Serial Section Transmission Electron Microscopy Research Articles

Inter-individual stereotypy of the Platynereis larval visual connectome.

Developmental programs have the fidelity to form neural circuits with the same structure and function among individuals of the same species. It is less well understood, however, to what extent entire neural circuits of different individuals are similar. Previously, we reported the neuronal connectome of the visual eye circuit from the head of a Platynereis dumerilii larva (Randel et al., 2014). We now report a full-body serial section transmission electron microscopy (ssTEM) dataset of another larva of the same age, for which we describe the connectome of the visual eyes and the larval eyespots. Anatomical comparisons and quantitative analyses of the two circuits reveal a high inter-individual stereotypy of the cell complement, neuronal projections, and synaptic connectivity, including the left-right asymmetry in the connectivity of some neurons. Our work shows the extent to which the eye circuitry in Platynereis larvae is hard-wired.

Induction of Hippocampal Synapses on Functionalized Micropatterns

We developed a novel presynaptic neuronal preparation in culture. Using microstructured coverslips functionalized with protein domains of synaptic cell adhesion molecules (SCAMs) as artificial postsynapse we are able to induce hippocampal neurons to form hybrid synapses, i.e. purely presynaptic structures ‘en face’ directly onto the coverslip.Axons growing over such micropatterns, functionalized with SCAM domains by click chemistry, form large flat varicosities. 4Pi microscopy revealed the presence of several presynaptic markers like the synaptic vesicle protein Synaptophysin1 or the active zone scaffold proteins RIM1/2, while postsynaptic staining against e.g. PSD 95, HOMER and the AMPA receptor was absent. Serial section transmission electron microscopy (TEM) as well as focused ion beam scanning electron microscopy (FIB-SEM) confirmed that these varicosities harbor a few hundred synaptic vesicles in several clusters near and at the bottom membrane and show typical active zone hallmarks at the bottom membrane. We found that isolated presynaptic varicosities are maintained and stable for at least two weeks allowing transfection and expression of fluorescent probes such as fusion constructs of the pH sensitive pHluorin protein. PHluorin is quenched at the acidic pH within vesicles and becomes fully fluorescent upon fusion. Neurons transfected with synaptophysin-pHluorin could be imaged after ten to 14 days in vitro by total internal reflection fluorescence microscopy (TIRFM). Where varicosities were formed on the functionalized micropatterns, we observed single fusion events, visible as diffraction-limited spots, on stimulation with single action potentials.Thus, this novel purely presynaptic preparation allows us to monitor the dynamics of presynaptic exo- and endocytosis in unprecedented detail.

Ultrastructural analysis of Caenorhabditis elegans cilia.

In addition to organizing centrosomes, centrioles have an evolutionarily conserved role as basal bodies in the formation of cilia. The discovery of a range of human genetic disorders linked to cilia dysfunction has led to a resurgence of interest in this cellular organelle. The nematode Caenorhabditis elegans possesses several unique features (highly stereotypical morphology, dispensability of cilia for organismal viability and fertility) that make it an attractive model to study cilia assembly and function. However, both the adult worm and the embryo present particular challenges for electron microscopy (EM), which remains the gold standard for high-resolution morphological studies. Here, we present a step-by-step guide for the ultrastructural analysis of C. elegans cilia, including optimized protocols for standard chemical fixation as well as high-pressure freezing/freeze substitution and further processing for serial-section transmission electron microscopy and electron tomography.

Three-dimensional volume imaging with electron microscopy toward connectome.

Ultrastructural analyses with electron microscopy have provided indispensable information to understand physiology and pathology of the nervous system. Recent advancement in imaging methodology paved the way for complete reconstruction of the neuronal connection map in the central nervous system, which is termed 'connectome' and would provide key insights to understand the functions of the brain. The critical advancement includes serial ultrastructural observation with scanning electron microscopy (SEM) instead of conventional serial sectioning transmission electron microscopy along with specific tissue preparation methods to increase heavy metal deposition for efficient SEM imaging. The advanced imaging methods using SEM have distinct advantages and disadvantages in multiple aspects, such as resolution and imaging speed, and should be selected depending on the observation conditions, such as target tissue sizes, required spatial resolution and necessity for re-observation. Dealing with the huge dataset remained to be a major obstacle, and automation in segmentation and 3D reconstruction would be critical to understand neuronal circuits in a larger volume of the brain. Future improvement in acquisition and analyses of the morphological data obtained with the advanced SEM imaging is awaited to elucidate the significance of whole connectome as the structural basis of the consciousness, intelligence and memory of a subject.

