhibitor</td>
			<td>Life technologies</td>
			<td>EO-0381</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DEPC</td>
			<td>Sigma</td>
			<td>40718</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Sigma</td>
			<td>T8532</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>43815</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Sodium Deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S5016</td>
			<td>Sucrose gradient preparation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima LE-80K</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Rotor</td>
			<td>Beckman Coulter</td>
			<td>SW 41 Ti</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Polyallomer tube</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Density Gradient Fractionation System&#160;</td>
			<td>Teledyne Isco</td>
			<td>67-9000-176</td>
			<td>Sucrose gradient fractionation</td>
		</tr>
		<tr>
			<td>AFM</td>
			<td>Asylum Research</td>
			<td>Cypher</td>
			<td>Polysome visualization</td>
		</tr>
	</tbody>
</table>

peering at brain polysomes with atomic force microscopy

平移机械， 即核糖体或多聚核糖体是细胞中最大和最复杂的细胞质机械之一。多核糖体，核糖体，mRNA的，几种蛋白质和非编码RNA形成的，代表集成，其中平移控制发生平台。然而，尽管核糖体已被广泛研究，多核糖的组织仍然缺乏全面的了解。因而很多努力，需要以阐明多核糖体组织和可嵌入的翻译控制的任何新颖的机制。原子力显微镜（AFM）是一种类型的扫描探针显微镜的，允许在纳米级的分辨率采集3D图像。相比电子显微镜（EM）技术，原子力显微镜的主要优点之一是，它能够获得几千无论在空气中和溶液的图像，使该样本被接近生理条件下维持，而无需任何染色和固定的亲cedures。这里，对从鼠脑和它们对云母基板沉积多核糖的准确纯化的详细协议进行说明。该协议允许在空气和液体多核糖成像AFM和它们重建为三维物体。为了补充低温电子显微镜（冷冻电镜），该方法可以方便地用于系统的分析多核糖体和研究他们的组织。

