Name: serial block-face scanning electron microscopy (sbem) for the study of dendritic spines
Uploaded: 2021-10-02T14:30:00
Description: Watch this Scientific Journal Video about Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines at JoVE.com

SBEM成像、光显微镜成像和电子显微镜样品制备是利用作为Nencki实验生物学研究所成像核心设施的成像组织和功能实验室的设备进行的。
为了准备图1，使用了鼠标（Souris_02）和 https://smart.servier.com/ 小瓶的图像。
这项工作得到了国家科学中心（波兰）授予KR的赠款Opus（UMO-2018/31/B/NZ4/01603）的支持。

这项研究是根据Nencki研究所的指导方针和地方道德委员会的许可进行的。这些研究是根据欧洲共同体理事会1986年11月24日指令（86/609/EEC）、波兰《动物保护法》进行的，并得到了华沙第一个地方道德委员会的批准。尽了一切努力尽量减少使用的动物数量及其痛苦。
注意：以下所有程序必须在实验室烟雾罩中执行。由于使用的试剂具有危险性。需要采取个人安全措施，如手套?...

<ol>
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2010年10月，迪林克描述的主要NCMIR方法有许多变化。基本原则保持不变，但根据所研究的材料类型，将实施轻微更改。以前曾描述过，不同的树脂可用于嵌入标本的SBEM，例如在植物的情况下，Spurr的树脂是首选的，因为它的低粘度，允许更好地渗透通过细胞壁22，23。此外，各种缓冲可用于固定（例如，可可二甲酸酯缓冲液、HEPES 或磷?...

