The authors have no acknowledgements.

注：所有涉及使用动物的程序进行，根据当地的法规。我们的工作是经悉尼当地卫生区动物福利委员会。允许程序项目许可文档中描述，并按照指引，以确保动物在任何时候都幸福。确保遵守有关的地方进行工作的国家动物实验的立法。 为准备EM固色剂1.协议<ol><li> 100ml中的固定剂的，通过加入2多聚甲醛粉末至25ml在一个锥形瓶中蒸馏水，加​​热至65ºC，用磁力搅拌器不断搅拌制备的多聚甲醛。 注：戊二醛，多聚甲醛和二甲胂酸钠缓冲都是有害物质，必须始终在通风柜处理。 </li><li>热，在65ºC为2分钟，然后允许冷却。如果需要的话，加氢氧化钠溶液dropwISE边搅拌至完全明确的解决方案。 注：氢氧化钠是危险的，小心处理。 </li><li>加入10毫升EM级库存戊二醛，将50ml的0.2M二甲胂酸钠缓冲液和2克蔗糖和搅拌用磁力搅拌器。添加1.0M的氯化钙2 2毫升</li><li>调节至pH 7.4之前使溶液至100毫升蒸馏水。最终固定剂中含有2.5％戊二醛，2％多聚甲醛，2毫摩尔氯化钙 ，2％蔗糖的0.1M二甲胂酸盐缓冲液中。确保总渗透压为440 mOsmol /升。使用内准备24小时。 </li></ol>肝2.灌注固定<ol><li>温馨灌注缓冲和定于35 - 37ºC。放血的缓冲液和固定液的正确的温度有助于确保组织的完整性。 </li><li>麻醉的动物用单次腹膜内注射50毫克/千克氯胺酮和5mg / kg的甲苯噻嗪。 </li><li>一旦动物是下深度麻醉，用后肢撤退和尾掐评估，A Y切口，钝性结束剪刀在动物的腹部，露出肝脏和门静脉。 注：确保设备清洁，避免标本微生物和一般的碎片，这将很容易看到SEM污染。 </li><li>扎两个非常松散的缝线门静脉周围1近侧至肝脏，在其他更远侧到肝脏。 </li><li>导管插入门静脉与适当大小的IV套管（18克的老鼠，22克的老鼠）.Tighten缝线固定套管。 </li><li>开始灌注用磷酸盐缓冲盐水加热到37ºC，并在10厘米的H 2 O的压力。 5至20毫升的PBS通常需要抽血肝脏。 注意：不要让气泡进入通过连接到他们的文物引起继发栓塞和压力套管管肝脏;损害肝细胞，SInusoids和LSEC窗孔发生，如果灌注压力过高。 </li><li>切断的腹部和胸部腔静脉，以允许缓冲从肝脏自由退出。这可以防止高背压损坏LSECs。 </li><li>一旦肝脏是免费的血液，用EM固定液更换PBS和灌注约5分钟，直到肝脏硬化，很苍白。 </li><li>停止灌注和削减固定肝脏中转化为1 - 2 mm 3的街区用锋利的手术刀。 </li><li>后修复组织EM固定液24 - 72小时，在4ºC。 注：在一般情况下，underfixation导致比overfixation更多的文物。 </li><li>更改固定液以下后修复期为存储0.1M的二甲胂酸钠缓冲液，4ºC。 注：存储在这种方式标本将被保存为一个显著一段时间（最长12个月），但及时的辅助固定和审查建议以获得最佳效果。 </li></ol> 3.针Perfusion 注：针固定是灌注固定的一个变种，涉及到注入固定液直接进入肝脏活检组织。这样做的目的是为固定剂可以通过血管，以抽血它们和公开所有的组织块以固定剂冲洗。必须小心，以保持注射压力低，以避免气压伤18,19。 <ol><li>取出一小片来自动物或受肝脏和冲洗的生理盐水，以清除血液和碎片。 </li><li>绘制生理盐水注射进装有细针头（通常胰岛素注射器）。 </li><li>注入生理盐水非常缓慢进入肝脏的血，直到被刷新出来的组织。多次注射可能是必需的。 </li><li>绘制对EM固定液到装配有细规针（通常是胰岛素注射器）另一个注射器。 </li><li>注入固定液非常缓慢进入肝脏，直到它变得坚硬和棕褐色。多次喷射之间可以需要。 </li><li>用锋利的手术刀2毫米3个街区-切肝固定为1。在孵育固定液EM为24 - 72小时4℃。开始二次固定前需清洗的0.1M的二甲胂酸钠缓冲液的3倍。 </li></ol> 4.准备扫描电子显微镜<ol><li>洗涤样品在0.1M二甲胂酸钠缓冲液3次。要修复脂类，交固定在0.1M二甲胂酸钠缓冲液将样品在2％四氧化锇进行2小时。 注：完全清洗是非常重要的，因为戊二醛和四氧化锇交叉反应，生成SEM文物。 </li><li>冲洗样品中增加的乙醇浓度开始脱水。第一冲洗在50％乙醇5分钟;然后3次，每次5分钟，用70％的乙醇;冲洗3次，每次5分钟，用90％的乙醇;冲洗2次，每次5分钟，在100％乙醇;最后漂洗2次，在100％乙醇（分子筛）10分钟。 </li><li>从样本中删除所有乙醇<html>和六甲基二带（HMDS）代替通风柜，离开10分钟。