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Volume flattening process. (A) Isosurface of a single mouse brain coronal section. The two boundary surfaces are delimited by two sets of points (in green and in blue). This 3D plot clearly shows the section unevenness that is corrected for during the flattening step. (B) XYZ view of a warped mouse brain coronal section within the IMOD viewer. The volume density has been converted from RGB to gray scale. Two objects consisting of a set of points (respectively in green and in blue) model the spatial variation of the section boundaries. These points are used as references during the flattening procedure. The specimen aspect ratio was modified and stretched along the z direction to allow the manual positioning of the marker points in the viewer. (C) XYZ view of a flattened mouse brain coronal section within the IMOD viewer. A warped section is submitted to local compressions/expansions along z the direction, forcing the specimen boundaries onto two parallel planes. The transformation leaves the section volume unchanged.
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Recent advances in high-throughput technology facilitate massive data collection and sharing, enabling neuroscientists to explore the brain across a large range of spatial scales. One such form of high-throughput data collection is the construction of large-scale mosaic volumes using light microscopy (Chow et al., 2006; Price et al., 2006). With th...
Citations
... Hence, automated, rapid, and adaptable atlas segmentation tools are still lacking but mandatory, for instance, when dealing with the segmentation of so-called single brain slices (devoid of 3D reference) needing to locate the 2D plane of each slice within a 3D atlas template volume. As the mouse brain has an elongated shape, most of the studies observe mouse brains in the coronal incidence (Bohland et al., 2010;Berlanga et al., 2011;Renier et al., 2016;Vandenberghe et al., 2016;Staeger et al., 2020), and we therefore focused on this incidence. Three parameters enable the exact location of a single slice plane within the atlas volume: (1) the z-position of the slice along the rostrocaudal (antero-posterior, AP) axis orthogonal to the coronal plane; ...
Conventional histology of the brain remains the gold standard in the analysis of animal models. In most biological studies, standard protocols usually involve producing a limited number of histological slices to be analyzed. These slices are often selected into a specific anatomical region of interest or around a specific pathological lesion. Due to the lack of automated solutions to analyze such single slices, neurobiologists perform the segmentation of anatomical regions manually most of the time. Because the task is long, tedious, and operator-dependent, we propose an automated atlas segmentation method called giRAff, which combines rigid and affine registrations and is suitable for conventional histological protocols involving any number of single slices from a given mouse brain. In particular, the method has been tested on several routine experimental protocols involving different anatomical regions of different sizes and for several brains. For a given set of single slices, the method can automatically identify the corresponding slices in the mouse Allen atlas template with good accuracy and segmentations comparable to those of an expert. This versatile and generic method allows the segmentation of any single slice without additional anatomical context in about 1 min. Basically, our proposed giRAff method is an easy-to-use, rapid, and automated atlas segmentation tool compliant with a wide variety of standard histological protocols.
... In skeletal muscles, lipid molecules are stored both extramyocellularly (in the connective tissue between muscle fibres) and intramyocellularly (as lipid droplets within skeletal muscle fibers) [15]. The intramyocellular lipids (IMCLs), composed mainly of triglycerides, are known to be metabolically active, providing an important energy source for skeletal muscle cells during exercise. ...
... The status of tolerance for glucose was assessed by the OGTT: A 25% glucose solution (2 g kg −1 ) was administered via orogastric tube feeding following 6-hour fasting (water available ad libitum) [21], with venous blood glucose assayed before and at 15,30,60, and 120 minutes after the glucose administration using the Bayer Contour glucose meter (Ascensia Diabetes Care Holdings AG, Basel, Switzerland). Blood samples were collected from the tail vein of the animals. ...
... While this phenomenon results from a range of tissue processing protocols including dehydration, drying, paraffin embedding and coverslipping; conversely, appropriate fixation and embedding conditions protect the sections against collapsing 1,9,12-14 . If the section deformation is homogenous, it can be corrected by stretching of sample image in the Z direction 15 . Differential deformation of tissue sections in the Z direction is however more problematic because this cannot be simply corrected during postprocessing. ...
