什么是系列连续切片成像?
系列连续切片扫描电子显微术(SBEM、SBSEM 和 SBFSEM)是从样品中重复获取高分辨率 3D 图像的一种方式。此方法尤擅在 X,Y,Z 坐标中进行纳米级分辨率的大视野成像。SBEM 通常依赖于扫描电子显微镜 (SEM) 中安装的原位超薄切片机。SEM 将收集切面图像,然后超薄切片机切割样品,暴露要成像的下一层,逐层切割,每层薄至 15 nm。
SBEM 的优势
功能 | 优势 |
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批量检验多个量级的结构 | 保留整个大数据集的细节层次 |
实现无中断的高产出 | 有助于加快样品分析和减少人为错误 |
展现所观察超微结构的特征 | 让您能够从一个样品中得到更全面的结果 |
控制切片和获取流程的各个方面 | 提供充分的灵活性,允许针对给定样品进行优化 |
消除截面损失、损坏和畸变 | 避免禁止的流程来修正损坏和畸变 |
用途
神经系统科学 | 细胞生物学 | 材料科学 | 其他 |
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神经系统科学
对 15,625 μm³ (25 x 25 x 25 μm) 体积数据集中的树突进行 3D 重建,该数据集包含 3View® 系统生成的 500 个小鼠小脑序列图像。树突结构(绿色)、终扣(黄色)和囊泡(红色)。插页图像,从左上方顺时针方向:树突共焦图像;渲染为体积模型的线框曲线;带突触的超高分辨率树突棘模型;显示线框曲线的图像。
细胞生物学:补充共焦
在无血清介质中饥饿 2 小时的 HeLa 细胞(能稳定表达刻蚀坐标玻璃底平皿盖玻片上生长的 LC-GFP)以及共焦显微镜识别的相关细胞的图像。然后,细胞经过原位处理以用于电子显微术,使用氢氟酸溶解盖玻片上的环氧树脂。再次在树脂坯块和 3View 系统生成的连续序列图像中识别细胞。
细胞生物学:发展
左上:高分辨率小鼠肾脏 8192 x 8192 像素图像,像素大小为 1.5 nm。上排中间:大视野小鼠肾脏 8192 x 8192 像素图像,像素大小为 80 nm。右:高压冷冻技术制备的秀丽隐杆线虫;高分辨率小鼠肾脏 4096 x 4096 像素图像,像素大小为 25 nm。样品由加州大学伯克利分校的 Kent McDonald 提供。左下:小鼠坐骨神经 2048 x 2048 像素图像,像素大小为 5 nm。下排中间:小鼠坐骨神经轴突 3D 可视化图像。
材料科学
左上和左下:3View 系统生成的铝表面阳极处理层图像。中间:3View 系统生成的带有锰颗粒的铝合金 3D 可视化图像。3D 数据集包含像素大小为 15 nm 且切割厚度为 15 nm 的 1,000 个 1024 x 1024 序列图像。使用 3D 可视化插件在 DigitalMicrograph® 中创建的 3D 模型。
系列连续切片成像的工作流程
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第 1 步:样品制备 固定典型生物样品,使用造影剂对样品进行着色,将样品嵌入树脂中使其保持稳定。 |
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第 2 步:安装并转移到 SEM 中 将样品修整为需要的大小,固定在铝销上,可通过溅射镀膜方法镀上一层金薄膜。将样品销放在 3View 系统中,与金刚石刀接触,门关闭并收回。 |
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第 3 步:优化成像 用户选择合适的电子束条件,类似于标准 SEM 所用的条件,但同时应考虑 Z 方向上的效应。选择合适的放大倍数、像素数和停留时间,以达到理想的图像分辨率、视野和获取时间。 |
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第 4 步:自动成像 用户可自行选择电子束条件来获取序列图像。生成每个图像之前,切割刀都会剥离一层样品表面。 |
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第 5 步:分析 图像堆叠形成 3D 数据集,可使用 DigitalMicrograph® 或第三方软件处理和查看。此时即可进行分段和定量。 |
DigitalMicrograph,或称为 Gatan Microscopy Suite,驱动您的电子相机和其他附件以支持一系列重要应用,包括断层扫描、原位、谱学和衍射成像等。
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Three-dimensional reconstruction of skeletal muscle extracellular matrix ultrastructure
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Resolution of three dimensional structure of components of the glomerular filtration barrier
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Zhang, N.; Kolesnikov, A. V.; Jastrzebska, B.; Mustafi, D.; Sawada, O.; Maeda, T.; Genoud, C.; Engel, A.; Kefalov, V. J.; Palczewski, K.
Starborg, T.; Kalson, N. S.; Lu, Y.; Mironov, A.; Cootes, T. F.; Holmes, D. F.; Kadler, K. E.
Paridaen, J. T. M. L.; Wilsch-Bräuninger, M.; Huttneremail, W. B.