从组织样品中除去HMDS，并允许蒸发。 注：HMDS是吸湿性强冷的时候，因此允许使用的最终脱水行程之前达到室温。 </li><li>可选，立即装入样品或将它们放入干燥器中，直到准备安装的扫描电镜。 </li></ol> 5.安装注：正确的样品安装将最大限度的提供SEM分析下明确界定血窦的数量。 <ol><li>标记金属SEM存根的基极与应用双面导电碳胶带的存根的顶部。 </li><li>使用解剖显微镜来识别与用于随后的SEM最好血窦表面可视化的样品。向上放置该表面上的截线的碳胶带表面。 </li><li>坚持标本坚决存根，样品现在已经准备好了溅射镀膜。 </li></ol>ove_title“&gt; 6。涂料注：涂层在溅射镀膜机的理由有导电金属（金或铂）的致密的膜试样的试样和从电子束损伤保护它。如果涂层是感兴趣太厚结构可以被遮蔽。 <ol><li>地方存根成的自动微溅射镀膜机的真空室，45秒设置模式为旋转和自动溅射涂覆。使用铂涂层更精细的细节，但是金也是合适的。 </li><li>涂抹一层薄薄的碳涂料以所安装的肝标本的外表面，小心不要涂覆正弦曲面。 </li><li>一旦标本都涂有铂和碳​​油漆已干，他们已经准备好观察的SEM。样品可以在干燥器中储存。 </li></ol> 7.使用扫描电子显微镜<ol><li>地方标本进入扫描电子显微镜，并返回显微镜至真空。&lt;/ LI&gt; <li>使用的样本可视化标准显微镜操作程序通过在开始低倍率，逐渐增加的放大倍率。 </li><li>确保显微镜为工作距离，光点尺寸和散光适当调整。 </li><li>检查固定的完整性。如果LSEC是从缺口较自由，并包含窗孔测量30 - 250 nm的直径，这将通常表明试样制备已经成功。 </li><li>如果正弦充满空隙或缺乏穿孔的试样准备尚未成功或有显著病理。在这种情况下，片具有非常优良的剃刀刀片的样品，并且切割表面重新涂复并成功地固定正弦分析。 </li><li>大约在15,000-20,000×倍率选择平坦的表面LSEC是免费的碎片具有良好的固定和其中窗孔清晰可见（通常至少含有10的窗孔）。 </li><li>保存至少10血窦图像每肝，来自各块的不同区域，使用每肝的至少两个块。采样10的显微照片以15,000 - 每只动物20000×放大倍数通常是足够的窗孔的量化是代表整个肝脏的。 </li></ol> 8.分析注意：可从美国国立卫生研究院免费下载的ImageJ软件来定量测定开窗直径和频率（ <a href="http://imagej.nih.gov/ij">www.imagej.nih.gov/ij</a> ）。 <ol><li>使用嵌入在图像（ 屏幕快照1）的比例尺打开图像J和设置比例。 </li><li>使用多边形工具中的ImageJ，跟踪绕LSEC的整个平面区域包括有孔和defenestrated领域。不要只跟踪筛板，因为这会错误地夸大孔隙度。排除任何大的碎片是在细胞表面上，可能是obscuriNG窗孔。 </li><li>为了测量直径开窗，通过选择行工具跟踪通过每个开窗的最长直径线，然后按“M”来衡量的路线，和“d”（画）永久画上的图片就行了。这条线被定义为开窗直径。该生产线的长度现在可在结果框中的ImageJ（ 屏幕打出2）。 </li><li>测量这些都是LSEC细胞质超过250纳米的较大的孔，以及窗孔所有间隙。间隙往往假象反射灌注或定影不良，但是间隙也可以作为疾病和毒性的结果。对于缺口的数据将被排除在开窗参数计算分析后。 </li><li>差距是不是圆形的区域应该通过跟踪周围的空白，并计算其在ImageJ的面积计算。 </li><li>从ImageJ的结果框中的数据粘贴到Excel电子表格的furthe- [R分析。 </li></ol> 9.计算<ol><li>平均直径开窗平均=所有开窗直径（不包括空白，其中差距≥250纳米）。 </li><li>开窗面积=πR2，其中r，半径，是从各个窗孔直径相关（r = D / 2），不包括间隙算出。 </li><li>孔隙率（％）=（Σ（πR2）/分析总面积- Σ（间隙的面积=））（微米2））×100。 </li><li>开窗频率=开窗总数/（总面积分析-Σ（空白区域）（微米2）。 </li></ol> 10.介绍开窗数据注：只要有可能，出版物，包括开窗数据的量化应包括下列资料<ol><li>开窗直径，以声明证实什么用的地方直径边界定义开窗（通常间50 - 250纳米），开窗频率（数个/μm2）和孔隙率（％）。 </li><li>声明确认是否间隙已被包括在分析中，开窗直径和肝脏，块，图像和窗孔的数目的频度分布曲线应包括在分析中。 </li></ol>