中枢神经系统中的大部分兴奋突触位于树突状脊柱上 - 神经元膜的小突起。这些突起形成密闭生化隔间，控制细胞内信号转导。树突状脊柱和突触的结构可塑性与突触功效的功能变化密切相关，突触功效的变化是学习和记忆1、2等重要过程的基础。需要注意的是，电子显微镜 （EM） 是唯一能够确定树突状脊柱是否有突触前输入的技术。EM 分辨率还需要研究超结构细节，如后合成密度 （PSD） 的形状，代表突触的后合成部分，或树突脊柱的尺寸，以及轴突的大小和形状。此外，通过 EM，可以可视化突触及其周围环境。
由于成像和计算技术的进步，可以重建整个神经回路。体积电子显微镜技术，如串行节传输电子显微镜（sSTEM）、串行块面扫描电子显微镜（SBEM）和聚焦离子束扫描电子显微镜（FIB-SEM）等，常用于神经元电路重建3。
在我们的研究中，SBEM方法成功地用于研究小鼠海马和有机性脑切片4，5的样本中树突状脊柱和PSD的结构可塑性。SBEM基于扫描电子显微镜室6、7、8、9内安装微型超微缩微切除器。对样品块顶部进行成像，然后由超微切除器在指定深度切割样品，露出新的块面，再次成像，然后重复过程 8。因此，仅留下块面图像，而已切割的切片则丢失为碎屑。这就是为什么SBEM被称为破坏性技术，这意味着它不可能再次映像同一个地方。但是，破坏性块上方法的优点是它们不会遭受扭曲问题和部分丢失，这些问题和部分损失会显著影响数据质量和数据分析3。此外，SBEM还提供了在高分辨率3下成像一个相对较大的视场（>0.5毫米×0.5毫米）的可能性。
要使用 SBEM，由于用于获取图像的背散射电子探测器，因此必须按照专用的、高度对比的协议准备样品。我们在这里展示如何根据Dierinck10（国家显微镜和成像研究中心（NCMIR）方法开发的程序，使用20世纪80年代8、11年代开发的减少的硫化硫化物（rOTO）污渍来进行样品制备。此外，我们引入了两步固定方法，采用温和的固定灌注，允许使用相同的大脑进行轻显微镜和SBEM的免疫荧光研究。
在协议中，小鼠大脑主要用温和的固定剂固定，然后切成两半，一个半球固定后，准备免疫荧光（IF），而另一个半球用于EM研究（图1）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62712/62712fig01.jpg"> 图1。树突脊柱工作流程的示意图，为 SBEM 分析做准备。 老鼠被牺牲，并灌注了温和的原发性固定剂。大脑被切成两半，一个半球被免疫荧光（IF）专用固定，冷冻保护，使用低温统计器切片，并处理IF研究，而另一个半球被固定后与EM固定，切片与振动器和准备EM研究。用于SBEM研究的大脑切片进行了对比，平嵌在树脂中，然后海马的CA1区域被安装到针脚上，并用SBEM成像（图1）。视频中展示了黄色框中突出显示的协议部分。 <a href="https://www.jove.com/files/ftp_upload/62712/62712fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthetic:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine/xylazine mixture (Ketamina/Sedazin)</td>
			<td>Biowet Pulawy, Pulawy, Poland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital (Morbital)</td>
			<td>Biowet Pulawy, Pulawy, Poland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixatives:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (GA)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>G5882</td>
			<td>Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>POCH, Gliwice, Poland</td>
			<td>575283115</td>
			<td>pure p.a.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>441244</td>
			<td>prilled, 95%</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), pH 7.4</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>P4417-50TAB</td>
			<td>tablets</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>S5881</td>
			<td>reagent grade, <img alt="Equation" src="/files/ftp_upload/62712/62712symbol.png" />98%, pellets (anhydrous)</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>S3264</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (NaH2PO4)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>S3139</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Large blunt/blunt curved scissors (~14.5 cm)</td>
			<td>Fine Science Tools, Foster City, CA, USA</td>
			<td>14519-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-spatula (double 2&#34; flat ends, one rounded, one tapered to 1/8&#34;)</td>
			<td>Fine Science Tools, Foster City, CA, USA</td>
			<td>10091-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle tip, 15 GA, blunt (perfusion needle)</td>
			<td>KD Medical GmbH Hospital Products, Berlin, Germany</td>
			<td>KD-FINE 900413</td>
			<td>1.80 x 40 mm</td>
		</tr>
		<tr>
			<td>Pair of fine (Graefe) tweezers</td>
			<td>Fine Science Tools, Foster City, CA, USA</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Lead Fluid</td>
			<td>BQ80S</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic vials</td>
			<td>Profilab, Warsaw, Poland</td>
			<td>534.02</td>
			<td>plastic vials with blue cap for tissue storage, 20 ml, 31 x 48 mm</td>
		</tr>
		<tr>
			<td>Straight iris scissors (~9 cm)</td>
			<td>Fine Science Tools, Foster City, CA, USA</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain slices preparation for EM:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>NEST, Rahway, NJ, USA</td>
			<td>712001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylic glue</td>
			<td>Fenedur, Montevideo, Uruguay</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>72632</td>
			<td>20 ml Scintillation Vial, a pack of 100</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>612-4545</td>
			<td>LDPE, disposable, 7.5 ml</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Wilkinson Sword, London, UK</td>
			<td></td>
			<td>Classic double edge safety razor blades</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Swann-Morton, Sheffield, UK</td>
			<td>No. 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Microsystems, Vienna, Austria</td>
			<td>Leica VT1000 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain slices preparation for IF:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>NEST, Rahway, NJ, USA</td>
			<td>701101</td>
			<td></td>
		</tr>
		<tr>
			<td>Criostat</td>
			<td>Leica Microsystems, Vienna, Austria</td>
			<td>Leica CM 1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol</td>
			<td>Bioshop, Burlington, Canada</td>
			<td>ETH001</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-profile disposable blade 819</td>
			<td>Leica Biosystems Inc., USA</td>
			<td>14035838925</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Swann-Morton, Sheffield, UK</td>
			<td>No. 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide (NaN3)</td>
			<td>POCH, Gliwice, Poland</td>
			<td>792770426</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>POCH, Gliwice, Poland</td>
			<td>772090110</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue freezing medium for cryosectioning, OCT-Compound</td>
			<td>Leica Biosystems, Switzerland</td>
			<td>14020108926</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunostaining:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>NEST, Rahway, NJ, USA</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Post Synaptic Density Protein 95 Antibody</td>
			<td>Merck-Millipore, Burlington, MA, USA</td>
			<td>MAB1598</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss, G&#246;ttingen, Germany</td>
			<td></td>
			<td>Zeiss Spinning Disc microscope (63 &#215; oil objective, NA 1.4, pixel size 0.13 &#181;m &#215; 0.13 &#181;m)</td>
		</tr>
		<tr>
			<td>Cover slide</td>
			<td>Menzel Glaser, Braunschweig, Germany</td>
			<td>B-1231</td>
			<td>24 x 60 mm</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>A31570</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G Mounting Medium, with DAPI</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>00-4959-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Thermo Scientific, Waltham, MA, USA</td>