Background: Obesity-related metabolic disorders are among the leading causes of morbidity and mortality in the developed world. Structural and functional changes in skeletal muscles and the microvasculature are critically involved in the mediation of obesity-related insulin resistance. A shift from expression of slow to fast type myosin heavy chain isoforms in slow twitch muscles has been shown to contribute to reduced insulin sensitivity in obesity, while in fast-twitch weight-bearing muscles no fibre type shifting was observed. Furthermore, in obesity, accumulation of lipid droplets in skeletal muscle fibres can also contribute to insulin resistance. However, it is not yet clear how intramyocellular lipid accumulation and fibre type changes are associated. Moreover, in advanced obesity with insulin resistance, reduced capillary network density in skeletal muscles and impaired capillary recruitment has been demonstrated. On the other hand, in the early stage of obesity with insulin resistance, an increased functional vascular response to insulin has been described. However, it remains unclear whether such functional vascular response is associated with morphological alterations in the capillary network, or if the switch of fibre types toward fast type isoforms also occurs in fast-twitch, non-weight-bearing muscles in the early stages of obesity with insulin resistance.
Methods: The study was carried out using eighteen 54-week-old C57BL/6JOlaHsd mice, divided into two study groups of nine mice each: the high fat diet-induced obese and the standard diet-treated lean groups. Insulin resistance status was assessed by the oral glucose tolerance test and fasting glucose measurements. We determined the capillary network characteristics using 3D analysis of 100 µm thick transverse sections of gluteus maximus muscle, and employed immunofluorescent techniques to mark the capillary endothelium and the basement membrane of capillaries and muscle fibres, which were then captured with a confocal microscope and analysed with the Ellipse software. We used the indirect immunohistochemical method to determine the myosin heavy chain isoform expression in muscle fibres. Antibodies against myosin heavy chain isoforms type 1, 2a, 2x/d and 2b were used to determine the expression of individual myosin heavy chain isoforms in successive 10 µm thick sections of gluteus maximus, gastrocnemius, plantaris and soleus muscles. We performed analysis of intramyocellular lipid content using 10 µm thick sections of gastrocnemius, plantaris and soleus muscles stained with Sudan Black B, and calculated the lipid content index as 100 times the ratio of the cross-sectional area of muscle fibre occupied by lipid droplets to the cross-sectional area of muscle fibre. The deformations of skeletal muscle thick tissue sections in horizontal plane were determined by comparison of gluteus maximus muscle fibre diameters in 10 µm thick native tissue sections to 100 µm thick fixed and immunofluorescently labelled tissue sections, respectively.
Results: Compared to the standard diet-treated lean mice, mice fed on a high fat diet had a significantly increased body mass (p = 0.0001) and basal glycaemia, and decreased glucose tolerance (p < 0.05). The obese mice also showed denser capillary network of the gluteus maximus muscle compared to lean mice. Compared to the lean mice, capillary length per muscle fibre length, and capillary length per muscle fibre surface area were significantly larger around small muscle fibres (< 40 µm) (p < 0.05) in the obese mice, while there were no significant differences around large fibres in both groups. Other capillary characterization indices such as tortuosity, anisotropy and fibre diameter did not significantly differ between the study groups. In the slow-twitch soleus and fast-twitch non-weight-bearing gluteus maximus muscles, we noted a shift towards fast type myosin heavy chain isoform expression in the obese mice (p < 0.05), while in the weight-bearing fast-twitch gastrocnemius and intermediate plantaris muscles there were no significant differences in myosin heavy chain expression between the two study groups. Moreover, in obese mice muscle fibre size and intramyocellular lipid content were significantly increased in type 2a and 2x/d muscle fibres (p<0.05), with greater prominence in the fast- and intermediate-twitch than slow-twitch skeletal muscles. During the preparation of thick transverse sections of skeletal muscle for confocal microscopy, we demonstrated significant dilation of the sections in horizontal direction (p < 0.001) and shrinkage in the axial direction (p < 0.001). In addition, we noted a positive correlation between the magnitude of horizontal dilation and the magnitude of shrinkage in the axial direction (r = 0.493, p < 0.01), the latter being more pronounced in transversely than obliquely cut tissue sections.
