Sample Preparation and Warping Accuracy for Correlative Multimodal Imaging in the Mouse Olfactory Bulb Using 2-Photon, Synchrotron X-Ray and Volume Electron Microscopy

Integrating physiology with structural insights of the same neuronal circuit provides a unique approach to understanding how the mammalian brain computes information. However, combining the techniques that provide both streams of data represents an experimental challenge. When studying glomerular column circuits in the mouse olfactory bulb, this approach involves e.g., recording the neuronal activity with in vivo 2-photon (2P) calcium imaging, retrieving the circuit structure with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) and/or serial block-face scanning electron microscopy (SBEM) and correlating these datasets. Sample preparation and dataset correlation are two key bottlenecks in this correlative workflow. Here, we first quantify the occurrence of different artefacts when staining tissue slices with heavy metals to generate X-ray or electron contrast. We report improvements in the staining procedure, ultimately achieving perfect staining in ∼67% of the 0.6 mm thick olfactory bulb slices that were previously imaged in vivo with 2P. Secondly, we characterise the accuracy of the spatial correlation between functional and structural datasets. We demonstrate that direct, single-cell precise correlation between in vivo 2P and SXRT tissue volumes is possible and as reliable as correlating between 2P and SBEM. Altogether, these results pave the way for experiments that require retrieving physiology, circuit structure and synaptic signatures in targeted regions. These correlative function-structure studies will bring a more complete understanding of mammalian olfactory processing across spatial scales and time

Registration of phase contrast images in propagation-based X-ray phase tomography

International audienceX-ray phase tomography aims at reconstructing the 3D electron density distribution of an object. It offers enhanced sensitivity compared to attenuation-based X-ray absorption tomography. In propagation-based methods, phase contrast is achieved by letting the beam propagate after interaction with the object. The phase shift is then retrieved at each projection angle, and subsequently used in tomographic reconstruction to obtain the refractive index decrement distribution, which is proportional to the electron density. Accurate phase retrieval is achieved by combining images at different propagation distances. For reconstructions of good quality, the phase-contrast images recorded at different distances need to be accurately aligned. In this work, we characterise the artefacts related to misalignment of the phase-contrast images, and investigate the use of different registration algorithms for aligning in-line phase-contrast images. The characterisation of artefacts is done by a simulation study and comparison with experimental data. Loss in resolution due to vibrations is found to be comparable to attenuation-based computed tomography. Further, it is shown that registration of phase-contrast images is nontrivial due to the difference in contrast between the different images, and the often periodical artefacts present in the phase-contrast images if multilayer X-ray optics are used. To address this, we compared two registration algorithms for aligning phase-contrast images acquired by magnified X-ray nanotomography: one based on cross-correlation and one based on mutual information. We found that the mutual information-based registration algorithm was more robust than a correlation-based method

Targeted positioning of quantum dots inside 3D silicon photonic crystals observed by synchrotron X-ray fluorescence tomography

We perform X-ray fluorescence tomography of a 3D photonic band gap crystal made from silicon with embedded quantum dot nanocrystals. We obtain the position of the quantum dots with a resolution of 50nm

Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences

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 labeled 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 or serial blockface scanning electron microscopy, 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 femtosecond laser (fs laser) device to prepare microscopic samples (1) of an optimized geometry for synchrotron x-ray tomography as well as (2) for volume electron microscopy applications and compatible with correlative multimodal imaging workflows that link both imaging modalities

An in vitro model for the development of mature bone containing an osteocyte network

Bone is a dynamic tissue that remodels continuously in response to local mechanical and chemical stimuli. This process can also result in maladaptive ectopic bone in response to injury, yet pathological differences at the molecular and structural levels are poorly understood. A number of in vivo models exist but can often be too complex to allow isolation of factors which may stimulate disease progression. A self-structuring model of bone formation is presented using a fibrin gel cast between two calcium phosphate ceramic anchors. Femoral periosteal cells, seeded into these structures, deposit an ordered matrix that closely resembles mature bone in terms of chemistry (collagen:mineral ratio) and structure, which is adapted over a period of one year in culture. Raman spectroscopy and X-ray diffraction confirm that the mineral is hydroxyapatite associated with collagen. Second-harmonic imaging demonstrates that collagen is organized similarly to mature mouse femora. Remarkably, cells differentiated to the osteocyte phase are linked by canaliculi (as demonstrated with nano-computed tomography) and remained viable over the full year of culture. It is demonstrated that novel drugs can prevent ossification in constructs. This model can be employed to study bone formation in an effort to encourage or prevent ossification in a range of pathologies

Interconnectivity Explains High Canalicular Network Robustness between Neighboring Osteocyte Lacunae in Human Bone