Three-dimensional structure analysis and percolation properties of a barrier marine coating
Chen, B.; Guizar-Sicairos,M.; Xiong,G.; Shemilt, L.; Diaz, A.; Nutter, J.; Burdet,N.; Huo,S.; Mancuso, J.; Monteith,A.; Vergeer,F.; Burgess,A.; Robinson, I.
Anttonen, T.; Kirjavainen, A.; Belevich, I.; Laos, M.; Richardson, W. D.; Jokitalo, E.; Brakebusch, C.; Pirvola, U.
Müllner, T.; Zankel, A.; Mayrhofer, C.; Reingruber, H.; Höltzel, A.; Lv, Y.; Svec, F.; Tallarek, U.
Investigation of dealloying by ultra-high-resolution nanotomography
Hashimoto, T.; Curioni, M.; Zhou, X.; Mancuso, J.; Skeldon, P.; Thompson, G. E.
Schwartz, C. L.; Heumann, J. M.; Dawson, S. C.; Hoenger,A.
Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity
Pinheiro, I.; Margueron, R.; Shukeir, N.; Eisold, M.; Fritzsch, C.; Richter, F. M.; Mittler, G.; Genoud, C.; Goyama, S.; Kurokawa, M.; Son, J.; Reinberg, D.; Lachner, M.; Jenuwein, T.
Puhka, M.; Joensuu, M.; Vihinen, H.; Belevich, I.; Jokitalo, E.
Zhuravleva, E.; Gut, H.; Hynx, D.; Marcellin, D.; Bleck, C. K.; Genoud, C.; Cron, P.; Keusch, J. J.; Dummler, B.; Esposti, M. D.; Hemmings, B. A.
Azeloglu, E.; Stothers, M.; Deerinck, T. J.; Falkenberg, C.; Chen, Y.; He, J. C.; Hone, J. C.; Leslie, L. M.; Ellisman, M. H.; Iyengar, R.
Extrusion of misfolded and aggregated proteins--a protective strategy of aging neurons?
Doehner, J.; Genoud, C.; Imhof, C.; Krstic, D.; Knuesel, I.
3D segmentation of SBFSEM images of neuropil by a graphic model over supervoxel boundaries
Andres, B.; Koethe, U.; Kroeger, T.; Helmstaedter, M.; Briggman, K. L.; Denk, W.; Hamprecht, F. A.
Differential modulation of retinal degeneration by Ccl2 and Cx3cr1 chemokine signalling
Luhmann, U. F.; Lange, C. A.; Robbie, S.; Munro, P. M.; Cowing, J. A.; Armer, H. E.; Luong, V.; Carvalho, L. S.; MacLaren, R. E.; Fitzke, F. W.; Bainbridge, J.W.; Ali, R. R.
Beam deceleration for block-face scanning electron microscopy of embedded biological tissue
Ohta, K.; Sadayama, S.; Togo, A.; Higashi, R.; Tanoue, R.; Nakamura, K.
Multivesicular exocytosis in rat pancreatic beta cells
Hoppa, M. B.; Jones, E.; Karanauskaite, J.; Ramracheya, R.; Braun, M.; Collins, S. C.; Zhang, Q.; Clark, A.; Eliasson, L.; Genoud, C.; Macdonald, P. E.; Monteith, A. G.; Barg, S.; Galvanovskis, J.; Rorsman, P.
Structural neurobiology: Missing link to a mechanistic understanding of neural computation
Denk, W.; Briggman, K. L.; Helmstaedter, M.
Giuly, R. J.; Martone, M. E.; Ellisman, M. H.
Volume electron microscopy for neuronal circuit reconstruction
Briggman, K. L.; Bock, D. D.
Yan, J.; Walker, C. G.; O'sullivan, M. J.; Bushong, E. A.; Ellisman, M. H.; Hoshijima, M.; Rajagopal, V.
High contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy
Tapia, J. C.; Kasthuri, N.; Hayworth, K. J.; Schalek, R.; Lichtman, J. W.; Smith, S. J.; Buchanan, J.
Modern electron microscopy methods for C. elegans
Hall, D. H.; Hartwieg, E.; Nguyen, K. C.
Ou, H. D.; Kwiatkowski, W.; Deerinck, T. J.; Noske, A.; Blain, K. Y.; Land, H. S.; Soria, C.; Powers, C. J.; May, A. P.; Shu, X.; Tsien, R. Y.; Fitzpatrick, J. A. J.; Long, J. A.; Ellisman, M. H.; Choe, S.; O'Sheaemail, C. C.
Mustafi, D.; Kevany, B. M.; Genoud, C.; Okano, K.; Cideciyan, A. V.; Sumaroka, A.; Roman, A. J.; Jacobson, S. G.; Engel, A.; Adams, M. D.; Palczewski, K.
Motskin, M.; Müller, K. H.; Genoud, C.; Monteith, A. G.; Skepper, J. N.
Kalson, N. S.; Holmes, D. F.; Herchenhan, A.; Lu, Y.; Starborg, T.; Kadler, K. E.
Wong, J. I. S.; Baddeley, D.; Bushong, E. A.; Ellisman, M.; Hoshijima, M.; Soeller, C.
