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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-016-0122-8) contains supplementary material, which is available to authorized users.

A targeted 3D EM and correlative microscopy method using SEM array tomography

“…In the published AT procedure, the array of sections is moved to the rear edge of the coverslip where it touches the interface between the glass and the water (Micheva and Smith, 2007). To attach the sections to support, water is drained from the side of the knife boat using a syringe (Micheva and Smith, 2007), or the coverslip is pulled out of the trough using a micromanipulator (Horstmann et al, 2012;Wacker et al, 2016). In our hands, this method compromises the control of water during draining.…”

Q&A: Array tomography

“…These attributes establish AT as an ideal choice for the most demanding analyses of diverse cellular architectures within mature and developing tissues, including brain. This essay will draw examples mainly from neuroscience, but AT methods are also finding many cell and tissue biology applications outside of neuroscience [ 1 – 13 ].…”

“…Various implementations and applications of AT are described in detail by excellent recent reviews [ 1 – 3 , 6 , 14 – 16 ]. Features common to all AT implementations include: (A) physical ultrathin serial sectioning of a fixed, resin-embedded specimen, (B) collection of the resulting serial sections to form an array on a solid substrate, (C) staining and digital imaging of the resulting serial section array by fluorescence microscopy (FM-AT) and/or electron microscopy (EM-AT), and (D) computational stitching of the resulting two-dimensional image tiles into coherent volumetric images.…”

“…Use of FM/EM-AT (Figs. 2 , 6 and 7 ) offers unique opportunities to combine the molecular discrimination strengths of fluorescence microscopy with the unrivaled structural resolution of electron microscopy [ 1 , 2 , 6 , 11 , 14 , 15 , 17 , 27 – 37 ]. While other approaches to correlative light and electron microscopy of individual specimens have proven extremely useful [ 38 ], AT alone offers a path to perfect registration of fluorescence and SEM voxels over extended volumes.…”

A Device for Ribbon Collection for Array Tomography with Scanning Electron Microscopy

“…However, the machine used in the ATUM method is expensive. Horstmann et al [ 4 ] and Wacker et al [ 10 ] have also reported a device for silicon substrate positioning. In their approach, the silicon substrate is clipped using forceps attached to a custom-built manipulator and a long ribbon is pulled out in a longitudinal direction from the water.…”