Some OFF bipolar cell types make contact with both rods and cones in macaque and mouse retinas.

This study compared the types of OFF bipolar cells found in the macaque retina with those found in the mouse retina and determined whether these OFF bipolar cells make direct contacts with both rods and cones by serial section transmission electron microscopy. We performed scatter plots and cluster analysis of the morphological variables of their axon terminals such as the stratification level, the arbor thickness, the arbor area, and the number of ribbons. Five OFF bipolar cell types, including the recently discovered DB3b type, were identified in the macaque retina. The macaque OFF bipolar cell types FMB, DB1, DB2, DB3a, and DB3b corresponded to the mouse OFF bipolar cell types 2, 1, 4, 3a, and 3b, respectively. In addition to contacting rod bipolar cells, ~7% of rods in the macaque retina made basal contacts exclusively with one cell type, DB3b, whereas 18% of rods in the mouse retina made basal contacts with one or two of types, 3a, 3b, and 4. Approximately 3% of mouse rods were divergently connected to two OFF bipolar cells of different types, but macaque rods were solely connected to one OFF bipolar cell. Rod-rod gap junctions were localized at rod cell bodies and axons in the outer nuclear layer in both macaque and mouse retinas. The direct rod-OFF bipolar connection system is slightly more developed in the mouse retina than in the macaque retina, possibly as a fine-tuned adaptation to nocturnal conditions. This one-step direct synaptic pathway from rods to OFF bipolar cells may enhance the response speed to OFF light stimuli compared with more indirect pathways via rod-cone gap junctions (a two-step pathway) and via rod bipolar and AII amacrine cells (a three-step pathway).

Membrane dynamics at the nuclear exchange junction during early mating (one to four hours) in the ciliate Tetrahymena thermophila.

Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila. Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.

Neuronal connectome of a sensory-motor circuit for visual navigation.

Animals use spatial differences in environmental light levels for visual navigation; however, how light inputs are translated into coordinated motor outputs remains poorly understood. Here we reconstruct the neuronal connectome of a four-eye visual circuit in the larva of the annelid Platynereis using serial-section transmission electron microscopy. In this 71-neuron circuit, photoreceptors connect via three layers of interneurons to motorneurons, which innervate trunk muscles. By combining eye ablations with behavioral experiments, we show that the circuit compares light on either side of the body and stimulates body bending upon left-right light imbalance during visual phototaxis. We also identified an interneuron motif that enhances sensitivity to different light intensity contrasts. The Platynereis eye circuit has the hallmarks of a visual system, including spatial light detection and contrast modulation, illustrating how image-forming eyes may have evolved via intermediate stages contrasting only a light and a dark field during a simple visual task.

Synapses lacking astrocyte appear in the amygdala during consolidation of pavlovian threat conditioning

There is growing evidence that astrocytes, long held to merely provide metabolic support in the adult brain, participate in both synaptic plasticity and learning and memory. Astrocytic processes are sometimes present at the synaptic cleft, suggesting that they might act directly at individual synapses. Associative learning induces synaptic plasticity and morphological changes at synapses in the lateral amygdala (LA). To determine whether astrocytic contacts are involved in these changes, we examined LA synapses after either threat conditioning (also called fear conditioning) or conditioned inhibition in adult rats by using serial section transmission electron microscopy (ssTEM) reconstructions. There was a transient increase in the density of synapses with no astrocytic contact after threat conditioning, especially on enlarged spines containing both polyribosomes and a spine apparatus. In contrast, synapses with astrocytic contacts were smaller after conditioned inhibition. This suggests that during memory consolidation astrocytic processes are absent if synapses are enlarging but present if they are shrinking. We measured the perimeter of each synapse and its degree of astrocyte coverage, and found that only about 20-30% of each synapse was ensheathed. The amount of synapse perimeter surrounded by astrocyte did not scale with synapse size, giving large synapses a disproportionately long astrocyte-free perimeter and resulting in a net increase in astrocyte-free perimeter after threat conditioning. Thus astrocytic processes do not mechanically isolate LA synapses, but may instead interact through local signaling, possibly via cell-surface receptors. Our results suggest that contact with astrocytic processes opposes synapse growth during memory consolidation.