			<td>AGAA00008</td>
			<td>SuperFrost</td>
		</tr>
		<tr>
			<td>Normal donkey serum (NDS)</td>
			<td>Jackson ImmunoResearch Laboratories, West Grove, PA, USA</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>JWElectronic, Warsaw, Poland</td>
			<td>KL-942</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonT X-100 Reagent Grade</td>
			<td>Bioshop, Burlington, Canada</td>
			<td>TRX506</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron microsocpy sample preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate(II) trihydrate</td>
			<td>POCH, Gliwice, Poland</td>
			<td>746980113</td>
			<td></td>
		</tr>
		<tr>
			<td>Aclar 33C Film</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>50425</td>
			<td>Fluoropolymer Film embedding sheet</td>
		</tr>
		<tr>
			<td>DMP-30, 2,4,6-Tris(dimethylaminomethyl)phenol</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>T58203</td>
			<td>Epoxy embedding medium accelerator</td>
		</tr>
		<tr>
			<td>Durcupan ACM single component A, M</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>44611</td>
			<td>Durcupan ACM single component A, M epoxy resin</td>
		</tr>
		<tr>
			<td>Durcupan ACM single component B</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>44612</td>
			<td>Durcupan ACM single component B, hardener 964</td>
		</tr>
		<tr>
			<td>Durcupan ACM single component D</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>44614</td>
			<td>Durcupan ACM single component D , plasticizer</td>
		</tr>
		<tr>
			<td>Ethyl alcohol absolute</td>
			<td>POCH, Gliwice, Poland</td>
			<td>64-17-5</td>
			<td>Ethyl alcohol absolute 99.8 % pure P.A.-BASIC</td>
		</tr>
		<tr>
			<td>Genlab laboratory oven</td>
			<td>Wolflabs, York, UK</td>
			<td>Mino/18/SS</td>
			<td>Oven Genlab MINO/18/SS 18l volume, no fan circulation, no digital display, standard temperature gradient, standard recovery rate, no timer, 250&#176;C maximum temperature, 240V electrical supply</td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>A-9256</td>
			<td>reagent grade, <img alt="Equation" src="/files/ftp_upload/62712/62712symbol.png" />98% (HPLC)</td>
		</tr>
		<tr>
			<td>Lead (II) nitrate</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>467790</td>
			<td><img alt="Equation" src="/files/ftp_upload/62712/62712symbol.png" />99.95% trace metals basis</td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>75632</td>
			<td>for electron microscopy, 4% in H2O</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Elmetron, Zabrze, Poland</td>
			<td>CP-5-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>BioSan, J&#243;zef&#243;w, Poland</td>
			<td>Multi Bio RS-24</td>
			<td>rotator Multi Bio RS-24</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>S5881</td>
			<td>reagent grade, <img alt="Equation" src="/files/ftp_upload/62712/62712symbol.png" />98%, pellets (anhydrous)</td>
		</tr>
		<tr>
			<td>Sunflower mini shaker</td>
			<td>Grant bio, Shepreth,UK</td>
			<td>PD-3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Millipore, Burlington, MA, USA</td>
			<td>SLGP033NB</td>
			<td>0,22 &#181;m pore size</td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>88535</td>
			<td>purum p.a., for electron microscopy, <img alt="Equation" src="/files/ftp_upload/62712/62712symbol.png" />99.0% (N)</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Serva, Heidelberg, Germany</td>
			<td>77870</td>
			<td>Uranyl acetate&#183;2H2O, research grade</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>WSL, Swietochlowice, Poland</td>
			<td>LWT</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen mounting for SBEM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>734-2782</td>
			<td>96-well plates, round bottom, non treated</td>
		</tr>
		<tr>
			<td>AM Gatan 3View stub handling tweezers</td>
			<td>Micro to Nano, Haarlem, Netherlands 
			Netherlands</td>
			<td>50-001521</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular</td>
			<td>OPTA-TECH, Warsaw, Poland</td>
			<td>X2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Conductive glue</td>
			<td>Chemtronics, Georgia, USA</td>
			<td>CW2400</td>
			<td>conductive eopxy</td>
		</tr>
		<tr>
			<td>Gatan 3View sample pin stubs</td>
			<td>Micro to Nano, Haarlem, Netherlands 
			Netherlands</td>
			<td>10-006003</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>P7793</td>
			<td>roll size 20&#160;in. &#215; 50&#160;ft</td>
		</tr>
		<tr>
			<td>Pelco conductive silver paint</td>
			<td>Ted Pella, Redding, CA, USA</td>
			<td>16062-15</td>
			<td>PELCO&#174;&#160;Conductive Silver Paint, 15g</td>
		</tr>
		<tr>
			<td>Razor blades double edge</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>72000</td>
			<td>Stainless Steel &#34;PTFE&#34; coated. PERSONNA brand .004&#34; thick, wrapped individually, 250 blades in a box.</td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Zeiss, Oberkochen, Germany</td>
			<td></td>
			<td>Sigma VP with Gatan 3View2 chamber, acceleration voltage 2.5 kV, variable pressure 5 Pa, aperture 20 &#181;m, dwell time 6 &#181;s, slice thickness 60 nm, magnification 15 000 x, image resolution 2048 x 2048 pixels, pixel size 7.3 nm</td>
		</tr>
		<tr>
			<td>trim 90&#176; diamond knife</td>
			<td>Diatome Ltd., Nidau, Switzerland</td>
			<td>DTB90</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica Microsystems, Vienna, Austria</td>
			<td>Leica ultracutR</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>webpage</td>
			<td></td>
			<td>tutorials</td>
		</tr>
		<tr>
			<td>FijiJ</td>
			<td>https://fiji.sc/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopy Image Browser</td>
			<td>http://mib.helsinki.fi/</td>
			<td></td>
			<td>http://mib.helsinki.fi/tutorials.html</td>
		</tr>
		<tr>
			<td>Reconstruct</td>
			<td>https://synapseweb.clm.utexas.edu/software-0</td>
			<td></td>
			<td>https://synapseweb.clm.utexas.edu/software-0)</td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Adult 3-month old and&#160;20&#177;1&#160;month old&#160;female&#160;Thy1-GFP(M) mice (Thy1-GFP +/-) (Feng et al.,2000) which express GFP in a sparsely distributed population of glutamatergic neurons. Animals were bred as heterozygotes with the C57BL/6J background in the Animal House of the Nencki Institute of Experimental Biology.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