整个小鼠大脑的蔗糖梯度多聚核糖分析 有可能从由多核糖体分析，其中根据它们的重量及尺寸分离大分子细胞裂解物纯化核糖。与多核糖体分析，从培养的细胞或组织，如在本实施例获得的细胞质裂解物，装载到一个线性蔗糖梯度和加工通过超速离心根据其沉降系数以分开40S和60S亚基，80S核糖体和多核糖体游离的RNA（ 图1上图）。在254nm样品和馏分收集的吸光度使在核糖体或核糖具有不同数目每转录的核糖体的富集的蔗糖级分的分离。的级分可以被收集，等分为原子力显微镜成像并储存在-80℃（ 图1下面板）。 从AFM在空气中观察到小鼠大脑本地多聚核糖体露出紧核糖体的相互作用 图像采集是利用AC模式（也称为轻敲模式）执行的。这种方式特别适合于成像软样品（如多核糖体），因为针尖 - 样品的相互作用和相应的剪切力大大减小。以这种方式两个针尖与样品被保留。原子力显微镜成像允许以高分辨率采集的数据，与依赖于各种因素，如测量条件，那种尖端，和样品的性质精确值。在本文所述的条件为约4-6纳米的横向分辨率和0.1-0.2纳米的垂直分辨率是可以实现的。云母蔗糖等分的沉积之后，AFM提供单个多核糖的描述那显示为紧凑的核糖体（ 图2A，C和C）的簇。通过横截面分析（ 图2D），核糖体峰的高度是根据什么空气干燥27后在多核糖体人类核糖体先前观察被约14纳米。的中心到由线轮廓确定的核糖体的中心的距离是23纳米，这与核糖体的溶液中的37，38的尺寸一致。 每从单一馏分多核糖的核糖体的数目表示单分散分布 图3A报告了获得来自蔗糖梯度的一小部分被吸收在云母上的样品获得的典型的AFM图像。插图示出了单个多聚核糖体的数字变焦，用适于ribos的识别的倍率omes在多核糖体。面板B显示了核糖体识别与RiboPick宏后相同的图像。红色圆圈（见插图）标记核糖体，和一个渐进号码（青色）是用来帮助核糖体的关联与特定多核糖坐标。每多核糖的核糖体的数目的频率分布分析其组分＃10（参见图1，下图中，对应于中等分子量多核糖体（MMW）），这清楚地显示了一个单峰（ 图C，灰色条）。实验分布装有该混合物在5.8核糖体/多核糖体中心，以1.3的核糖体/多核糖体的标准偏差的高斯曲线（黑线）。 <img alt="图1" src="/files/ftp_upload/53851/53851fig1.jpg" /> 对于整个鼠脑的蔗糖梯度沉降图1.代表吸收轮廓。（高p从P27小鼠大脑获得头面板）一个多核糖体细胞质裂解和到线性蔗糖梯度（15-50％蔗糖加载[W / V]）。这是可能观察到相应的吸光度RiboNucleoParticles的254纳米（RNP）清峰，核糖体亚基（40S和60），核糖体（80S）和多核糖体。以避免重复冷冻和解冻的云母的AFM沉积之前样 ​​品的周期，它是方便的分成等份都核糖体级分和多核糖体级分（下图）与不同数量的每转录的核糖体（ 即，低，中，和高分子模量多核糖体（LMW，MMW和HMW，分别））。等分试样可以储存在-80℃，只要两年。 <img alt="图2" src="/files/ftp_upload/53851/53851fig2.jpg" /> 图2.大脑多核糖体图像通过轻敲模式原子力显微镜（A）MMW大脑聚AFM图像示例萨姆云母吸收后采用线性灰色刻度。 0-0.5毫微米灰色的背景，0.5-2纳米，2-10纳米黄色为核糖体:（B）的（A）中所示的相同的图像是使用不同的Z范围的颜色比例描绘。在这种情况下，更好的视觉对比度为低和高的Z-Range对象获得的。一个典型的毫米波多核糖与横截面轮廓（虚线白线）的（C）的放大倍数。通过横截面轮廓（C）中定义的两个核糖体（D）的高度轮廓示出的14纳米的核糖体的高度。该数值与先前在AFM观察到在核糖27人的核糖体兼容。 <img alt="图3" src="/files/ftp_upload/53851/53851fig3.jpg" /> 图3.计数每在毫米波大脑多核糖多核糖的核糖体的数目的实施例。该repre（A）的AFM图像货物内的ImageJ的宏，RiboPick的输入端。 （B）的各核糖体（红色圆圈）的位置手动拾取每多核糖每个核糖体，RiboPick标记在图像中之后。 （C）将所得到的每多核糖的核糖体的数目可以绘制为可装配有高斯曲线分布。在本实施例考虑多核糖的数目是164，从9个独立的图像获得。与高斯曲线拟合返回多核糖在毫米波馏分人口的平均值（＃核糖体/多核糖体5.8±1.3，R 2 = 0.99）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 缓冲 </td><td> 组成 </td><td> 应用 </td></tr><tr><td>裂解液</td><td>的10mM的Tris-HCl，pH值7.5 </td><td>裂解物的制备（1.1） </td></tr><tr><td></td><td> 10毫米氯化钠</td><td></td></tr><tr><td></td><td> 10毫米氯化镁2 </td><td></td></tr><tr><td></td><td> 1％的Triton-X100 </td><td></td></tr><tr><td></td><td> 1％的钠脱氧胆酸</td><td></td></tr><tr><td></td><td> 0.4 U / ml的RNA酶抑制剂</td><td></td></tr><tr><td></td><td> 1毫摩尔DTT </td><td></td></tr><tr><td></td><td> 0.2mg / ml的放线菌酮</td><td></td></tr><tr><td></td><td> 5 U / ml的DNA酶I </td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td> 50％的蔗糖溶液</td><td> 50％（重量/体积）蔗糖</td><td>蔗糖梯度制备（1.2） </td></tr><tr><td></td><td> 100毫米氯化钠</td><td></td></tr><tr><td></td><td> 10毫米氯化镁2 </td><td></td></tr><tr><td></td><td>的10mM Tris /盐酸pH为7.5 </td><td></td></tr><tr><td></td><td></td><td></td></tr> &lt;TR&gt; <td> 15％的蔗糖溶液</td><td> 15％（重量/体积）蔗糖</td><td>蔗糖梯度制备（1.2） </td></tr><tr><td></td><td> 100毫米氯化钠</td><td></td></tr><tr><td></td><td> 10毫米氯化镁2 </td><td></td></tr><tr><td></td><td>的10mM Tris /盐酸pH为7.5 </td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td>镍溶液</td><td> 1 mM的硫酸镍4 </td><td>原子力显微镜样品制备（2） </td></tr><tr><td></td><td></td><td></td></tr><tr><td>缓冲区AFM </td><td> 100毫米氯化钠</td><td>原子力显微镜样品制备（2） </td></tr><tr><td></td><td> 10毫米氯化镁2 </td><td></td></tr><tr><td></td><td> 100μg/ ml的环己酰亚胺</td><td></td></tr><tr><td></td><td>的10mM Hepes </td><td></td></tr><tr><td></td><td colspan="2"> 3％（重量/体积）蔗糖，pH值= 7.4 </td></tr><tr><td></td><td></td><td></td></tr><tr><td>洗液</td><td> DEPC水</td><td>原子力显微镜样品制备（2） </td></tr><tr><td></td><td> 100μg/ ml的环己酰亚胺</td><td></td></tr></tbody></table> 表1.缓冲器。

Watch this Scientific Journal Video about Peering at Brain Polysomes with Atomic Force Microscopy at JoVE.com

Peering at Brain Polysomes with Atomic Force Microscopy

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

在脑核糖凝视与原子力显微镜

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

peering-at-brain-polysomes-with-atomic-force-microscopy

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.3K 次观看.  Fondazione Bruno Kessler. While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid. 

视频: Peering at Brain Polysomes with Atomic Force Microscopy

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

依赖国家对TMS的:一看动机幻视行为

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

科研

神经科学

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

聚焦离子束铣削和脑组织的扫描电子显微镜

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

实验脱髓鞘和小鼠脊髓髓鞘再生由溶血卵磷脂的焦点注射

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

慢性压迫性损伤小鼠步态的自动分析

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.