Fast extraction of neuron morphologies from large-scale SBFSEM image stacks
Lang, S.; Drouvelis, P.; Tafaj, E.; Bastian, P.; Sakmann, B.
Mun, J. Y.; Jeong, S. Y.; Kim, J. H.; Han, S. S.; Kim, I. H.
Basement membrane changes in capillaries of the ageing human retina
Powner, M. B.; Scott, A.; Zhu, M.; Munro, P. M.; Foss, A. J.; Hageman, G. S.; Gillies, M. C.; Fruttiger, M.
Cadherin-9 regulates synapse-specific differentiation in the developing hippocampus
Williams, M. E.; Wilke, S. A.; Daggett, A.; Davis, E.; Otto, S.; Ravi, D.; Ripley, B.; Bushong, E. A.; Ellisman, M. H.; Klein, G.; Ghosh, A.
High-accuracy neurite reconstruction for high-throughput neuroanatomy
Helmstaedter, M.; Briggman, K. L.; Denk, W.
Role of elastin anisotropy in structural strain energy functions of arterial tissue
Rezakhaniha, R.; Fonck, E.; Genoud, C.; Stergiopulos, N.
Modeling of flow in a polymeric chromatographic monolith
Koku, H.; Maier, R. S.; Czymmek, K. J.; Schure, M. R.; Lenhoff, A. M.
Quantitative Characterisation of microfiltration membranes by 3D reconstruction
Reingruber, H.; Zankel, A.; Mayrhofer, C.; Poelt, P.
Shu, X.; Lev-Ram, V.; Deerinck, T. J.; Qi, T.; Ramko, E. B.; Davidson, M. W.; Jin, Y. Ellisman, M. H.; Tsien, R. Y.
Wiring specificity in the direction-selectivity circuit of the retina
Briggman, K. L.; Helmstaedter, M.; Denk, W.
Nguyen, J. V.; Soto, I.; Kim, K.; Bushong, E. A.; Oglesby, E.; Valiente-Soriano, F. J.; Yang, Z.; Davis, C. O.; Bedont, J. L.; Son, J. L.; Wei, J. O.; Buchman, V. L.; Zack, D. J.; Vidal-Sanz, M.; Ellisman, M. H.; Marsh-Armstrong, N.
Environmental scanning electron microscopy (ESEM)--a versatile tool in studying plants.
Stabentheiner, E.; Zankel A.; Pölt P.
Jurrus, E.; Paiva, A. R.; Watanabe, S.; Anderson, J. R.; Jones, B. W.; Whitaker, R. T.; Jorgensen, E. M.; Marc, R. E.; Tasdizen, T.
Reconstruction of a neuron from SBFSEM: tools, reliability, accuracy and efficiency
Drouvelis, P.; Kurz, T.; Bastian, P.; Sakmann, B.; Lang, S.
Deerinck, T. J.; Bushong, E. A.; Lev-Ram, V.; Shu, X.; Tsien, R. Y.; Ellisman, M. H.
Structure-function studies of blood and air capillaries in chicken lung using 3D electron microscopy
West, J. B.; Fu, Z.; Deerinck, T. J.; Mackey, M. R.; Obayashi, J. T.; Ellisman, M.H.
Imaging transient blood vessel fusion events in zebrafish by correlative volume electron microscopy
Armer, H. E. J.; Mariggi, G.; Png, K. M. Y.; Genoud, C.; Monteith, A. G.; Bushby, A. J.; Gerhardt, H.; Collinson, L. M.
Saetzler, K.; McCanny, P.; Rodriguez, E.P.; Horstmann, H.; Bruno, R.M.; Denk, W.
Axon tracking in serial block-face scanning electron microscopy
Jurrus, E.; Hardy, M.; Tasdizen, T.; Fletcher, P. T.; Koshevoy, P.; Chien, C. B.; Denk, W.; Whitaker, R.
Mishchenko, Y.
Three-dimensional reconstruction methods for Caenorhabditis elegans ultrastructure
Müller-Reichert, T.; Mancuso, J.; Lich, B.; McDonald, K.
Ultramicrotomy in the ESEM, a versatile method for materials and life sciences
Zankel, A.; Kraus, B.; Poelt, P.; Schaffer, M.; Ingolic, E.
Genoud, C.; Mancuso, J.; Monteith, S.; Kraus, B.
Contour-propagation algorithms for semi-automated reconstruction of neural processes
Macke, J. H.; Maack, N.; Gupta, R.; Denk, W.; Schölkopf, B.; Borst, A.
Segmentation of SBFSEM volume data of neural tissue by hierarchical classification
Andres, B.; Köthe, U.; Helmstaedter, M.; Denk, W.; Hamprecht, F. A.
Alpha-herpesvirus infection induces the formation of nuclear actin filaments
Feierbach, B.; Piccinotti, S.; Bisher, M.; Denk, W.; Enquist, L. W.
Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure
Denk, W.; Horstmann, H.