serial block-face scanning electron microscopy (sbem) for the study of dendritic spines

三维电子显微镜（3D EM）提供了分析具有纳米级分辨率的树突脊柱形态参数的可能性。此外，树突状脊柱的某些特征，如脊柱体积和突触后密度 （PSD） （表示突触的突触后部分）、突触前终端的存在以及光滑的内质视网膜或非典型形式的 PSD（例如多内侧脊柱），只能使用 3D EM 进行观察。通过使用串行块面扫描电子显微镜 （SBEM），可以比执行传统串行分割时更容易、更可重复的方式获得 3D EM 数据。在这里，我们展示如何准备小鼠海马样本进行SBEM分析，以及如何将此协议与树突状脊柱的免疫荧光研究相结合。轻度固定灌注使我们能够在大脑的一半进行轻度显微镜免疫荧光研究，而另一半则为SBEM做好准备。这种方法减少了用于研究的动物数量。

使用上述高对比度方法，可以获得小鼠脑组织的良好分辨率图像。SBEM 技术提供的大量视野有助于精确选择感兴趣的区域。海马CA1区域的大图像被采取测量地层径向（SR）的长度（图2A），并设置成像精确在中心（图2B）。接下来，获取了图像堆，并细分了感兴趣的对象，如树突、树突、突触的后合成密度（图2C，D）。...

Watch this Scientific Journal Video about Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines at JoVE.com

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

连续块面扫描电子显微镜 （SBEM） 应用于图像和分析穆林海马体中的树突状脊柱。

用于登德里脊柱研究的串行块面扫描电子显微镜 （SBEM）

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...

