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Apical dendrites of principal neurons can be traced in SXRT datasets a Volume of a mouse olfactory bulb obtained with SXRT, virtually sliced sagittally, displaying all main histological layers. Below, SXRT (b) and low-resolution SBEM ((50 nm)³ voxels) (c) of the highlighted region. d Apical dendrites traced in both imaging modalities by 3 independent tracers (grey lines: SBEM ground truth consensus, coloured lines: SXRT individual traces). e Traceable length of all SXRT dendrites and of the correctly linked ones. Traceable length measures the dendrite length through which SXRT tracing is within 12 µm (lost threshold) away from the paired EM tracing. Each dot represents one cell’s apical dendrite. The box covers the 25 to 75% percentile range; the middle bar represents the median value (printed above); the whiskers extend to the most extreme data points that are not an outlier (defined as outside of the 1.5× interquartile range). The grey dashed line marks the lost threshold used. Source data for (e) are provided as a Source Data file. onl, olfactory nerve layer; gl, glomerular layer; epl, external plexiform layer; mcl, mitral cell layer; ipl, inner plexiform layer.
Source publication
Understanding the function of biological tissues requires a coordinated study of physiology and structure, exploring volumes that contain complete functional units at a detail that resolves the relevant features. Here, we introduce an approach to address this challenge: Mouse brain tissue sections containing a region where function was recorded usi...
Citations
... However, achieving this requires establishing sample preparation protocols compatible with multiple imaging methods. One effective approach involves using light microscopy to capture a fluorescent-based labeled region of interest in a biological soft tissue sample (as demonstrated by Bosch et al., 2022). Subsequently, the sample is fixed and stained with heavy metals for high-resolution imaging at the selected site, revealing fine structural details. ...
Optical microscopy has revolutionized the field of biology, enabling researchers to explore the intricate details of biological structures and processes with unprecedented clarity. Over the past few decades, significant strides have been made in tailoring optical microscopy techniques to meet the specific needs of biologists (Schermelleh , 2019; Prakash , 2022). From sample preparation to hardware designs and software requirements, improvements have been driven by the goal of enhancing imaging capabilities and facilitating quantitative analysis.
... In recent years, with the continuous development and optimization of electron microscope imaging technology, big data storage, image processing technology and computer hardware technology, volume electron microscopy(vEM) has developed rapidly. vEM allows directly depict 3D ultrastructure of organisms, cells [1][2][3][4] and tissues 5,6 , especially has been widely used in connectomics studies of neurons [7][8][9][10] . The physical scale studied by vEM is also getting larger and larger, from the nanoscale to tens of microns and even hundreds of microns 9,11 . ...
... Therefore, we use the FSC-0.5 standard, which is more suitable for situations where the signal is much more significant than the noise 68 . (10) where and are Fourier transform of EMformer reconstructed volume and ground truth volume respectively at radius r in Fourier space; is the conjugator of ...
Volume electron microscopy (vEM) has become the rapidly developed technique to study the 3D architecture of biological specimen such as cells, tissues, and organs at nanometer resolution, which collects series of electron micrographs of axial sequential sections and reconstructs the 3D volume, providing fruitful information of cellular ultrastructural spectrum. This technique is currently suffering from the anisotropic resolution between lateral (x, y) and axial (z) directions and the loss/damage of sections. Here, we develop a new algorithm, EMformer, based on video transformer model, to boost the axial resolution and then achieve an isotropic reconstruction of vEM. By learning the high resolution axis structures and utilizing the 3D continuity of biological structures, EMformer can recovery the axial information and repair the random loss/damage sections based on self-supervision strategy, achieving a higher resolution than existing methods, which is validated for both simulated FIB-SEM dataset and experimental ssTEM dataset. Besides visual validation, the segmentation efficiency and statistical precision of various ultrastructures, e.g. neurons, mitochondria, vesicle, and membrane bilayers, also prove a better performance of EMformer. Therefore, using EMformer, we could achieve isotropic reconstruction from anisotropic axial sampling, which will increase the throughput of vEM to study a large scale of biological architectures.