<ol>
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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=Sinusoidal+ultrastructure+evaluated+during+the+revascularization+of+regenerating+rat+liver.">Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.</a> Hepatology. 33 (9), 363-378 (2001).</li><li>Morsiani, E., Mazzoni, M., Aleotti, A., Gorini, P., Ricci, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+sinusoidal+wall+permeability+and+liver+fatty+change+after+two-thirds+hepatectomy:+An+ultrastructural+study+in+the+rat.">Increased sinusoidal wall permeability and liver fatty change after two-thirds hepatectomy: An ultrastructural study in the rat.</a> Hepatology. 21 (2), 539-544 (1995).</li><li>Straub, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic-stimulated+liver+sinusoidal+capillarization+in+mice+requires+NADPH+oxidase&#8211;generated+superoxide.">Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase&#8211;generated superoxide.</a> J Clin Invest. 118 (12), 3980-3989 (2008).</li><li>Barber&#225;-Guillem, E., Arrue, J. M., Ballesteros, J., Vidal-Vanaclocha, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+in+endothelial+cells+of+developing+rat+liver+in+the+transition+from+fetal+to+postnatal+life.">Structural changes in endothelial cells of developing rat liver in the transition from fetal to postnatal life.</a> JJ Ultrastruct Mol Struct Res. 97 (1-3), 197-206 (1986).</li><li>Jamieson, H. 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=Caloric+restriction+reduces+age-related+pseudocapillarization+of+the+hepatic+sinusoid.">Caloric restriction reduces age-related pseudocapillarization of the hepatic sinusoid.</a> Exp Gerontol. 42 (4), 374-378 (2007).</li><li>Cogger, V. 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=Hyperlipidemia+and+surfactants:+the+liver+sieve+is+a+link.">Hyperlipidemia and surfactants: the liver sieve is a link.</a> Atherosclerosis. 189 (2), 273-281 (2006).</li><li>Cogger, V. 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=The+effects+of+oxidative+stress+on+the+liver+sieve.">The effects of oxidative stress on the liver sieve.</a> J Hepatol. 41 (3), 370-376 (2004).</li><li>Cogger, V. 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=Three-dimensional+structured+illumination+microscopy+of+liver+sinusoidal+endothelial+cell+fenestrations.">Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations.</a> J Struct Biol. 171 (3), 382-388 (2010).</li><li>Braet, F., de Zanger, R., Seynaeve, C., Baekeland, M., Wisse, 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+comparative+atomic+force+microscopy+study+on+living+skin+fibroblasts+and+liver+endothelial+cells.">A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells.</a> J Electron Microsc. 50 (4), 283-290 (2001).</li><li>Monkemoller, 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=Imaging+fenestrations+in+liver+sinusoidal+endothelial+cells+by+optical+localization+microscopy.">Imaging fenestrations in liver sinusoidal endothelial cells by optical localization microscopy.</a> Phys Chem Chem Phys. 16 (24), 12576-12581 (2014).</li></ol>