Osteocytes are the most frequent bone cells connected with each other through cell processes within tiny tubular-shaped canaliculi. The so-called osteocyte lacunar-canalicular network (LCN) plays a crucial role in bone remodeling and mineral homeostasis. Given the critical nature of these functions, it is herein hypothesized that the LCN must be structurally "overengineered" to provide network resilience. This hypothesis is tested by characterizing canalicular networks in human bone at the fundamental "building-block" level of LCN formed by two adjacent osteocytes. As the hierarchical micro- and macroscale structure of bone is influenced by anatomical location, subjected loads, and growth rate, three distinct tissue types are studied. These include femur, jaw, and heterotopic ossification (HO), a rapidly forming mineralized tissue found in soft tissue compartments following severe trauma. It is found that the LCNs at the fundamental level are composed of hundreds of canalicular segments but of only few separated groups of linked canaliculi (canalicular clusters), resulting in a strongly pronounced interconnectivity. Fluid permeability simulations on intact and artificially altered LCN suggest that the function of the LCN is not only to optimize rapid and efficient access to bone mineral, but also to maintain high permeability when inevitable local interruption of canaliculi occurs.Peer reviewe

Functional and multiscale 3D structural investigation of brain tissue through correlative in vivo physiology, synchrotron microtomography and volume electron microscopy

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 using in vivo 2-photon calcium imaging were stained, dehydrated, resin-embedded and imaged with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT). SXRT provided context at subcellular detail, and could be followed by targeted acquisition of multiple volumes using serial block-face electron microscopy (SBEM). In the olfactory bulb, combining SXRT and SBEM enabled disambiguation of in vivo-assigned regions of interest. In the hippocampus, we found that superficial pyramidal neurons in CA1a displayed a larger density of spine apparati than deeper ones. Altogether, this approach can enable a functional and structural investigation of subcellular features in the context of cells and tissues

Reptile-like physiology in Early Jurassic stem-mammals

Despite considerable advances in knowledge of the anatomy, ecology and evolution of early mammals, far less is known about their physiology. Evidence is contradictory concerning the timing and fossil groups in which mammalian endothermy arose. To determine the state of metabolic evolution in two of the earliest stem-mammals, the Early Jurassic Morganucodon and Kuehneotherium, we use separate proxies for basal and maximum metabolic rate. Here we report, using synchrotron X-ray tomographic imaging of incremental tooth cementum, that they had maximum lifespans considerably longer than comparably sized living mammals, but similar to those of reptiles, and so they likely had reptilian-level basal metabolic rates. Measurements of femoral nutrient foramina show Morganucodon had blood flow rates intermediate between living mammals and reptiles, suggesting maximum metabolic rates increased evolutionarily before basal metabolic rates. Stem mammals lacked the elevated endothermic metabolism of living mammals, highlighting the mosaic nature of mammalian physiological evolution. Modern mammals are endothermic, but it has not been clear when this type of metabolism evolved. Here, Newham et al. analyse tooth and bone structure in Early Jurassic stem-mammal fossils to estimate lifespan and blood flow rates, which inform about basal and maximum metabolic rates, respectively, and show these stem-mammals had metabolic rates closer to modern ectothermic reptiles than to endothermic mammals.Peer reviewe

Reply to: Revisiting life history and morphological proxies for early mammaliaform metabolic rates

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Interconnectivity explains high canalicular network robustness between neighboring osteocyte lacunae in human bone

Osteocytes are the most frequent bone cells connected with each other through cell processes within tiny tubular-shaped canaliculi. The so-called osteocyte lacunar-canalicular network (LCN) plays a crucial role in bone remodeling and mineral homeostasis. Given the critical nature of these functions, it is herein hypothesized that the LCN must be structurally “overengineered” to provide network resilience. This hypothesis is tested by characterizing canalicular networks in human bone at the fundamental “building-block” level of LCN formed by two adjacent osteocytes. As the hierarchical micro- and macroscale structure of bone is influenced by anatomical location, subjected loads, and growth rate, three distinct tissue types are studied. These include femur, jaw, and heterotopic ossification (HO), a rapidly forming mineralized tissue found in soft tissue compartments following severe trauma. It is found that the LCNs at the fundamental level are composed of hundreds of canalicular segments but of only few separated groups of linked canaliculi (canalicular clusters), resulting in a strongly pronounced interconnectivity. Fluid permeability simulations on intact and artificially altered LCN suggest that the function of the LCN is not only to optimize rapid and efficient access to bone mineral, but also to maintain high permeability when inevitable local interruption of canaliculi occurs.DFG, 372486779, SFB 1340: In vivo Visualisierung der pathologisch veränderten Extrazellulärmatrix „Matrix in Vision“TU Berlin, Open-Access-Mittel – 202