... 24% replied that their largest dataset yet was larger than a Terabyte. Outside of this survey, the literature also contains several published vEM datasets that are well in the hundreds of Terabytes in size (Bosch et al., 2022;Consortium et al., 2021;Hildebrand et al., 2017;Loomba et al., 2022;Scheffer et al., 2020;Xu et al., 2021;Zheng et al., 2018). These numbers show that the vEM community uniquely and routinely deals with very large 3D image datasets. ...
The growing size of EM volumes is a significant barrier to findable, accessible, interoperable, and reusable (FAIR) sharing. Storage, sharing, visualization and processing are challenging for large datasets. Here we discuss a recent development toward the standardized storage of volume electron microscopy (vEM) data which addresses many of the issues that researchers face. The OME-Zarr format splits data into more manageable, performant chunks enabling streaming-based access, and unifies important metadata such as multiresolution pyramid descriptions. The file format is designed for centralized and remote storage (e.g., cloud storage or file system) and is therefore ideal for sharing large data. By coalescing on a common, community-wide format, these benefits will expand as ever more data is made available to the scientific community.
... As a result, only isolated micro-circuits have been mapped in mammalian brains [9], [14]- [17]. It has been recently demonstrated that synchrotron-based Xray nano-holography (XNH) can resolve neuronal wiring at larger scales than EM [18], [19], albeit with slightly lower resolution. XNH is particularly well-suited to map long-range connections, which are composed of thick, myelinated axons in the white-matter regions of the brain (Fig. 1a). ...
The wiring and connectivity of neurons form a structural basis for the function of the nervous system. Advances in volume electron microscopy (EM) and image segmentation have enabled mapping of circuit diagrams (connectomics) within local regions of the mouse brain. However, applying volume EM over the whole brain is not currently feasible due to technological challenges. As a result, comprehensive maps of long-range connections between brain regions are lacking. Recently, we demonstrated that X-ray holographic nanotomography (XNH) can provide high-resolution images of brain tissue at a much larger scale than EM. In particular, XNH is wellsuited to resolve large, myelinated axon tracts (white matter) that make up the bulk of long-range connections (projections) and are critical for inter-region communication. Thus, XNH provides an imaging solution for brain-wide projectomics. However, because XNH data is typically collected at lower resolutions and larger fields-of-view than EM, accurate segmentation of XNH images remains an important challenge that we present here. In this task, we provide volumetric XNH images of cortical white matter axons from the mouse brain along with ground truth annotations for axon trajectories. Manual voxel-wise annotation of ground truth is a time-consuming bottleneck for training segmentation networks. On the other hand, skeleton-based ground truth is much faster to annotate, and sufficient to determine connectivity. Therefore, we encourage participants to develop methods to leverage skeleton-based training. To this end, we provide two types of ground-truth annotations: a small volume of voxel-wise annotations and a larger volume with skeleton-based annotations. Entries will be evaluated on how accurately the submitted segmentations agree with the ground-truth skeleton annotations.
... Next morning, fixative was washed and the OB slab was then imaged at a 2-photon microscope ( 29 , Scientifica Multiphoton VivoScope, coupled with a SpectraPhysics MaiTai DeepSee laser tuned to 940 nm). Slabs were then stained with heavy metals using an established ROTO protocol 5,28 . Subsequently, samples were dehydrated with increasing ethanol solutions (75%, 90%, 2x100%), transferred to propylene oxide, and infiltrated with hard epon mixed with propylene oxide in increasing concentrations (25%, 50%, 75%, 2x100%). ...
... Synchrotron imaging was performed at the ID19 microtomography beamline at ESRF as described before 5 . This imaging modality benefits from the cylindrical geometry of the sample. ...
... SBF-SEM imaging was performed as described previously 5,27 . Volumes were acquired on a 3View2-Zeiss Merlin SBF-SEM under high vacuum (1.5 kV, 0.5 nA, 0.5 μs/pixel, 10 nm pixels, resulting in a surface dose of 15.6 e -/nm 2 ) using an OnPoint (Gatan) back-scattered electron detector. ...
Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently-labelled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods such as synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) or serial blockface scanning electron microscopy (SBF-SEM) might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging.
Here we showcase the utility of a novel femtosecond laser device to prepare microscopic samples (1) of an optimised geometry for synchrotron X-ray microscopy as well as (2) for subsequent volume electron microscopy applications, embedded in a wider correlative multimodal imaging workflow ( Fig. 1 ).
... In addition, the segmentation and manual curation turn out to be time-consuming for such large datasets, even with an established analysis pipeline. However, the ability of 3D electron microscopy to image tissue in an unbiased fashion (Kornfeld et al., 2017;Schmidt et al., 2017;Svara et al., 2018;Motta et al., 2019;Karimi et al., 2020;Morgan and Lichtman, 2020;Hua et al., 2021) could thereby generate unexpected results and trigger further studies using, for example, X-ray computed tomography (Bosch et al., 2022). Nevertheless, our work complements prior 3D EM studies [P2, P3, P4, P6, and P9 (Holcomb et al., 2013); P7, P21 (Thomas et al., 2019); P21, P180, P540, and P720, this study] providing a framework for studying connectomic and morphomic changes in the MNTB over the lifespan of rodents. ...
The medial nucleus of the trapezoid body (MNTB) is an integral component of the auditory brainstem circuitry involved in sound localization. The giant presynaptic nerve terminal with multiple active zones, the calyx of Held (CH), is a hallmark of this nucleus, which mediates fast and synchronized glutamatergic synaptic transmission. To delineate how these synaptic structures adapt to reduced auditory afferents due to aging, we acquired and reconstructed circuitry-level volumes of mouse MNTB at different ages (3 weeks, 6, 18, and 24 months) using serial block-face electron microscopy. We used C57BL/6J, the most widely inbred mouse strain used for transgenic lines, which displays a type of age-related hearing loss. We found that MNTB neurons reduce in density with age. Surprisingly we observed an average of approximately 10% of poly-innervated MNTB neurons along the mouse lifespan, with prevalence in the low frequency region. Moreover, a tonotopy-dependent heterogeneity in CH morphology was observed in young but not in older mice. In conclusion, our data support the notion that age-related hearing impairments can be in part a direct consequence of several structural alterations and circuit remodeling in the brainstem.
... While high energy X-ray beams are widely used for medical practice, their penetrating power is far too strong for cells and ex vivo biological samples. Synchrotron produces relatively "soft" X-rays with energy in the 0.1-1 keV range and fits better for cell and tissue-level research [54,214]. Unlike most electron or fluorophore-based microscopic imaging methods, synchrotron-based XRM can be used to image cellular and sub-cellular architectures without complex staining or risk of sample damage [215]. ...
Analyzing the complex structures and functions of brain is the key issue to understanding the physiological and pathological processes. Although neuronal morphology and local distribution of neurons/blood vessels in the brain have been known, the subcellular structures of cells remain challenging, especially in the live brain. In addition, the complicated brain functions involve numerous functional molecules, but the concentrations, distributions and interactions of these molecules in the brain are still poorly understood. In this review, frontier techniques available for multiscale structure imaging from organelles to the whole brain are first overviewed, including magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), serial-section electron microscopy (ssEM), light microscopy (LM) and synchrotron-based X-ray microscopy (XRM). Specially, XRM for three-dimensional (3D) imaging of large-scale brain tissue with high resolution and fast imaging speed is highlighted. Additionally, the development of elegant methods for acquisition of brain functions from electrical/chemical signals in the brain is outlined. In particular, the new electrophysiology technologies for neural recordings at the single-neuron level and in the brain are also summarized. We also focus on the construction of electrochemical probes based on dual-recognition strategy and surface/interface chemistry for determination of chemical species in the brain with high selectivity and long-term stability, as well as electrochemophysiological microarray for simultaneously recording of electrochemical and electrophysiological signals in the brain. Moreover, the recent development of brain MRI probes with high contrast-to-noise ratio (CNR) and sensitivity based on hyperpolarized techniques and multi-nuclear chemistry is introduced. Furthermore, multiple optical probes and instruments, especially the optophysiological Raman probes and fiber Raman photometry, for imaging and biosensing in live brain are emphasized. Finally, a brief perspective on existing challenges and further research development is provided.
