Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues

BMC Cell Biology

Methodology Article

Marlene Thaler¹  Irene Wacker²  Rasmus R. Schröder³  Stefan Hillmer⁴  Waldemar Spomer⁵  Ulrich Gengenbach⁵  Andreas Hofmann⁵ 

[1] Carl Zeiss Microscopy GmbH, Carl-Zeiss Str. 22, D-73447, Oberkochen, Germany;Cryo Electron Microscopy, Centre for Advanced Materials, Universität Heidelberg, Im Neuenheimer Feld 267, D-691120, Heidelberg, Germany;HEiKA, Heidelberg Karlsruhe Research Partnership, Heidelberg, Germany;Cryo Electron Microscopy, Centre for Advanced Materials, Universität Heidelberg, Im Neuenheimer Feld 267, D-691120, Heidelberg, Germany;HEiKA, Heidelberg Karlsruhe Research Partnership, Heidelberg, Germany;Cryo Electron Microscopy, CellNetworks, BioQuant, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 267, D-691120, Heidelberg, Germany;Electron Microscopy Core Facility, Universität Heidelberg, Im Neuenheimer Feld 345, D-69120, Heidelberg, Germany;HEiKA, Heidelberg Karlsruhe Research Partnership, Heidelberg, Germany;Institute for Applied Computer Science, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany;

关键词: Array tomography;    Serial sectioning;    Section libraries;    Hierarchical imaging;    Large volume 3D reconstruction;   

DOI  :  10.1186/s12860-016-0122-8

 received in 2016-05-18, accepted in 2016-11-29,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundImaging large volumes such as entire cells or small model organisms at nanoscale resolution seemed an unrealistic, rather tedious task so far. Now, technical advances have lead to several electron microscopy (EM) large volume imaging techniques. One is array tomography, where ribbons of ultrathin serial sections are deposited on solid substrates like silicon wafers or glass coverslips.ResultsTo ensure reliable retrieval of multiple ribbons from the boat of a diamond knife we introduce a substrate holder with 7 axes of translation or rotation specifically designed for that purpose. With this device we are able to deposit hundreds of sections in an ordered way in an area of 22 × 22 mm, the size of a coverslip. Imaging such arrays in a standard wide field fluorescence microscope produces reconstructions with 200 nm lateral resolution and 100 nm (the section thickness) resolution in z.By hierarchical imaging cascades in the scanning electron microscope (SEM), using a new software platform, we can address volumes from single cells to complete organs. In our first example, a cell population isolated from zebrafish spleen, we characterize different cell types according to their organelle inventory by segmenting 3D reconstructions of complete cells imaged with nanoscale resolution. In addition, by screening large numbers of cells at decreased resolution we can define the percentage at which different cell types are present in our preparation. With the second example, the root tip of cress, we illustrate how combining information from intermediate resolution data with high resolution data from selected regions of interest can drastically reduce the amount of data that has to be recorded. By imaging only the interesting parts of a sample considerably less data need to be stored, handled and eventually analysed.ConclusionsOur custom-designed substrate holder allows reproducible generation of section libraries, which can then be imaged in a hierarchical way. We demonstrate, that EM volume data at different levels of resolution can yield comprehensive information, including statistics, morphology and organization of cells and tissue. We predict, that hierarchical imaging will be a first step in tackling the big data issue inevitably connected with volume EM.

【 授权许可】

CC BY   
© The Author(s). 2016