Conclusions: We found a selective increase in capillarisation around small muscle fibres of the more oxidative fibre types in our obese insulin-resistant mice, which could be an early compensatory mechanism ameliorating obesity-related insulin resistance. Our findings further suggest that in obesity with insulin resistance, both slow- and fast-twitch muscles exhibit the tendency for a shift toward fast type myosin heavy chain isoforms, and that increased weight bearing may condition the resistance of fast-twitch muscles to fibre type shifting. Our results also reaffirm that in obesity, intramyocellular lipid accumulation is specific for both skeletal muscle and fibre types, with greater prominence in fast-twitch muscles and muscle fibre types 1, 2a and 2x/d. Finally, our study also provide evidence on horizontal dilation and axial shrinkage of thick transverse sections of skeletal muscle. The magnitude of the former was partially dependent on the latter, suggesting that even though axial shrinkage can be corrected by calibration, histological protocols should be optimised to minimize the axial collapse that could cause horizontal dilation.
... Myelin staining can provide 3D information about the local nerve fiber distribution by 3D volume reconstruction of a series of brain sections. CLSM achieves 0.2 μm in-plane resolution 25 . Myelin immunostaining can further reveal differences in the myelination pattern between healthy and diseased brains 26 . ...
The structural connectivity of the brain has been addressed by various imaging techniques such as diffusion weighted magnetic resonance imaging (DWMRI) or specific microscopic approaches based on histological staining or label-free using polarized light (e.g., three-dimensional Polarized Light Imaging (3D-PLI), Optical Coherence Tomography (OCT)). These methods are sensitive to different properties of the fiber enwrapping myelin sheaths i.e. the distribution of myelin basic protein (histology), the apparent diffusion coefficient of water molecules restricted in their movements by the myelin sheath (DWMRI), and the birefringence of the oriented myelin lipid bilayers (3D-PLI, OCT). We show that the orientation and distribution of nerve fibers as well as myelin in thin brain sections can be determined using scanning small angle neutron scattering (sSANS). Neutrons are scattered from the fiber assembly causing anisotropic diffuse small-angle scattering and Bragg peaks related to the highly ordered periodic myelin multilayer structure. The scattering anisotropy, intensity, and angular position of the Bragg peaks can be mapped across the entire brain section. This enables mapping of the fiber and myelin distribution and their orientation in a thin brain section, which was validated by 3D-PLI. The experiments became possible by optimizing the neutron beam collimation to highest flux and enhancing the myelin contrast by deuteration. This method is very sensitive to small microstructures of biological tissue and can directly extract information on the average fiber orientation and even myelin membrane thickness. The present results pave the way toward bio-imaging for detecting structural aberrations causing neurological diseases in future.
... The mechanical preparation of thin sections from frozen or formalin-fixed paraffin-embedded (FFPE) 3D specimens, with subsequent staining, imaging, and image reconstruction, has been considered the standard method for visualization at the cellular resolution [8,9]. However, the troublesome and time-consuming steps of these preparation procedures often cause the potential loss of sections, which may result in imprecise spatial reconstruction, leading to partial or incomplete sample analysis and incompatibility with high-throughput screening (HTS). ...
... However, critical limitations exist in the immunostaining and visualization of such 3D models due to two major inherent obstacles: (1) the limited penetration of macromolecules such as staining antibodies and (2) light scattering. Mechanical sectioning is an available alternative [8,9]; however, the laborious and skill-demanding procedure is unfavorable, and 3D rendering from 2D sections is unreliable [25,26]. In this study, we successfully applied TOC to microtumor samples cultured on minipillar array chips. ...
Simple Summary
Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) recapitulates solid tumors in vivo. This microtumor model is particularly useful for multiplex phenotypic analysis, but requires tissue optical clearing (TOC) for 3D visualization. We developed a transfer-free 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with the TOC method. Our method succeeded in improving immunostaining and optical transmission in each TS as well as the entire microtumor specimen. The utility of this method was demonstrated by showing phenotypic changes, such as increased levels of membrane protrusion, single-cell dissemination, and ECM remodeling, and changes in the expression of epithelial–mesenchymal transition–related proteins and drug-induced apoptosis in TSs of human pancreatic cancer cells co-cultured with cancer-associated fibroblasts and M2-type tumor-associated macrophages.