... Other studies have improved the precision and ease of targeting by leveraging external 3D maps from light or X-ray microscopy (Karreman et al., 2016;Karreman et al., 2017;Xu et al., 2021;Musser et al., 2021;Bushong et al., 2015;Ronchi et al., 2021). X-ray offers many advantages for EM targeting -for example, it is readily compatible with standard EM sample preparation methods (Bosch et al., 2022;Kuan et al., 2020;Bushong et al., 2015;Karreman et al., 2017) and provides images that highlight similar structures to volume EM (although at a lower resolution) (Bosch et al., 2022;Kuan et al., 2020;Bushong et al., 2015;Musser et al., 2021). Laboratory micro-CT systems are becoming ever more popular and can provide an isotropic resolution of about 1 micron, with scan times in the range of a few hours (Withers et al., 2021;Karreman et al., 2016). ...
... Other studies have improved the precision and ease of targeting by leveraging external 3D maps from light or X-ray microscopy (Karreman et al., 2016;Karreman et al., 2017;Xu et al., 2021;Musser et al., 2021;Bushong et al., 2015;Ronchi et al., 2021). X-ray offers many advantages for EM targeting -for example, it is readily compatible with standard EM sample preparation methods (Bosch et al., 2022;Kuan et al., 2020;Bushong et al., 2015;Karreman et al., 2017) and provides images that highlight similar structures to volume EM (although at a lower resolution) (Bosch et al., 2022;Kuan et al., 2020;Bushong et al., 2015;Musser et al., 2021). Laboratory micro-CT systems are becoming ever more popular and can provide an isotropic resolution of about 1 micron, with scan times in the range of a few hours (Withers et al., 2021;Karreman et al., 2016). ...
... Laboratory micro-CT systems are becoming ever more popular and can provide an isotropic resolution of about 1 micron, with scan times in the range of a few hours (Withers et al., 2021;Karreman et al., 2016). In addition, there is increasing access to synchrotrons for X-ray imaging, which can provide resolutions in the range of hundreds or even tens of nanometres, and scan times in the range of minutes (depending on the resolution required) (Withers et al., 2021;Bosch et al., 2022;Kuan et al., 2020). X-ray imaging therefore offers a fast, non-destructive method for obtaining 3D maps of the internal features of a sample for targeting. ...
Volume electron microscopy (EM) is a time-consuming process – often requiring weeks or months of continuous acquisition for large samples. In order to compare the ultrastructure of a number of individuals or conditions, acquisition times must therefore be reduced. For resin-embedded samples, one solution is to selectively target smaller regions of interest by trimming with an ultramicrotome. This is a difficult and labour-intensive process, requiring manual positioning of the diamond knife and sample, and much time and training to master. Here, we have developed a semi-automated workflow for targeting with a modified ultramicrotome. We adapted two recent commercial systems to add motors for each rotational axis (and also each translational axis for one system), allowing precise and automated movement. We also developed a user-friendly software to convert X-ray images of resin-embedded samples into angles and cutting depths for the ultramicrotome. This is provided as an open-source Fiji plugin called Crosshair. This workflow is demonstrated by targeting regions of interest in a series of Platynereis dumerilii samples.
... For example, elements with a higher atomic number tend to have both higher X-ray attenuation and higher electron scattering properties. Therefore, many of the same biological structures are visible with both techniquesespecially when contrasted with heavy metals -and images can have a very similar appearance 45,46,[285][286][287] . Tomography works by taking 2D images of a sample from many different angles and combining these via a reconstruction algorithm to give a 3D tomogram. ...