The authors have nothing to disclose.

精确地和可重复地测量所述肝窦内皮的状态的能力是在理解这些高度特化的细胞的生物学的一个重要步骤。如结构照明显微镜32，原子力显微镜33和d- STORM（直接随机光学Reconstrucion显微镜）34更新的技术将赋予重要的信息对这些细胞在体外的形态，但SEM仍然是主要的方法来可视化和在测量它们的结构原位 。 最关键和技术挑战性的一?...

肝窦内皮细胞（LSECs）是高度分化的内皮细胞排列在肝窦的壁。 LSECs均穿有穿孔是非diaphragmed，跨细胞孔径为50-250纳米。高达LSECs的表面的20％被覆盖穿孔，这通常是在几十组到几百称为筛板1-3（ 图1）。开窗使血浆和血液和肝细胞之间nanosubstrates的转移，创造一个高效的超滤系统。窗孔是动态的结构 - 无论它们的尺寸和/或数量可以响应于各种生理状态，药物和疾病而改变。例如，穿孔较大在禁食比在进食状态4; 2-二- iodoamphetamine增加开窗号; 5,6-和尺寸的减小和穿孔的数目每细胞发生衰老和许多疾病状态7-13 </sup&gt;。窗孔的尺寸和数量的精确测量是理解如何LSEC形态是由各种疾病，中毒性和生理状态的影响重要;它们对肝功能的影响;和用于显影开窗调制治疗干预1。 窗孔的研究是困难的。窗孔的直径在于传统光学显微镜的分辨率下面，所以无论是在完整的肝组织或培养LSECs以前只观察用电子显微镜已经成为可能。扫描电子显微镜（SEM）已最经常被用于研究开窗尺寸，频率和孔隙率（LSEC膜是受窗孔穿孔的百分比），因为SEM允许观测大面积的数千内皮表面和测量，如果不是几万窗孔的。尽管它的效用，这是从报告的结果的SEM为基础的研究为LSEC参数如开窗大小，数量，频率和孔隙率有很大的不同在文献中（ 表1）。 开窗和筛板是脆弱的结构，其合同，打破，扩张或标本制备过程中凝聚，从而精心加工是需要保持其完整性。升高灌注压14;固定液和缓冲区15的不正确的渗透压;固定不充分或固定时间;和后固定脱水和干燥的速度是处理用于SEM的所有领域，可能产生伪影，与保存超微结构的干扰（ 图2）。窗孔的损失（&#39;抛出窗外&#39;）和开窗收缩可发生定影不良的结果，从而降低开窗直径和孔的孔隙率。方法，提高试样的保存用于SEM分析先前已经描述15-17和将要讨论的这里，关于如何提高标本保存额外的提示。检体保存的主要目标是从血窦除去血液，使得LSEC的表面可以可视化，并避免从任一高压或延迟固定的LSEC损坏。固定剂经门静脉全肝灌注是肝脏固定的首选方法。如在详细描述别处16,18灌注必须在低压力下进行（例如，10厘米H 2 O），以避免压力相关灌注艺术品和损坏LSEC，典型表现内于细胞膜一样大的差距。然而，合理的固定通常可以使用从人类和动物的肝活检的针灌注获得，如在别处19进行详细描述。该技术包括直接注射入固定液的组织，直到血冲出样品和组织是坚定的和固定的。样品电子显微镜的固定需要被法律约束ormed尽快以下血流停止，以防止发生由于肝脏极其迅速自溶过程的结果超微结构改变。 我们还提出，最大限度地减少包含伪影，和标准化窗孔的测量图像分析的方法。变化血窦为显微照片的选择，人工制品的图像分析，并且小区区域的测量孔隙度和开窗频率已导致在已发表的结果重大差异。评估和窗孔和数据呈现的最低要求的测量标准化的做法没有得到明确解决在文献中以前4,10,20-31。