Abstract
Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) represents a microtumor model that recapitulates human solid tumors in vivo, and is useful for 3D multiplex phenotypic analysis. However, the low efficiency of 3D culture and limited 3D visualization of microtumor specimens impose technical hurdles for the evaluation of TS-based phenotypic analysis. Here, we report a 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with a tissue optical clearing (TOC) method for high-content phenotypic analysis of microtumors. To prove the utility of this method, phenotypic changes in TSs of human pancreatic cancer cells were determined by co-culture with cancer-associated fibroblasts and M2-type tumor-associated macrophages. Significant improvement was achieved in immunostaining and optical transmission in each TS as well as the entire microtumor specimen, enabling optimization in image-based analysis of the morphology, structural organization, and protein expression in cancer cells and the ECM. Changes in the invasive phenotype, including cellular morphology and expression of epithelial–mesenchymal transition-related proteins and drug-induced apoptosis under stromal cell co-culture were also successfully analyzed. Overall, our study demonstrates that a minipillar array chip combined with TOC offers a novel system for 3D culture-to-3D visualization of microtumors to facilitate high-content phenotypic analysis.
... 1,9,[12][13][14] If the section deformation is homogenous, it can be corrected by stretching of sample image in the Z direction. 15 Differential deformation of tissue sections in the Z direction is however more problematic because this cannot be simply corrected during postprocessing. Such deformation can occur as a consequence of inhomogeneous sample composition, 10 particular sample processing techniques where samples are processed on the slides, 16 or the use of particular embedding and sectioning methods. ...
Certain morphological parameters of the skeletal muscle tissue can be better understood via 3D considerations. Fluorescent confocal microscopy of thick tissue sections is a well‐established method for visualising and measuring skeletal muscle fibres and surrounding capillaries in 3D. However, thick tissue sections are prone to deformations which may significantly influence some stereological and morphometric results like muscle fibre diameter and capillary length, but not dimensionless parameters like object number and Euler‐Poincaré characteristics. To better understand this phenomenon, we studied the horizontal deformation of thick (100 µm) transverse skeletal muscle sections, by comparing the muscle fibre diameters measured on thick sections to muscle fibre diameters measured on thin (10 µm) sections of the same sample. Diameter changes were further correlated with shrinkage in the Z direction (axial shrinkage) and deviation of the muscle fibre preferential axis from the Z‐axis. We showed that the thick sections dilated in horizontal and shrunk in Z direction, and that the magnitude of horizontal dilation was associated with the magnitude of shrinkage in the Z direction. The latter was more pronounced in transversely than obliquely cut tissue sections. The results emphasise that even when shrinkage in the Z direction can be corrected using calibration, it is important to optimise histological protocols to minimise the Z‐axis collapse that could cause horizontal dilation.
Lay description
In skeletal muscle research, 3D analysis is especially important for studying the microvasculature. Laser scanning confocal microscopy of skeletal muscle thick tissue sections is a well‐established method for visualising and measuring skeletal muscle fibres and surrounding capillaries in 3D. However, such sections are prone to deformations which may significantly influence the study results. To better understand this phenomenon, we studied the horizontal deformation of thick transverse skeletal muscle sections. We compared the average muscle fibre diameters measured on thick skeletal muscle sections, thin fixed skeletal muscle sections and immunohistochemically stained thin skeletal muscle sections with the muscle fibre diameters measured on thin native skeletal muscle sections of the same sample, with the latter condition serving as the standard diameters (ie the control condition). We further studied the association among muscle fibre diameter changes, shrinkage of the thick skeletal muscle sections in the Z direction and their sectioning angle. We showed that the thick skeletal muscle sections dilated in the horizontal direction and shrunk in the Z direction, and that the magnitude of horizontal dilation was associated with the magnitude of shrinkage in Z direction. The shrinkage in the Z direction was more pronounced in transversely than obliquely cut tissue sections. These results emphasise that even when shrinkage in the Z direction can be corrected using Z‐axis calibration, it is very important to optimise histological protocols to minimise the Z‐axis collapse that could cause horizontal dilation in order to enhance the integrity of study results.
