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Morphological Quantification of Nuclei and Mitochondria in Serial Block-face Scanning Electron Microscopy Images
串行块面扫描电子显微成像检测细胞核和线粒体的形态特征   

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本实验方案简略版
The Journal of Experimental Medicine
Jul 2014

Abstract

Serial Block-face Scanning Electron Microscopy (SBF-SEM or 3D-EM) is a powerful tool to study biological structure in ultrastructural level. Quantification of cellular ultrastructure is useful to providing biological information. This technique requires not only high quality of tissue fixation and ideal sample embedding to preserve structures, but also delicate 3D image scanning and post-processing of images. We have adapted previous method to optimize the EM technique to detect and study cellular ultrastructure. Here we present the method to embed samples for 3D-EM technique and to quantify the morphological parameters of nucleus and mitochondria.

Part I. Tissue embedding for 3D-EM images

Keywords: Electron microscopy (电子显微镜), Mitochondria (线粒体), Nuclus (核), Morphology (形态学)

Materials and Reagents

  1. Adult mouse brain / spinal cord
  2. 4% Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  3. Glutaraldehyde solution (GA) (Sigma-Aldrich, catalog number: G5882 )
  4. Sodium Cacodylate Buffer (0.4 M, pH 7.2) (Electronic Microscopy Science, catalog number: 11654 )
  5. Phosphate buffered saline (PBS) (1x Solution, Fisher BioReagents) (Fisher Scientific, catalog number: BP2438-4 )
  6. Potassium ferricyanide (III) (Sigma-Aldrich, catalog number: 702587 )
  7. Thiocarbohydrazide (TCH) (Electronic Microscopy Science, catalog number: 21900 )
  8. Osmium tetroxide 4% solution (Electronic Microscopy Science, catalog number: 19150 )
  9. Uranyl acetate (UA) (Electron Microscopy Sciences, catalog number: 22400 )
  10. Lead Nitrate (Crystalline/Certified ACS), Fisher Chemical (Fisher Scientific, catalog number: L62-100 )
  11. L-aspartic acid solution (Sigma-Aldrich, catalog number: A9256 )
  12. Ethanol, 200 proof (100%), USP, DeconTM Labs (Fisher Scientific, catalog number: 07-678-004 )
  13. Acetone (Electronic Microscopy Science, catalog number: 10010)
  14. EMbed-812 kit (Electronic Microscopy Sciences, catalog number: 14120 )
  15. EMS Molded Flat Embedding Mold (Electronic Microscopy Sciences, catalog number: 70905-01 )

Equipment

  1. Vibratome (Leica, catalog number: Leica VT1000S )
  2. Zeiss Sigma VP SEM (Zeiss) with Gatan 3View (Gatan)


    Figure 1. Zeiss Sigma VP SEM (Zeiss) with Gatan 3View (Gatan)