Inhibitory and multisynaptic spines, and hemispherical synaptic specialization in the posterodorsal medial amygdala of male and female rats

The density of dendritic spines is sexually dimorphic and variable throughout the female estrous cycle in the rat posterodorsal medial amygdala (MePD), a relevant area for the modulation of reproductive behavior in rats. The local synaptic activity differs between hemispheres in prepubertal animals. Here we used serial section transmission electron microscopy to produce 3D reconstructions of dendritic shafts and spines to characterize synaptic contacts on MePD neurons of both hemispheres in adult males and in females along the estrous cycle. Pleomorphic spines and nonsynaptic filopodia occur in the MePD. On average, 8.6% of dendritic spines received inputs from symmetric gamma-aminobutyric acid (GABA)-immunoreactive terminals, whereas 3.6% received two synaptic contacts on the spine head, neck, or base. Presynaptic terminals in female right MePD had a higher density of synaptic vesicles and docked vesicles than the left MePD, suggesting a higher rate of synaptic vesicle release in the right MePD of female rats. In contrast, males did not show laterality in any of those parameters. The proportion of putative inhibitory synapses on dendritic shafts in the right MePD of females in proestrus was higher than in the left MePD, and higher than in the right MePD in males, or in females in diestrus or estrus. This work shows synaptic laterality depending on sex and estrous cycle phase in mature MePD neurons. Most likely, sexual hormone effects are lateralized in this brain region, leading to higher synaptic activity in the right than in the left hemisphere of females, mediating timely neuroendocrine and social/reproductive behavior.

Serial Block-Face Imaging and its Potential for Reconstructing Diminutive Cell Systems: A Case Study from Arthropods

Until recently, three-dimensional reconstruction on an ultrastructural level was only possible using serial section transmission electron microscopy (ssTEM). However, ssTEM is highly challenging and prone to artifacts as, e.g., section loss and image distortions. New methods, such as serial block-face scanning electron microscopy (SBFSEM) overcome these limitations and promise a high lateral resolution. However, little is known about the usability of SBFSEM in diminutive, but highly complex cellular systems. We used spider sperm (~3 µm in diameter), which fulfills these conditions, to analyze the potential of SBFSEM compared with ssTEM. Our data suggest that the resolution obtained by SBFSEM allows depicting structures on a cellular level and is sufficient to discriminate subcellular components, but is highly dependent on previous staining procedures and electron density of the target structures.

Automated Detection of Synapses in Serial Section Transmission Electron Microscopy Image Stacks

We describe a method for fully automated detection of chemical synapses in serial electron microscopy images with highly anisotropic axial and lateral resolution, such as images taken on transmission electron microscopes. Our pipeline starts from classification of the pixels based on 3D pixel features, which is followed by segmentation with an Ising model MRF and another classification step, based on object-level features. Classifiers are learned on sparse user labels; a fully annotated data subvolume is not required for training. The algorithm was validated on a set of 238 synapses in 20 serial 7197×7351 pixel images (4.5×4.5×45 nm resolution) of mouse visual cortex, manually labeled by three independent human annotators and additionally re-verified by an expert neuroscientist. The error rate of the algorithm (12% false negative, 7% false positive detections) is better than state-of-the-art, even though, unlike the state-of-the-art method, our algorithm does not require a prior segmentation of the image volume into cells. The software is based on the ilastik learning and segmentation toolkit and the vigra image processing library and is freely available on our website, along with the test data and gold standard annotations (http://www.ilastik.org/synapse-detection/sstem).

Ultrastructure and phylogeny of Theleodinium calcisporum gen. et sp. nov., a freshwater dinoflagellate that produces calcareous cysts