... The analysis of cell type or marker protein distribution in fixed frozen or paraffin-embedded biological 3D samples typically uses tissue sectioning followed by immunohistological staining, and confocal laser scanning microscopy (CLSM). Due to the time-consuming preparation, potential loss of tissue sections, and the cumbersome reconstruction of spatial 3D-information, such samples are mostly analyzed only partially (Leong, 2004;Berlanga et al., 2011;Marchevsky and Wick, 2015). In addition, this method is destructive and not compatible with high throughput. ...
Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 μm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-to-noise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols – in particular, for screening purposes – clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.
... Because serial passage of LNC led to reduced expression and loss of nuclear Pax6 staining and other neural crest markers, we wondered whether such a change was correlated with the loss of the neural progenitor status defined by neurosphere formation and neuroglial differentiation potential. LNC from 4 regions and CSC were serially passaged and seeded at the same density of 5 × 10 3 /cm 2 in poly-HEMA coated 12-well in the neurosphere medium containing 1.6% of methylcellulose for 7 days 32 . Spheres emerged with an increasing size (Fig. 3A, representative P4 and P10 LNC from Region A). ...
... Neurosphere formation. Single cells of both LNC or CSC expanded at different passages were plated at cell density of 5000/cm 2 on anti-adhesive poly-HEMA in 12 well-plate for 6 days in neural stem cell medium (NSCM) consisting of 20 ng/ml EGF, 20 ng/ml FGF2, 2% NSCM supplement, and 1.6% methylcellulose 32 . Sphere formation was monitored by phase microscope and spheres with the size of greater than 50 μm in diameter were counted throughout the entire 12-well on day 6 by Zeiss Axio-Observer Z1 Motorized Inverted Microscope (Carl Zeiss, Thornwood, NY). ...
On ocular surface, corneal epithelial stem cells (SC) reside in limbus between cornea and conjunctiva. Pax6, an evolutionally conserved transcription factor essential for eye development, is expressed in post-natal corneal and limbal epithelia progenitors (LEPC) but not in underlying stroma. Because Pax6 is transiently expressed in developing corneal stroma and a subset of limbal and corneal stromal progenitors, we examined the role of Pax6 in limbal niche cells (LNC) in maintaining the phenotype of neural crest (NC) progenitors to support LEPC. Our results showed that nuclear Pax6 staining was found in freshly isolated LNC but not corneal stromal cells. Serial passaged LNC resulted in gradual loss of nuclear Pax6 (46 kDa) staining and neural crest progenitor status defined by the expression of embryonic SCs and NC markers, neurosphere formation, and differentiation into neurons, oligodendrocytes and astrocytes. Gain of function of 46 kDa Pax6 in late-passaged LNC resulted in nuclear Pax6 staining and promotion of the aforementioned NC progenitor status. In an in vitro reunion assay, early passaged LNC and late passaged LNC with overexpression of Pax6 inhibited the expression of corneal epithelial differentiation marker and promoted holoclone by LEPC. Therefore, expression of nuclear 46 kDa Pax6 in LNC plays an important developmental role in maintaining NC progenitor status to support self-renewal of corneal epithelial SCs in the limbal niche.
... If this data is not available, then the vignetting effect can be eliminated by cropping the images to only keep the relatively flat illumination area at the center of each tile [98]. Alternatives are estimating flat-field from the dataset, either by averaging all images within the dataset [12,99], by averaging only the tiles within the agarose background [16], or by performing low-frequency high-pass filtering of the illumination intensity [100]. A recent method, BaSiC [101], is a more advanced signal model to perform retrospective background and shading correction. ...
... Indeed, this imaging and slicing configuration introduces a very limited amount of tissue deformations. This eliminates the resource-intensive and time-consuming processing steps that are necessary with conventional serial histology, such as slice flattening [88], or complex deformable tissue registrations between slices [99,[115][116][117][118]. In fact, some SBH systems [10,15,82] rely only on the accuracy of the motorized stage and on the slicing performance of the system and do not even perform axial registration between consecutive slices. ...