Procedure

  1. Mouse is perfused intracardially with 20 ml PBS in 1-2 min followed by 50ml 4% PFA with 1% GA in 5 min.
  2. Mouse brain/spinal column is dissected out and post-fixed by 4% PFA with 1% GA for 24 h at 4 °C.
  3. Peel the spinal cord from spinal column.
  4. Fixed brain/spinal cord sections are made with 100 μm thickness on a vibratome.
  5. Brain/spinal cord sections are cut into 0.2 x 0.2 cm2 tissue blocks depending on the interesting area.
  6. Tissue blocks are put in 1.5 ml Eppendorf tubes and incubated in 1ml cold (4 °C) 0.1 M cacodylate buffer for 3 times for 5 min each on ice.
  7. Right before use, make fresh OsO4-ferrocyanide buffer by mixing 3% potassium ferricyanide in 0.2 M cacodylate buffer and an equal volume of 4% aqueous osmium tetroxide. The tissue blocks are incubated in this solution for 1 h on ice.
  8. During the one hour incubation, freshly prepare the fresh thiocarbohydrazide solution. Add 0.1 g thiocarbohydrazide (TCH) to 10 ml ddH2O and place in a 60 °C oven for 1 h. Agitate the buffer by swirling gently every 10 min to facilitate dissolving. Filter this solution through a 0.22 μm Millipore syringe filter right before use.
  9. At the end of the incubation in 0.2 ml OsO4-ferrocyanide, the tissues are incubated in ddH2O for 3 times for 5 min each at room temperature.
  10. Remove ddH2O in sample tubes and add TCH to sample tube directly after its incubation in 60 °C. Tissue blocks are incubated in TCH for 20 min at room temperature.
  11. Tissue blocks are incubated in 1ml ddH2O for 3 times for 5 min each, and then incubated in freshly prepared 0.2 ml 2% osmium tetroxide for 30 min at room temperature.
  12. Tissue blocks are incubated in 1 ml ddH2O for 3 times for 5 min each, and then incubated in 1% uranyl acetate overnight in 4 °C.
  13. The next day, prepare lead aspartate solution freshly. Firstly, prepare 0.03 M Aspartic Acid buffer. Briefly, add 0.2 g L-Aspartic Acid in 50 ml ddH2O. Adjust pH value to 5.5, and then put the Aspartic Acid solution at 60 °C until Aspartic Acid is totally dissolved. Secondly, prepare lead aspartate solution. Dissolve 0.066 g of lead nitrate in 10 ml aspartic acid solution. Put lead solution in a 60 °C oven for 30 min.
  14. Tissue blocks are incubated in 1ml ddH2O for 3 times for 5 min each, and then incubated in lead solution at 60 °C for 30 min.
  15. Tissue blocks are incubated in 1ml ddH2O for 3 times for 5 min each at room temperature, and then dehydrated using ice-cold solutions of freshly prepared 20%, 60%, 80%, 90%, 100% ethanol for 5 min each step.
  16. Tissue blocks are incubated in acetone for 10 min at room temperature.
  17. During acetone incubation, prepare 100% Epon solution using Epon-812 embedding kit as follows: mix EMbed-812 10 ml, Dodecenylsuccinic anhydride (DDSA) 8 ml, Methyl-5-Norbornene-2, 3-Dicarboxylic Anhydride (NMA) ml and 4 2, 2-dimethoxypropane (DMP-30) 0.4 ml together very well. To prepare 50% Epon solution, mix equal volumes of of 100% Epon with 100% acetone.
  18. Tissue blocks are incubated in 50% Epon solution for 2 h at room temperature.
  19. Tissue blocks are placed in 100% Epon solution on shaker overnight at room temperature.
  20. The next morning, prepare new 100% Epon solution using the recipe in 17. Transfer the tissues into new Epon solution, and then put them on shaker for 2 h at room temperature.
  21. Transfer Epon to Plastic Flat Embedding Mold and polymerize tissue blocks in a 60 °C oven overnight.
  22. Cut the samples put from Epon blocks and try to remove all the Epon surrounding the samples.
    Note: It’s better to cut all the Epon away since Epon will cause charging during the scanning. The best size for the tissue block after the cutting is 1*1*2 mm3, but it’s very technique demanding and people seldom reach this standard.
  23. SBF-SEM images were acquired using a Zeiss Sigma VP SEM with Gatan 3 View. Serial image stacks of images at 100nm steps were obtained by sectioning (48 x 48 x 20) µm3 tissue blocks (length x width x depth) at a resolution of 8192 x 8192 pixels.
    Note: Steps 1-20 should be done under a fume hood.

Representative data



Figure 2. A representative single SBF-SEM image from a stack images showing nucleus and mitochondria in microglia. ↑: Mitochondria; ★: Nucleus.