Craveiro S.C., Pandeirada M.S., Daugbjerg N., Moestrup Ø. and Calado A.J. 2013. Ultrastructure and phylogeny of Theleodinium calcisporum gen. et sp. nov., a freshwater dinoflagellate that produces calcareous cysts. Phycologia 52: 488–507. DOI: 10:2216/13–152.1A freshwater photosynthetic dinoflagellate isolated from a shallow lake near Aveiro, Portugal, was examined by light microscopy, scanning electron microscopy (SEM) and serial-section transmission electron microscopy (TEM), and characterized genetically. Cells were small, spherical to slightly elongated, and had a projecting, nearly cylindrical, apical pore. The chloroplasts were yellowish-brown, arranged near the surface of the cell, and had up to four pyrenoids surrounded by starch sheaths. The cells had a peridinioid plate pattern with Kofoidian plate formula pp, cp, x, 3′ (seldom 4′), 2a, 7′′, 6c, 5s (6s?), 5′′′, 2′′′′. A small extruded peduncle was observed by SEM in cells with intact membranes. A microtubular basket, made of about 46 microtubules disposed in four rows, was seen in the ventral area in connection with the cytoplasmic extension that made the peduncle. The flagellar apparatus was typical of a peridinioid with two roots associated with each of the basal bodies and a layered connective linking the proximal ends of roots 1 and 4. In dense cultures, this organism produced a round resting cyst with a thick wall covered by irregular calcified elements. Energy dispersive X-ray analysis (EDS) analysis of the cysts showed calcium as the most abundant element. A total of 3048 nucleotides of the nuclear ribosomal operon were sequenced and used in a phylogenetic analysis that placed this organism as a sister group to a clade of Scrippsiella species and the parasitic Duboscquodinium collinii. Theleodinium calcisporum gen. et sp. nov. is described for the first freshwater dinoflagellate reported to produce calcareous cysts.

The Microtubular Cytoskeleton of the Apusomonad Thecamonas, a Sister Lineage to the Opisthokonts

Apusomonads are of evolutionary interest because they are close relatives to the supergroup Opisthokonta, which contains both animals and fungi. There are no detailed morphological studies of 'amastigomonad' type apusomonads, such as Thecamonas trahens, despite this species having a sequenced genome. We use serial-section transmission electron microscopy and 3D reconstruction to examine the cell architecture and complete microtubular cytoskeleton of Thecamonas. Thecamonas has two flagella and an anteriorly projecting 'tusk'. The anterior basal body associates with one microtubular root, which travels leftward, and a non-root 'ribbon' of six microtubules that travels down the right side of the cell. The posterior basal body associates with three roots: an eight-membered right root, a doublet left root, and an intermediate singlet root. These rearrange into two bands, both on the left side of the cell. One comprises the left and singlet roots plus one right root microtubule. The other comprises the remaining right root microtubules. A splitting right root and supernumerary singlet root are also present in breviates, ancyromonads, and 'typical excavates', suggesting that these characters are ancestral for much of eukaryote diversity. If so, opisthokonts, and most or all living eukaryotes, probably arose from cells with complex microtubular cytoskeletons.

Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization

Collagen fibrils are the major tensile element in vertebrate tissues, in which they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess the formation of the fibrils, predominantly by measuring fibril diameter. Here we describe a protocol for measuring fibril diameter as well as fibril volume fraction, mean fibril length, fibril cross-sectional shape and fibril 3D organization, all of which are major determinants of tissue function. Serial-section TEM (ssTEM) has been used to visualize fibril 3D organization in vivo. However, serial block face-scanning electron microscopy (SBF-SEM) has emerged as a time-efficient alternative to ssTEM. The protocol described below is suitable for preparing tissues for TEM and SBF-SEM (by 3View). We describe how to use 3View for studying collagen fibril organization in vivo and show how to find and track individual fibrils. The overall time scale is ~8 d from isolating the tissue to having a 3D image stack.

Morphology, microanatomy and sequence data of Sclerolinum contortum (Siboglindae, Annelida) of the Gulf of Mexico.

Sclerolinum is a small genus of Siboglinidae (Annelida) living in an obligate mutualistic association with thiotrophic bacteria as adults. Its taxonomic position, based on morphology, has been controversial; however, molecular data point to a sister taxa relationship with vestimentiferans. 16S rRNA gene sequencing and comparative morphology revealed that the studied population from deep-sea hydrocarbon seeps of the Gulf of Mexico belongs to Sclerolinum contortum known from the Arctic Sea. Since no anatomical and microanatomical studies have been published yet, we conducted such a study on S. contortum using serial sectioning and light and transmission electron microscopy. We show that the Sclerolinum body, divided into a head, trunk, and opisthosoma, is very similar to that of the vestimentiferans, and therefore we propose that the body regions are homologous in both taxa.