... X-ray tomography can be done in the laboratory using laboratory-based X-ray systems or at large synchrotron facilities. In general, laboratory-based systems can reach submicron resolutions with scan times in the range of a few hours to a few days 44,283 , whereas synchrotrons can achieve resolutions of tens to hundreds of nanometres 283,285,287 (depending on the set-up chosen and the technique employed) with scan times in the range of seconds or minutes 283,285 , although the highest resolution scans still take several hours 287 . However, the benefits of synchrotron X-rays come at the cost of reduced accessibility, and care must be taken to avoid damage to the sample from high doses of X-rays 288,289 . ...
... X-ray tomography can be done in the laboratory using laboratory-based X-ray systems or at large synchrotron facilities. In general, laboratory-based systems can reach submicron resolutions with scan times in the range of a few hours to a few days 44,283 , whereas synchrotrons can achieve resolutions of tens to hundreds of nanometres 283,285,287 (depending on the set-up chosen and the technique employed) with scan times in the range of seconds or minutes 283,285 , although the highest resolution scans still take several hours 287 . However, the benefits of synchrotron X-rays come at the cost of reduced accessibility, and care must be taken to avoid damage to the sample from high doses of X-rays 288,289 . ...
... For example, elements with a higher atomic number tend to have both higher X-ray attenuation and higher electron scattering properties. Therefore, many of the same biological structures are visible with both techniquesespecially when contrasted with heavy metals -and images can have a very similar appearance 45,46,[285][286][287] . Tomography works by taking 2D images of a sample from many different angles and combining these via a reconstruction algorithm to give a 3D tomogram. ...
... X-ray tomography can be done in the laboratory using laboratory-based X-ray systems or at large synchrotron facilities. In general, laboratory-based systems can reach submicron resolutions with scan times in the range of a few hours to a few days 44,283 , whereas synchrotrons can achieve resolutions of tens to hundreds of nanometres 283,285,287 (depending on the set-up chosen and the technique employed) with scan times in the range of seconds or minutes 283,285 , although the highest resolution scans still take several hours 287 . However, the benefits of synchrotron X-rays come at the cost of reduced accessibility, and care must be taken to avoid damage to the sample from high doses of X-rays 288,289 . ...
... X-ray tomography can be done in the laboratory using laboratory-based X-ray systems or at large synchrotron facilities. In general, laboratory-based systems can reach submicron resolutions with scan times in the range of a few hours to a few days 44,283 , whereas synchrotrons can achieve resolutions of tens to hundreds of nanometres 283,285,287 (depending on the set-up chosen and the technique employed) with scan times in the range of seconds or minutes 283,285 , although the highest resolution scans still take several hours 287 . However, the benefits of synchrotron X-rays come at the cost of reduced accessibility, and care must be taken to avoid damage to the sample from high doses of X-rays 288,289 . ...
Life exists in three dimensions, but until the turn of the century most electron microscopy methods provided only 2D image data. Recently, electron microscopy techniques capable of delving deep into the structure of cells and tissues have emerged, collectively called volume electron microscopy (vEM). Developments in vEM have been dubbed a quiet revolution as the field evolved from established transmission and scanning electron microscopy techniques, so early publications largely focused on the bioscience applications rather than the underlying technological breakthroughs. However, with an explosion in the uptake of vEM across the biosciences and fast-paced advances in volume, resolution, throughput and ease of use, it is timely to introduce the field to new audiences. In this Primer, we introduce the different vEM imaging modalities, the specialized sample processing and image analysis pipelines that accompany each modality and the types of information revealed in the data. We showcase key applications in the biosciences where vEM has helped make breakthrough discoveries and consider limitations and future directions. We aim to show new users how vEM can support discovery science in their own research fields and inspire broader uptake of the technology, finally allowing its full adoption into mainstream biological imaging. Volume electron microscopy techniques are high-resolution imaging approaches that reveal the 3D structure of cells, tissues and small model organisms at nanometre resolution. This Primer introduces the different imaging modalities, specialized sample processing and key applications in biosciences.