大脑中的每个神经元通过大约7000个突触与其他神经元细胞相连。除了哺乳动物大脑中的突触主要位于膜的小突起上，称为树突状脊柱。突触和树突状脊柱等的可塑性，诸如学习和记忆过程等重要大脑功能。需要注意的是，只有电子显微镜才能全面了解树突状脊柱是否具有后合成输入。提供各种树突脊柱成像技术。然而，串行块面部扫描电子显微镜为高分辨率成像大量组织提供了独特的可能性。基于串行块的扫描电子显微镜技术需要特殊的样品制备协议，它涉及与重金属的强烈对比。在灌注过程中，我的主要固定使我们能够使用相同的大脑进行减轻电子显微镜。老鼠被牺牲，并灌注了温和的原发性固定剂。大脑被切成两半，一个半球被固定在免疫荧光专用固定剂，冷冻保护剂，使用低温统计切片，并处理免疫荧光研究。另一个半球被固定在电子显微镜固定，切片与振动器，并准备电子显微镜研究。用于基于序列块的扫描电子显微镜研究的大脑切片进行了对比，平嵌在树脂中，然后海马体的CA1区域被安装到针脚上，并用基于序列块的扫描电子显微镜成像。视频中展示了黄色框中突出显示的协议部分。取入已修复的组织，切成100片微生物片，用冷磷酸盐缓冲器清洗5次，每次3分钟。准备4%的四氧化硫和3%的铁氰化钾的一对一混合物。最终产品将变成棕色。将样品浸入这种混合物中。然后将样品放在冰上。从这个阶段开始，保护他们免受光线照射是很重要的。用轻轻的摇晃孵育它们一个小时。同时，准备 TCH 解决方案。混合 10 毫升双蒸馏水和 0.1 克 TCH。将其放入60摄氏度的烤箱中一小时。不时地挥动解决方案非常重要。准备好后，将其冷却至室温。现在用双蒸馏水清洗样品五次，每次三分钟，然后浸入过滤的TCH溶液中。切片将变黑。在室温下孵育20分钟。用双蒸馏水清洗样品五次，每次三分钟，用2%的四氧化硫化物孵育30分钟，这次是在室温下。再次，用双蒸馏水清洗样品五次，每次三分钟，并把它们放入过滤的1%或乙酸铵。在四摄氏度的一夜之间孵化它们。第二天，从 D-阿斯巴丁准备开始。将硝酸铅与预热至60摄氏度的阿斯酸溶液混合。将混合物放入60摄氏度的水浴中，用一个摩尔氢氧化钠将pH值调整到5.5。用铅阿斯巴甜关闭小瓶，在60摄氏度下离开30分钟。解决方案应该清晰。如果变成多云，必须丢弃它，需要准备一个新的。同时，用气体双蒸馏水清洗样品五次，每次三分钟，并将它们放在60摄氏度的烤箱中30分钟。将样品浸入新制备的铅阿斯巴酸盐溶液中，在60摄氏度的烤箱中孵育20分钟。准备环氧树脂。在添加加速器 DMP 30 之前，称重配料并混合树脂至少 30 分钟。用乙醇的分级稀释来准备病毒。然后将样品从烤箱中取出，然后用双蒸馏水再洗五次，每次三分钟。同时，将树脂与100%乙醇按一对一的比例混合，获得50%的树脂。混合好。在此之后，每次稀释乙醇时脱水样品五分钟。请记住，样品绝不能完全干燥。先在 50% 树脂中渗透样品 30 分钟，然后在 100% 树脂中渗透一小时，然后在一夜之间再次浸入新鲜的 100% 树脂中。第二天，将样品放入新鲜的100%树脂中一小时，然后嵌入氟聚合物嵌入片之间。准备已脱脂的嵌入片，并放置玻璃滑梯，用作支撑。将一块嵌入片放在玻璃滑梯上，在上面放一滴树脂，然后使用木棍小心地转移样品。用第二块盖住它。尽量避免气泡。要温柔，因为组织非常脆弱。小心地将另一个玻璃滑梯放在嵌入片的顶部，并在 70 摄氏度下将样品在烤箱中治愈至少 48 小时。分离层，用剃须刀刀片切开嵌入的样品的一块。将其转移到石蜡上。这将最大限度地降低因静电而丢失样品的危险。拿一个已经脱脂乙醇的铝针。混合导电环氧树脂井，并用少量环氧树脂将样品安装到销中。在 70 摄氏度下治疗导电环氧树脂 10 分钟。用钻石刀修剪样品块的每一侧，然后擦亮块的表面，直到组织暴露。使用导电涂料将样品磨到针脚上并治愈它。为了尽量减少充电，还应发现样品上涂有一层薄薄的黄金或金铂。将带样品的引脚放入基于串行块的扫描电子显微镜的腔室中。将样品对齐到刀上，然后关闭腔室并设置参数。收集一叠图像。使用上述方法，获得了小鼠脑组织的图像。海马体一个区域的大图像被拍摄，成像设置在地层半径图的中心。获得了一叠图像，并分割了感兴趣的对象，如树突状脊柱和突触。通过清晰可见的膜和突触囊泡以及保存完好的线粒体获得了高质量的组织形态。PSD 95抗体的免疫荧光研究证实，轻度原发性固定和传统固定4%PFA具有可比性。呈现协议允许获得脑组织图像，使用串行块为基础的扫描电子显微镜，高质量的组织形态学，和12个可见膜促进10正确的分割和重建。我的主要固定使使用相同的大脑分析PSD 95免疫荧光和电子显微镜成像树突脊柱成为可能。在这里，我们使用 PSE 9，500 萌芽。但是，其他也可以使用。

Research

JoVE Journal

Neuroscience

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

科研

神经科学

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

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 ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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

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

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 ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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

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

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 ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

Correlative Light and Electron Microscopy to Study Microglial Interactions with ÃƒÅ½Ã‚Â²-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

使用串行块面部扫描电子显微镜脑线粒体分析

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

用于登德里脊柱研究的串行块面扫描电子显微镜 （SBEM）

用于登德里脊柱研究的串行块面扫描电子显微镜（SBEM）