Three-dimensional reconstruction of electron micrographs reveals intrabulbar circuit differences between accessory and main olfactory bulbs

Three-dimensional (3D) reconstruction of synaptic arrangement on a particular dendrite provides essential information regarding neuronal properties and neural microcircuits. Unconventional synapses are particularly good candidates for such steric attribution. In main and accessory olfactory bulbs (MOBs and AOBs), there are dendrodendritic reciprocal synapses (RSs) between excitatory projection neurons and inhibitory interneurons. Although the fine structure and configuration of these synapses have been investigated in MOB, their characteristics in AOB were unknown. In this study, we performed 3D AOB reconstruction using serial section transmission electron microscopy. We found numerous RSs on primary dendrites from glomeruli to mitral/tufted (MT) cell somas. These synapses formed between dendritic shafts of MT cells and large dendritic spines, or so-called gemmules, of granule (Gr) cells. This indicates that chemical signals received by a glomerulus are regulated in the primary dendrite of an MT cell before reaching its soma. In MOB, RSs are located on secondary dendrites and act as lateral and self-inhibiting following mitral cell depolarization. Our results indicate that AOB intrabulbar microcircuitry is quite different from that in the MOB.

Introduction to Electron Microscopy

Event Abstract Back to Event Introduction to Electron Microscopy Moritz Helmstaedter1* 1 Max Planck Institute of Neurobiology, Germany Electron Microscopy has experienced a revival in the past decade, based on the goal of imaging neural tissue at the required scope and resolution for dense circuit mapping. The lecture will review the advantages and limitations of conventional serial sectioning transmission electron microscopy. It will then cover novel automated electron microscopy techniques that have fueled hopes to reconstruct neural circuits with substantial completeness, a methodological prerequisite for the neuroscientific research now termed “connectomics”. Keywords: Cortex, connectomics, introduction, electron, Microscopy Conference: Imaging the brain at different scales: How to integrate multi-scale structural information?, Antwerp, Belgium, 2 Sep - 6 Sep, 2013. Presentation Type: Lecture Topic: Image acquisition across different modalities Citation: Helmstaedter M (2013). Introduction to Electron Microscopy. Front. Neuroinform. Conference Abstract: Imaging the brain at different scales: How to integrate multi-scale structural information?. doi: 10.3389/conf.fninf.2013.10.00009 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Aug 2013; Published Online: 30 Aug 2013. * Correspondence: Dr. Moritz Helmstaedter, Max Planck Institute of Neurobiology, Munich, Germany, mhelmstaedter@neuro.mpg.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Moritz Helmstaedter Google Moritz Helmstaedter Google Scholar Moritz Helmstaedter PubMed Moritz Helmstaedter Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Serial Section Scanning Electron Microscopy (S3EM) on Silicon Wafers for Ultra-Structural Volume Imaging of Cells and Tissues

High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S3EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 µm3 volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S3EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S3EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation.

Stability of presynaptic vesicle pools and changes in synapse morphology in the amygdala following fear learning in adult rats

Changes in synaptic strength in the lateral amygdala (LA) that occur with fear learning are believed to mediate memory storage, and both presynaptic and postsynaptic mechanisms have been proposed to contribute. In a previous study we used serial section transmission electron microscopy (ssTEM) to observe differences in dendritic spine morphology in the adult rat LA after fear conditioning, conditioned inhibition (safety conditioning), or naïve control handling (Ostroff et al. [2010] Proc Natl Acad Sci U S A 107:9418-9423). We have now reconstructed axons from the same dataset and compared their morphology and relationship to the postsynaptic spines between the three training groups. Relative to the naïve control and conditioned inhibition groups, the ratio of postsynaptic density (PSD) area to docked vesicles at synapses was greater in the fear-conditioned group, while the size of the synaptic vesicle pools was unchanged. There was significant coherence in synapse size between neighboring boutons on the same axon in the naïve control and conditioned inhibition groups, but not in the fear-conditioned group. Within multiple-synapse boutons, both synapse size and the PSD-to-docked vesicle ratio were variable between individual synapses. Our results confirm that synaptic connectivity increases in the LA with fear conditioning. In addition, we provide evidence that boutons along the same axon and even synapses on the same bouton are independent in their structure and learning-related morphological plasticity.

High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy

Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high-resolution confocal microscopy, deconvolution and image analysis with NeuronStudio. Recent technical advancements include better preservation of tissue, resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically to identify both optimal resolution and the highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing three experimental groups with eight subjects each can take as little as 1 month if optimized for speed, or approximately 4-5 months if the highest resolution and morphometric detail is sought.