Part II. Quantification of nuclei and mitochondria in 3D-EM images

Software

  1. Fiji software (NIH; http://fiji.sc/Downloads)

Procedure

  1. Save the images of the same stack in one folder.
  2. Open Fiji software and drag the folder to Fiji software to open the images.
  3. Click Plugins/Registration / Linear stack Alignment with SIFT to produce an aligned stack images.
  4. Save the stack images as tiff format.
  5. Click File/New/TrakEM2 (blank), and then choose the location to store the TrakEM file.
  6. Drag the Aligned stack images in tiff format to TrakEM image procession station.
  7. In Template window, right click anything / Add new child / area list.
  8. In Project Objects window, right click Untitled / Add / New anything.
  9. In project Objects window, right click anything / Add / New area_list.
  10. Right click New area_list / Rename to rename the layer as nucleus.
  11. In project Objects window, right click anything / Add / New area_list.
  12. Right click New area_list / Rename to rename the layer as mitochondria.
  13. In image processing station, right click the stack images, choose display / calibration, change the Unit of Length as micron, and put the Pixel Width, Pixel Height and Voxel Depth in the window. The Pixel Width and Pixel Height should be calculated according to the image scanning settings. The Voxel Depth is the interval of each image in the stack. For example, if the interval of the images is 100 nm, the Voxel Depth should be 0.1 micron.
  14. In Layers window, choose all the layers and right click the layers, choose Scale Z and thickness, calculate the scale number, which should put in the winder and could make the interval of each layer equal to the Voxel Depth.
  15. In image processing station, two layers named nucleus and mitochondria should appear in the left of the window.
  16. Click nucleus layer, double click the eyedropper tool to choose the one color.
  17. Use pen tool to draw the outline of nucleus in each image of the stack and click shift + F to fill the nucleus area in each image to make 3D reconstruction of the nucleus
  18. In control penal of Fiji, click Analyze / measure to measure the parameters of the nucleus including volume.
  19. Quantification of nuclear shapes is conducted in 3D reconstructions. Nuclei were categorized as follows; Round: round shape and smooth surface with ratio of length/width≤1.5; Elongated: elongated or oval shape with length/width>1.5, and may have small indentations; Bilobulated: two connected lobes with single intervening large indentation; Irregular: complicated shape with corrugated surface, and may have multiple and variable sizable indentations.
  20. Click mitochondria layer, double click the eyedropper tool to choose one color other than nucleus.
  21. Use pen tool to draw the outline of the mitochondria in each image of the stack and click shift + F to fill the outline are in each image to make 3D reconstruction of the mitochondria
  22. In control panel of Fiji, click Analyze / measure to measure the parameters of the mitochondria including volume and max diameter (Length of the mitochondria).
  23. Go through all the stack images, choose five different images where the cross section area of the mitochondria could be representative of the median of the cross section area.
  24. In the five representative images, use line tool and Analyze / measure in the control panel to draw and measure length of an Axis and b Axis.
  25. Use Cross section area = πab. Save all the parameters in a excel file. Calculate the mean Cross section area by the formula: Sum of five Cross section areas / 5.
  26. Repeat steps 11-12 to add another layer for the second mitochondria.
  27. Repeat steps 20-25 to do the quantification of mitochondria.
  28. Repeat steps 26-27 until finish the quantification of all the mitochondria in the cell.
  29. Calculate the number of mitochondria and the total volume of mitochondria in the cell.

Acknowledgments

The authors would like to thank the members of the Dr. Richard M. Ransohoff’s Lab for help on this manuscript. This work was supported by the National Multiple Sclerosis Society.

References

  1. TrackEM2 User Manual: http://www.ini.uzh.ch/~acardona/trakem2_manual.html
  2. TrakEM2 tutorials: http://fiji.sc/TrakEM2_tutorials

简介

串行块面扫描电子显微镜(SBF-SEM或3D-EM)是研究超微结构水平生物结构的强大工具。 细胞超微结构的定量对于提供生物信息是有用的。 这种技术不仅需要高质量的组织固定和理想的样品嵌入以保存结构,而且还需要精细的3D图像扫描和图像的后处理。 我们改编以前的方法优化EM技术检测和研究细胞超微结构。 在这里我们提出嵌入样品的3D-EM技术和量化的核和线粒体的形态参数的方法。

第I部分:3D-EM图像的组织嵌入

关键字:电子显微镜, 线粒体, 核, 形态学

材料和试剂

  1. 成年小鼠脑/脊髓
  2. 4%多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  3. 戊二醛溶液(GA)(Sigma-Aldrich,目录号:G5882)
  4. 二甲胂酸钠缓冲液(0.4M,pH7.2)(Electronic Microscopy Science,目录号:11654)
  5. 磷酸盐缓冲盐水(PBS)(1x Solution,Fisher BioReagents)(Fisher Scientific,目录号:BP2438-4)
  6. 铁氰化钾(III)(Sigma-Aldrich,目录号:702587)
  7. 硫代碳酰肼(TCH)(Electronic Microscopy Science,目录号:21900)
  8. 四氧化锇4%溶液(Electronic Microscopy Science,目录号:19150)
  9. 醋酸铀(UA)(Electron Microscopy Sciences,目录号:22400)
  10. 硝酸铅(结晶/Certified ACS),Fisher Chemical(Fisher Scientific,目录号:L62-100)
  11. L-天冬氨酸溶液(Sigma-Aldrich,目录号:A9256)
  12. 乙醇,200标准(100%),USP,Decon Labs(Fisher Scientific,目录号:07-678-004)
  13. 丙酮(电子显微镜科学,目录号:10010)
  14. EMbed-812试剂盒(Electronic Microscopy Sciences,目录号:14120)
  15. EMS成型平嵌入模具(Electronic Microscopy Sciences,目录号:70905-01)

