Tissue gene expression profiling is performed on homogenates or on populations of isolated single cells to resolve molecular states of different cell types. In both approaches, histological context is lost. We have developed an in situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue. We demonstrate in situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections.

Bone strength and failure are increasingly thought to be due to ultrastructural properties, such as the morphology of the lacuno-canalicular network, the collagen fiber orientation and the mineralization on the nanoscale. However, these properties have not been studied in 3D so far. Here we report the investigation of the human bone ultrastructure with X-ray phase nanotomography, which now provides the required sensitivity, spatial resolution and field of view. The 3D organization of the lacuno-canalicular network is studied in detail over several cells in osteonal and interstitial tissue. Nanoscale density variations are revealed and show that the cement line separating these tissues is hypermineralized. Finally, we show that the collagen fibers are organized as a twisted plywood structure in 3D.

Axonal conduction velocity, which ensures efficient function of the brain network, is related to axon diameter. Noninvasive, in vivo axon diameter estimates can be made with diffusion magnetic resonance imaging, but the technique requires three-dimensional (3D) validation. Here, high-resolution, 3D synchrotron X-ray nano-holotomography images of white matter samples from the corpus callosum of a monkey brain reveal that blood vessels, cells, and vacuoles affect axonal diameter and trajectory. Within single axons, we find that the variation in diameter and conduction velocity correlates with the mean diameter, contesting the value of precise diameter determination in larger axons. These complex 3D axon morphologies drive previously reported 2D trends in axon diameter and g-ratio. Furthermore, we find that these morphologies bias the estimates of axon diameter with diffusion magnetic resonance imaging and, ultimately, impact the investigation and formulation of the axon structure–function relationship.

Imaging neuronal networks provides a foundation for understanding the nervous system, but resolving dense nanometer-scale structures over large volumes remains challenging for light (LM) and electron microscopy (EM). Here, we show that X-ray holographic nano-tomography (XNH) can image millimeter-scale volumes with sub-100 nm resolution, enabling reconstruction of dense wiring in Drosophila melanogaster and mouse nervous tissue. We performed correlative XNH and EM to reconstruct hundreds of cortical pyramidal cells, and show that more superficial cells receive stronger synaptic inhibition on their apical dendrites. By combining multiple XNH scans, we imaged an adult Drosophila leg with sufficient resolution to comprehensively catalog mechanosensory neurons and trace individual motor axons from muscles to the central nervous system. To accelerate neuronal reconstructions, we trained a convolutional neural network to automatically segment neurons from XNH volumes. Thus, XNH bridges a key gap between LM and EM, providing a new avenue for neural circuit discovery.

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.

In bone remodeling, maturation of the newly formed osteonal tissue is associated with a rapid primary increase followed by a slower secondary increase of mineralization. This requires supply and precipitation of mineral into the bone matrix. Mineral delivery can occur only from the extracellular fluid via interfaces such as the Haversian system and the osteocyte pore network. We hypothesized that in mineralization, mineral exchange is achieved by the diffusion of mineral from the lacunar-canalicular network (LCN) to the bone matrix, resulting in a gradual change in tissue mineralization with respect to the distance from the pore-matrix interface. We expected to observe alterations in the mass density distribution with tissue age. We further hypothesized that mineral exchange occurs not only at the lacunar but also at the canalicular boundaries. The aim of this study was, therefore, to investigate the spatial distribution of mass density in the perilacunar and pericanalicular bone matrix and to explore how these densities are influenced by tissue aging. This is achieved by analyzing human jawbone specimens originating from four healthy donors and four treated with high-dosage bisphosphonate using synchrotron radiation phase-contrast nano-CT with a 50-nm voxel size. Our results provide the first experimental evidence that mass density in the direct vicinity of both lacunae (p < 0.001) and canaliculi (p < 0.001) is different from the mean matrix mass density, resulting in gradients with respect to the distance from both pore-matrix interfaces, which diminish with increasing tissue age. Though limited by the sample size, these findings support our hypotheses. Moreover, the density gradients are more pronounced around the lacunae than around the canaliculi, which are explained by geometrical considerations in the LCN morphology. In addition, we speculate that mineral exchange occurs at all interfaces of the LCN, not only in mineralization but also in mineral homeostasis.

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Like the vertebrate spinal cord, the insect ventral nerve cord (VNC) mediates limb sensation and motor control. Here, we applied automated tools for electron microscopy (EM) volume alignment, neuron reconstruction, and synapse prediction to create a draft connectome of theDrosophilaVNC. To interpret the VNC connectome, it is crucial to know its relationship with the rest of the body. We therefore mapped the muscle targets of leg and wing motor neurons in the connectome by comparing their morphology to genetic driver lines, dye fills, and x-ray holographic nano-tomography volumes of the fly leg and wing. Knowing the outputs of the connectome allowed us to identify neural circuits that coordinate the wings with the middle and front legs during escape takeoff. We provide the draft VNC connectome and motor neuron atlas, along with tools for programmatic and interactive access, as community resources.