<table border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Name of material</td>
			<td style="width:100px;">Company</td>
			<td style="width:93px;">Catalogue Number</td>
			<td style="width:311px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">EM grade Glutaraldehyde</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C001</td>
			<td style="width:311px;">Store stock at -20 C until needed, avoid refreeze</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Paraformaldehyde powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>158127</td>
			<td style="width:311px;">Always prepare Paraformaldehyde fresh</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:155px;">Sodium Cacodylate powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C0250</td>
			<td style="width:311px;">Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH2O, used mostly at 0.1 M by preparing 1:2 dilution</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:155px;">Calcium Chloride</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C1016</td>
			<td style="width:311px;">Prepare 1 M CaCl2by dissolving powder in dH2O</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:155px;">Osmium tretroxide</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C011</td>
			<td style="width:311px;">Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Ethanol- Absolute</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>459836&#160;</td>
			<td style="width:311px;">100 % Ethanol must be high grade and stored with Molecular Sieve</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Other grades of Ethanol</td>
			<td style="width:100px;">Labtech</td>
			<td style="width:93px;">EL5</td>
			<td style="width:311px;">Prepare graded Ethanols with dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Hexamethyldisilazane</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>52619&#160;</td>
			<td style="width:311px;">Allow to reach room temperature before use</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:155px;">Cannulas</td>
			<td style="width:100px;">Terumo</td>
			<td style="width:93px;">TSROX1832C, TSROX2225C, TSROX2419C</td>
			<td style="width:311px;">18 G is suitable for most rats, 22 G is suitable for most mice, but it is good to have a few 24 G on hand in case of very small mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Conductive Carbon tape</td>
			<td style="width:100px;">ProSciTech</td>
			<td>IA0201</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Carbon Paint</td>
			<td style="width:100px;">ProSciTech</td>
			<td>I003</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Ketamine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be optained under licence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Xylazine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be obtained under licence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Molecular Sieve</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">208647</td>
			<td style="width:311px;">Removes water from the 100 % Ethanol</td>
		</tr>
	</tbody>
</table>

a standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

在低放大倍数的扫描电子显微镜初始可视揭示肝脏标本具有暴露面积大到足以观察许多大的肝血管和正弦波（ 图1A）的平坦表面。确保正确的肝块放置于安装存根是用于获得清晰的图像的正弦和 ​​肝脏的Glisson囊应避免这个原因（ 图1B）是必不可少的。增加放大率允许稠密脉管肝脏的仔细检查揭示该分割容器（ 图1C）的肝细胞的单细胞板。的肝导致图像质?...

Watch this Scientific Journal Video about A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy at JoVE.com

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

a-standardized-method-for-analysis-liver-sinusoidal-endothelial-cells

Research

JoVE Journal

Biology

14.2K 次观看.  University of Sydney and Concord Hospital. The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

视频: A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

科研

生物学

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

扫描浸软组织的电子显微镜可视化的细胞外基质

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

通过低温扫描电子显微镜研究光合膜内冻结，割断叶片组织的超分子组织

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

用化学干燥进行扫描电镜 (sem) 形态分析的原核生物和真核生物的制备

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

阵列断层扫描工作流程，用于使用扫描电子显微镜有针对性地采集体积信息

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...