Volume electron microscopy for neuronal circuit reconstruction
Highlights
► The dense analysis of neuronal circuits requires volume electron microscopy. ► We review new EM methods that provide increases in z-resolution and throughput. ► Tradeoffs between resolution, acquisition speed, and reliability are discussed. ► We argue wiring diagrams are necessary but not sufficient to understand circuits.
Introduction
Vertebrate and invertebrate nervous systems are densely packed with intertwining neuronal axons and dendrites and the synapses between them. The small physical size of these structures, as thin as 40–50 nm in diameter [1, 2], requires imaging by electron microscopy (EM), particularly when the goal is the dense reconstruction of neuronal circuits. By imaging volumes of brain using 3-dimensional EM, the details of neuronal shape and connectivity can be reconstructed. Importantly, and in contrast to fluorescence-based labeling approaches that require sparse labeling [3] or super-resolution optical imaging [4, 5] to resolve densely packed neurites, standard EM stains result in a relatively unbiased staining of all membranes and synapses in the neuropil [6]. This means that EM volumes can, in principle, be used to reconstruct the complete connectivity of a neuron with all its presynaptic and postsynaptic partners. Furthermore, this operation can be repeated for all the neurons in the volume, such that the connectivity of the neurons comprising a circuit — its wiring diagram or ‘connectome’ — can be extracted.
The main challenge in volume EM imaging is to acquire a data set of sufficient size, resolution, and completeness that the tortuous trajectories of axons and dendrites can be followed, and the chemical (and, ideally, electrical) synaptic connections identified. The necessary volume depends on the anatomical extent of the circuit to be characterized. For example, the volume of an entire adult nematode, Caenorhabditis elegans (approximately 50 μm × 50 μm × 1000 μm, [7]), is about 1% that of a single mouse cortical column (400 μm × 400 μm × 1000 μm, [8]). The required 3D voxel resolution depends on how fine the processes are that must be traced: the finer the process, the greater the required resolution to reliably follow it over a long distance. For example, dendritic spine necks in the mammalian central nervous system can be as fine as 40 nm in diameter [2], and the fine neurites of the fruit fly Drosophila melanogaster can be as thin as 50 nm in diameter [1]. The required completeness of the EM-imaged volume is related to required resolution: when image data are lost due to staining artifacts, a missed section, or some other glitch in the imaging process, the probability of ambiguities in the dataset increases, resulting in fine processes becoming lost or mixed up during tracing.
There is currently no ‘best’ volume EM imaging method. Rather, each of the available methods involves tradeoffs in size, resolution and completeness, and which method is most appropriate depends on the scientific questions under investigation. The field, however, is changing rapidly. Here we summarize current volume EM methods, focusing on those designed to answer questions about neuronal circuit structure, and offer suggestions about the types of circuit questions that each method is currently well suited to answer.