In recent years, multiple serial histology techniques were developed to enable whole rodent brain imaging in 3-D. The main driving forces behind the emergence of these imaging techniques were the genome-wide atlas of gene expression in the mouse brain, the pursuit of the mouse brain connectome, and the BigBrain project. These projects rely on the use of optical imaging to target neuronal structures with histological stains or fluorescent dyes that are either expressed by transgenic mice or injected at specific locations in the brain. Efforts to adapt the serial histology acquisition scheme to use intrinsic contrast imaging (ICI) were also put forward, thus leveraging the natural contrast of neuronal tissue. This review focuses on these efforts. First, the origin of optical contrast in brain tissue is discussed with emphasis on the various imaging modalities exploiting these contrast mechanisms. Serial blockface histology (SBH) systems using ICI modalities are then reported, followed by a review of some of their applications. These include validation studies and the creation of multimodal brain atlases at a micrometer resolution. The paper concludes with a perspective of future developments, calling for a consolidation of the SBH research and development efforts around the world. The goal would be to offer the neuroscience community a single standardized open-source SBH solution, including optical design, acquisition automation, reconstruction algorithms, and analysis pipelines.
... All the above measures which mitigate the 2D slice-specific artifacts and help its registration, in addition to being time consuming, are expensive and require a lot of experimental planning. Although, slicing thicker sections may be a plausible solution to avoid tissue tears [38], it constrains the subsequent staining and imaging procedures. One needs to ensure that the slicing thickness is in accordance with the penetration depth of the stain and depth of focus of the light microscope used. ...
Background:
Brain mapping research in most neuroanatomical laboratories relies on conventional processing techniques, which often introduce histological artifacts such as tissue tears and tissue loss.
New method:
In this paper we present techniques and algorithms for automatic registration and 3D reconstruction of conventionally produced mouse brain slices in a standardized atlas space. This is achieved first by constructing a virtual 3D mouse brain model from annotated slices of Allen Reference Atlas (ARA). Virtual re-slicing of the reconstructed model generates ARA-based slice images corresponding to the microscopic images of histological brain sections. These image pairs are aligned using a geometric approach through contour images. Histological artifacts in the microscopic images are detected and removed using Constrained Delaunay Triangulation before performing global alignment. Finally, non-linear registration is performed by solving Laplace's equation with Dirichlet boundary conditions.
Results:
Our methods provide significant improvements over previously reported registration techniques for the tested slices in 3D space, especially on slices with significant histological artifacts. Further, as one of the application we count the number of neurons in various anatomical regions using a dataset of 51 microscopic slices from a single mouse brain.
Comparison with existing method(s):
To the best of our knowledge the presented work is the first that automatically registers both clean as well as highly damaged high-resolutions histological slices of mouse brain to a 3D annotated reference atlas space.
Conclusions:
This work represents a significant contribution to this subfield of neuroscience as it provides tools to neuroanatomist for analyzing and processing histological data.
... Similarly, Jones et al. (2014) introduce an artifact as a fiducial mark from which section thickness can be estimated in FIB-SEM acquisitions under the assumption of planar sections. Berlanga et al. (2011) correct small volumes by evening out top and bottom surfaces that have been manually annotated by the user and transforming the whole series accordingly by a single transformation, which fails to capture varying thickness. Boergens and Denk (2013) reduce non-planar distortions during acquisition using measurements of the intensity of the ion beam to control the FIB-SEM milling process. ...
Motivation:
Serial section microscopy is an established method for detailed anatomy reconstruction of biological specimen. During the last decade, high resolution electron microscopy (EM) of serial sections has become the de-facto standard for reconstruction of neural connectivity at ever increasing scales (EM connectomics). In serial section microscopy, the axial dimension of the volume is sampled by physically removing thin sections from the embedded specimen and subsequently imaging either the block-face or the section series. This process has limited precision leading to inhomogeneous non-planar sampling of the axial dimension of the volume which, in turn, results in distorted image volumes. This includes that section series may be collected and imaged in unknown order.
Results:
We developed methods to identify and correct these distortions through image-based signal analysis without any additional physical apparatus or measurements. We demonstrate the efficacy of our methods in proof of principle experiments and application to real world problems.
Availability and implementation:
We made our work available as libraries for the ImageJ distribution Fiji and for deployment in a high performance parallel computing environment. Our sources are open and available at http://github.com/saalfeldlab/section-sort, http://github.com/saalfeldlab/z-spacing, and http://github.com/saalfeldlab/z-spacing-spark CONTACT: saalfelds@janelia.hhmi.org.
