Array tomography: trails to discovery

Abstract

Tissue slicing is at the core of many approaches to studying biological structures. Among the modern volume electron microscopy (vEM) methods, array tomography (AT) is based on serial ultramicrotomy, section collection onto solid support, imaging via light and/or scanning electron microscopy, and re-assembly of the serial images into a volume for analysis. While AT largely uses standard EM equipment, it provides several advantages, including long-term preservation of the sample and compatibility with multi-scale and multi-modal imaging. Furthermore, the collection of serial ultrathin sections improves axial resolution and provides access for molecular labeling, which is beneficial for light microscopy and immunolabeling, and facilitates correlation with EM. Despite these benefits, AT techniques are underrepresented in imaging facilities and labs, due to their perceived difficulty and lack of training opportunities. Here we point towards novel developments in serial sectioning and image analysis that facilitate the AT pipeline, and solutions to overcome constraints. Because no single vEM technique can serve all needs regarding field of view and resolution, we sketch a decision tree to aid researchers in navigating the plethora of options available. Lastly, we elaborate on the unexplored potential of AT approaches to add valuable insight in diverse biological fields.

Keywords: ATUM; array tomography; light microscopy; serial sectioning; ultramicrotomy; volume electron microscopy.

Figure 1:

Illustration of different volume EM options. The colors indicate the different approaches: block-face (FIB-SEM, blue; SBF-SEM, cyan), serial section TEM (ssTEM, orange) and AT (magenta), including transport modes and trail trajectories. Improvements of the AT pipeline are shown as hiking equipment, symbolizing automation options as well as optimization of mapping and image registration. Hidden regions of interest (ROI) and completed volume ultrastructural data sets are peaks to be reached (flag) dependent on the approach. All in all, block-face methods are the fastest way to reach the top, albeit along a fixed route. Traditional ssTEM provides the highest level of detail, but along a challenging trail that is not for the faint of heart. The AT hiker is well equipped to climb any trail (shorter or longer) to reach the peak and explore treasures along the way, e.g. hierarchical imaging options (rare flowers) or multimodality (native animal species).

Figure 2:

Immunofluorescence AT (IF-AT) versus EM. Both IF-AT and EM can be used to study myelinated axons in the mammalian brain [55]. With IF-AT, myelin is readily identified using myelin basic protein (MBP) immunolabeling, and mitochondria using MDH2 immunolabeling. With EM, these features are defined by their distinct ultrastructural appearance. Both approaches can quantify the prevalence and distribution of myelin within the tissue, as well as basic morphology. IF-AT can further analyze the molecular composition of these axons (e.g. neurotransmitter content, GABA) and their myelin, while EM provides the ultrastructural information to assess myelin or axonal integrity, as well as the tissue context to examine features such as the relationship between myelin and astrocytic processes. IF-AT and EM can be applied together as conjugate AT when both molecular and ultrastructural information is desired [37]. The method of choice varies depending on the scientific question.

Figure 3:

Proposed flow chart to support decision making between different vEM approaches for life science EM facilities with standard equipment. While there are physical constraints that make some techniques more suitable for specific research projects, the boundaries are quite flexible. Decision making is further impacted by technology accessibility and capacity, available expertise, and costs. In many cases, different approaches are equally well-suited and we only give suggestions for decision making. As an example, the criterium “sample restoration beneficial” refers to the advantage of methods that preserve the imaged sections, like AT or ssTEM, in case of precious samples (e.g. human biopsy or a tissue from a complex mouse model) that will potentially be used to answer several present and future scientific questions. Samples can be reimaged at different resolution regimes at a later time point, and shared around the world for alternative investigations. In case this is not needed, it might be more straightforward to apply block-face techniques. SBF-SEM provides restricted but not a “repetitive hierarchical imaging” option through the option to image at different resolution regimes during a run. In this chart we distinguish AT techniques according to their support material for section collection: rigid support AT (rsAT) and tape (ATUM) as this influences the block-face size and shape; when not specified, e.g. AT-SEM, either support material can be used. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), serial section TEM (ssTEM), focused ion beam (FIB), serial block-face (SBF), rigid support AT (rsAT), automated tape collecting ultramicrotomy (ATUM), correlated light and electron microscopy (CLEM), region of interest (ROI).