设备

  1. Vibratome(Leica,目录号:Leica VT1000S)
  2. Zeiss Sigma VP SEM(Zeiss)与Gatan 3View(Gatan)


    图1. Zeiss Sigma VP SEM(Zeiss)with Gatan 3View(Gatan)

程序

  1. 在1-2分钟内用20 ml PBS在心脏内灌注小鼠,然后在5分钟内用含有1%GA的50ml 4%PFA灌注。
  2. 解剖出小鼠脑/脊柱,并用4%PFA和1%GA在4℃下后固定24小时。
  3. 从脊柱剥离脊髓。
  4. 固定的脑/脊髓切片在振动器上制成100μm厚度。
  5. 根据感兴趣的区域,将脑/脊髓切片切成0.2×0.2cm 2的组织块。
  6. 将组织块置于1.5ml Eppendorf管中,并在1ml冷(4℃)0.1M二甲胂酸盐缓冲液中孵育3次,每次5分钟,在冰上。
  7. 在使用之前,通过将3%的铁氰化钾在0.2M二甲砷酸盐缓冲液和等体积的4%四氧化锇水溶液中混合,制备新鲜的OsO 4 - 亚铁氰化物缓冲液。将组织块在该溶液中在冰上孵育1小时。
  8. 在1小时温育期间,新鲜制备新鲜硫代卡巴酰肼溶液。将0.1g硫代卡巴酰肼(TCH)加入10ml ddH 2 O中,并在60℃烘箱中放置1小时。通过每10分钟轻轻摇动缓冲液以促进溶解。在使用前通过0.22μm微孔注射器过滤器过滤该溶液。
  9. 在0.2ml OsO 4 - 亚铁氰化物中温育结束时,将组织在室温下在ddH 2 O中孵育3次,每次5分钟。
  10. 去除样品管中的ddH 2 O并在60℃下孵育后直接将TCH加入样品管中。将组织块在室温下在TCH中孵育20分钟。
  11. 将组织块在1ml ddH 2 O中孵育3次,每次5分钟,然后在室温下在新制备的0.2ml 2%四氧化锇中温育30分钟。
  12. 将组织块在1ml ddH 2 O中孵育3次,每次5分钟,然后在4℃下在1%乙酸双氧铀中温育过夜。
  13. 第二天,新鲜制备天冬氨酸溶液。首先,制备0.03M天冬氨酸缓冲液。简言之,在50ml ddH 2 O中加入0.2g L-天冬氨酸。将pH值调节至5.5,然后将天冬氨酸溶液置于60℃直至天冬氨酸完全溶解。其次,制备天冬氨酸铅溶液。将0.066g硝酸铅溶于10ml天冬氨酸溶液中。将铅溶液在60℃的烤箱中30分钟。
  14. 将组织块在1ml ddH 2 O中孵育3次,每次5分钟,然后在60℃的铅溶液中温育30分钟。
  15. 将组织块在室温下在1ml ddH 2 O中孵育3次,每次5分钟,然后使用新鲜制备的20%,60%,80%,90%,100% 100%乙醇,每步5分钟。
  16. 将组织块在丙酮中在室温下温育10分钟。
  17. 在丙酮孵育期间,使用Epon-812包埋试剂盒制备100%Epon溶液,如下:将EMbed-812 10ml,十二碳烯基琥珀酸酐(DDSA)8ml,甲基-5-降冰片烯-2,3-二羧酸酐4 2,2-二甲氧基丙烷(DMP-30)0.4ml一起很好。为了制备50%Epon溶液,将等体积的100%Epon与100%丙酮混合
  18. 将组织块在50%Epon溶液中在室温下温育2小时。
  19. 将组织块在室温下置于摇床上的100%Epon溶液中过夜
  20. 第二天早上,使用配方在17中准备新的100%Epon溶液。将组织转移到新的Epon溶液中,然后在室温下在振荡器上放置2小时。
  21. 转移Epon到塑料扁平嵌入模具,并在60℃烘箱中聚合组织块过夜
  22. 切割从Epon块中取出的样本,并尝试删除样本周围的所有Epon 注意:最好切割所有的Epon,因为Epon将导致在扫描期间充电。 切割后组织块的最佳尺寸为1 * 1 * 2 mm 3 ,但这是非常技术要求,人们很少达到 标准。
  23. 使用具有Gatan 3视图的Zeiss Sigma VP SEM获得SBF-SEM图像。 通过以8192×8192像素的分辨率切割(48×48×20)μm<3>组织块(长度×宽度×深度)获得100nm步长的图像的连续图像堆叠。
    注意:步骤1-20应在通风橱下进行。