Section snippets
Acquisition techniques
The primary dichotomy between modern volume EM methods lays in the choice of widefield transmission electron microscopy (TEM)-based or scanning electron microscopy (SEM)-based techniques. Because TEM-based approaches rely on the imaging of those electrons that pass through a specimen, a requirement is the use of thin sections cut before imaging. SEM, in contrast, is typically used to image electrons backscattered from the surface of samples, allowing the surfaces of both thin sections and
Resolution and reliability
The lateral resolutions obtainable in TEMs remain unparalleled, with sub-nanometer resolutions easily achieved in modern TEMs. In practice, sub-nanometer imaging is overkill for the purposes of circuit reconstruction; a common pixel resolution in TEM is ∼2–4 nm (e.g. [11•, 12, 13]). Such resolutions are also achievable in SEMs where the size of the electron probe ultimately limits resolutions to 1–2 nm [22]. What resolution do we need for circuit reconstruction? If the minimum diameter of a
Acquisition speed
Because local neuronal circuits can span 3D volumes of at least hundreds of microns on a side, acquisition speed has become an increasingly important parameter (Figure 3). A TEM camera array (TEMCA, Figure 1a) enabled high acquisition rates through the use of multiple high frame rate cameras in combination with optimized stage motion and on-line image processing software [11•]. System throughput is largely determined by how quickly image frames can be read out from the cameras in the array, and
Post-acquisition alignment
Once a series of images has been acquired, an alignment step is necessary to stitch the images into a 3D volume. An advantage of block-face SEM imaging is the inherent registration that comes with acquiring images before sectioning; typically only a simple translational shift of images is required [24•]. Alignment is more complicated for ssTEM and ATUM-SEM sections, often requiring local warping algorithms to compensate for section stretching, folds in sections, and distortions that occur
Tissue preparation and correlative techniques
The electron dense staining of tissue primarily still relies on chemical compounds first described decades ago during the early years of biological EM. Stains based on high-Z number elements such as osmium, uranium, and lead are most common for each of the techniques described [30]. The use of en bloc staining methods [30] is, in particular, essential for the block-face methods in which post-staining of sections is not possible. But the art of staining is by no means dead. The development of
Current best applications and future outlook
Each volume EM method has a maximum section size. ATUM-SEM sections are supported by a tape substrate, and can therefore be as large as 2.5 mm × 6 mm (K.J. Hayworth, pers. comm.). Sections cut for TEM can be as large as 1 mm × 2 mm, with the possibility of longer sections if custom support grids (e.g. [14], p. 60) are used. SBEM, in its current implementation, is limited to tissue blocks of about 1 mm on a side [18••], although there is no fundamental technological limit to acquiring larger volumes. For
Is it worth it?
As neuroscientists interested in the structure of circuits, a common question we are asked is something along the lines of: ‘Will wiring diagrams tell us how circuits work?’ Our answer is, ‘Not on their own.’ We argue that wiring diagrams, in the absence of any other information about cell type, synapse type, firing dynamics, etc. are likely insufficient to define circuit function. However, we think wiring diagrams may be necessary to understand circuit function. At a minimum, wiring diagrams
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank W. Denk for useful comments and criticism, and G. Knott, K. Hayworth and J. Lichtman for providing images and imaging speed parameters.
References (42)
- et al.
Synaptic organization of columnar elements in the lamina of the wild type in Drosophila melanogaster
J Comp Neurol
(1991) - et al.
Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics
J Neurosci
(1989) - et al.
Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP
Neuron
(2000) - et al.
Imaging intracellular fluorescent proteins at nanometer resolution
Science
(2006) - et al.
Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission
Proc Natl Acad Sci U S A
(2000) - et al.
The Fine Structure of the Nervous System: Neurons and their Supporting Cells
(1991) - et al.
A novel mode of ecdysozoan growth in Caenorhabditis elegans
Evol Dev
(2002) - et al.
High-resolution 2-deoxyglucose mapping of functional cortical columns in mouse barrel cortex
J Comp Neurol
(1988) - et al.
Life inside a thin section: tomography
J Neurosci
(2008) - et al.
Uniform serial sectioning for transmission electron microscopy
J Neurosci
(2006)
Network anatomy and in vivo physiology of visual cortical neurons
Nature
An integrated micro- and macroarchitectural analysis of the Drosophila brain by computer-assisted serial section electron microscopy
PLoS Biol
Exploring the retinal connectome
Mol Vis
Principles and Practice of Electron Microscope Operation
Point-spread functions for backscattered imaging in the scanning electron microscope
J Appl Phys
Automating the collection of ultrathin serial sections for large volume TEM reconstructions
Microsc Microanal
New technique for ultra-thin serial brain section imaging using scanning electron microscopy
Microsc Microanal
Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure
PLoS Biol
SEM images of block faces, cut by a miniature microtome within the SEM — a technical note
Scan Electron Microsc
Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling
J Neurosci
Site-specific 3D imaging of cells and tissues with a dual beam microscope
J Struct Biol
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