代表数据



图2.来自显示小胶质细胞中的核和线粒体的堆叠图像的代表性单SBF-SEM图像。 ↑:Mitochondria; ★:核。


第二部分。 3D-EM图像中的核和线粒体的定量

软件

  1. 斐济软件(NIH; http://fiji.sc/Downloads

程序

  1. 将同一堆叠的图像保存在一个文件夹中。
  2. 打开斐济软件并将文件夹拖动到斐济软件以打开图像。
  3. 单击插件/注册/线性堆栈与SIFT对齐以生成对齐的堆栈图像。
  4. 将堆栈图像保存为tiff格式。
  5. 单击文件/新建/TrakEM2(空白),然后选择存储TrakEM文件的位置。
  6. 将tiff格式的对齐堆叠图像拖动到TrakEM图像处理站。
  7. 在模板窗口中,右键单击任何/添加新的子/区列表
  8. 在"项目对象"窗口中,右键单击"未命名/添加/新建任何内容"
  9. 在项目对象窗口中,右键单击任何/添加/新建区域列表。
  10. 右键单击新建area_list /重命名以将图层重命名为核。
  11. 在项目对象窗口中,右键单击任何/添加/新建区域列表。
  12. 右键单击新建area_list /重命名将该层重命名为线粒体。
  13. 在图像处理站中,右键单击堆叠图像,选择显示/校准,将长度单位更改为微米,并将像素宽度,像素高度和体素深度放在窗口中。 像素宽度和像素高度应根据图像扫描设置计算。 体素深度是堆叠中每个图像的间隔。 例如,如果图像的间隔为100nm,则体素深度应为0.1微米
  14. 在"图层"窗口中,选择所有图层,然后右键单击图层,选择"缩放Z"和"厚度",计算缩放数,应放在绕线器中,并使每个图层的间隔等于体素深度。
  15. 在图像处理站中,窗口左侧应出现两层命名为核和线粒体
  16. 点击核心层,双击滴管工具选择一种颜色。
  17. 使用笔工具绘制在堆栈的每个图像中的核的轮廓,并单击shift + F填充每个图像中的核区域,以使核的3D重建
  18. 在斐济的控制惩罚中,单击分析/测量以测量核的参数,包括体积
  19. 核形状的量化在3D重建中进行。核被分类如下;圆形:圆形和光滑表面,长/宽比≤1.5;细长的:长/宽> 1.5的细长或椭圆形,并且可具有小的凹痕;生叶:具有单个中间大缺口的两个连接的叶;不规则:具有波纹表面的复杂形状,并且可以具有多个和可变的大小 凹痕。
  20. 点击线粒体层,双击滴管工具选择除核以外的一种颜色。
  21. 使用笔工具绘制线框内的每个图像的线粒体的轮廓,并单击shift + F填充轮廓在每个图像,以使线粒体的三维重建
  22. 在斐济的控制面板中,单击分析/测量以测量线粒体的参数,包括体积和最大直径(线粒体的长度)。
  23. 通过所有的堆栈图像,选择五个不同的图像,其中线粒体的横截面积可以代表横截面面积的中值。
  24. 在五个代表图像中,使用线工具和控制面板中的分析/测量来绘制和测量轴和b轴的长度。
  25. 使用横截面积=πab。将所有参数保存在excel文件中。通过以下公式计算平均横截面积:五个横截面积/5的总和。
  26. 重复步骤11-12为第二个线粒体添加另一层。
  27. 重复步骤20-25做线粒体的定量。
  28. 重复步骤26-27,直到完成细胞中所有线粒体的定量。
  29. 计算细胞中线粒体的数量和线粒体的总体积

致谢

作者要感谢Richard M. Ransohoff博士实验室的成员对这份手稿的帮助。 这项工作由国家多发性硬化症协会支持。

参考文献

  1. TrackEM2用户手册: http://www.ini.uzh.ch/~acardona/trakem2_manual .html
  2. TrakEM2教程: http://fiji.sc/TrakEM2_tutorials

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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lu, H., Ohno, N. and Ransohoff, R. M. (2015). Morphological Quantification of Nuclei and Mitochondria in Serial Block-face Scanning Electron Microscopy Images. Bio-protocol 5(18): e1593. DOI: 10.21769/BioProtoc.1593.
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