Desert locusts (Schistocerca gregaria) feed with self-sharpening, scissor-like mandibles.

The mandibles of the desert locust Schistocerca gregaria (Forsskål, 1775) are digger-shovel-shaped mouthparts that are part of the locust's exoskeleton formed by the insect cuticle. The cuticle is a polymer-fibre composite, which supports, encases and protects the entire body. Mandibles experience heavy loading and wear due to direct contact with hard and abrasive food, just like teeth, their mineralized analogues in vertebrates. With dual-energy X-ray tomography, we image well-defined regions of zinc (Zn)-enriched cuticle at the mandible cutting edges and quantify the Zn concentrations in these regions. Zn is known to increase stiffness, hardness and wear resistance of the otherwise purely polymeric insect cuticle. In S. gregaria, the position of the Zn-enriched cutting-edge regions relative to one another suggests that the mandibles form a scissor-like cutting tool, which sharpens itself as the mouthparts shear past one another during feeding. Comparing the architecture of these purely polymeric mandibles with the mineralized incisors of rodents, we find fundamental design differences in cutting-tool structure and performance. Locusts' scissors and rodents' carving knives perform different functions, because they act on food that differs significantly in properties and shape: softer, sheet-like material in the case of locusts and harder bulk material in the case of rodents.

Intracellular Fate of Sub-Toxic Concentration of Functionalized Selenium Nanoparticles in Aggressive Prostate Cancer Cells.

Selenium 0 (Se0) is a powerful anti-proliferative agent in cancer research. We investigated the impact of sub-toxic concentrations of Se0 functionalized nanoparticles (SeNPs) on prostate cancer PC-3 cells and determined their intracellular localization and fate. An in-depth characterization of functionalized selenium nanoparticles composition is proposed to certify that no chemical bias relative to synthesis issues might have impacted the study. Selenium is an extremely diluted element in the biological environment and therefore requires high-performance techniques with a very low detection limit and high spatial resolution for intracellular imaging. This was explored with state-of-the-art techniques, but also with cryopreparation to preserve the chemical and structural integrity of the cells for spatially resolved and speciation techniques. Monodisperse solutions of SeNPs capped with bovine serum albumin (BSA) were shown to slow down the migration capacity of aggressive prostate cancer cells compared to polydisperse solutions of SeNPs capped with chitosan. BSA coating could prevent interactions between the reactive surface of the nanoparticles and the plasma membrane, mitigating the generation of reactive oxygen species. The intracellular localization showed interaction with mitochondria and also a localization in the lysosome-related organelle. The SeNPs-BSA localization in mitochondria constitute a possible explanation for our result showing a very significant dampening of the PC-3 cell proliferation capabilities. The purpose of the use of sublethal compound concentrations was to limit adverse effects resulting from high cell death to best evaluate some cellular changes and the fate of these SeNPs on PC-3. Our findings provide new insight to further study the various mechanisms of cytotoxicity of SeNPs.

Synchrotron X-rays reveal the modes of Fe binding and trace metal storage in the brown algae Laminaria digitata and Ectocarpus siliculosus.

Iron is accumulated symplastically in kelp in a non-ferritin core that seems to be a general feature of brown algae. Microprobe studies show that Fe binding depends on tissue type. The sea is generally an iron-poor environment and brown algae were recognized in recent years for having a unique, ferritin-free iron storage system. Kelp (Laminaria digitata) and the filamentous brown alga Ectocarpus siliculosus were investigated using X-ray microprobe imaging and nanoprobe X-ray fluorescence tomography to explore the localization of iron, arsenic, strontium, and zinc, and micro-X-ray absorption near-edge structure (µXANES) to study Fe binding. Fe distribution in frozen hydrated environmental samples of both algae shows higher accumulation in the cortex with symplastic subcellular localization. This should be seen in the context of recent ultrastructural insight by cryofixation-freeze substitution that found a new type of cisternae that may have a storage function but differs from the apoplastic Fe accumulation found by conventional chemical fixation. Zn distribution co-localizes with Fe in E. siliculosus, whereas it is chiefly located in the L. digitata medulla, which is similar to As and Sr. Both As and Sr are mostly found at the cell wall of both algae. XANES spectra indicate that Fe in L. digitata is stored in a mineral non-ferritin core, due to the lack of ferritin-encoding genes. We show that the L. digitata cortex contains mostly a ferritin-like mineral, while the meristoderm may include an additional component.

X-ray multiscale 3D neuroimaging to quantify cellular aging and neurodegeneration postmortem in a model of Alzheimer's disease.

PURPOSE: Modern neuroimaging lacks the tools necessary for whole-brain, anatomically dense neuronal damage screening. An ideal approach would include unbiased histopathologic identification of aging and neurodegenerative disease. METHODS: We report the postmortem application of multiscale X-ray phase-contrast computed tomography (X-PCI-CT) for the label-free and dissection-free organ-level to intracellular-level 3D visualization of distinct single neurons and glia. In deep neuronal populations in the brain of aged wild-type and of 3xTgAD mice (a triply-transgenic model of Alzheimer's disease), we quantified intracellular hyperdensity, a manifestation of aging or neurodegeneration. RESULTS: In 3xTgAD mice, the observed hyperdensity was identified as amyloid-ß and hyper-phosphorylated tau protein deposits with calcium and iron involvement, by correlating the X-PCI-CT data to immunohistochemistry, X-ray fluorescence microscopy, high-field MRI, and TEM. As a proof-of-concept, X-PCI-CT was used to analyze hippocampal and cortical brain regions of 3xTgAD mice treated with LY379268, selective agonist of group II metabotropic glutamate receptors (mGlu2/3 receptors). Chronic pharmacologic activation of mGlu2/3 receptors significantly reduced the hyperdensity particle load in the ventral cortical regions of 3xTgAD mice, suggesting a neuroprotective effect with locoregional efficacy. CONCLUSIONS: This multiscale micro-to-nano 3D imaging method based on X-PCI-CT enabled identification and quantification of cellular and sub-cellular aging and neurodegeneration in deep neuronal and glial cell populations in a transgenic model of Alzheimer's disease. This approach quantified the localized and intracellular neuroprotective effects of pharmacological activation of mGlu2/3 receptors.

Dynamics of particle network in composite battery cathodes.

Improving composite battery electrodes requires a delicate control of active materials and electrode formulation. The electrochemically active particles fulfill their role as energy exchange reservoirs through interacting with the surrounding conductive network. We formulate a network evolution model to interpret the regulation and equilibration between electrochemical activity and mechanical damage of these particles. Through statistical analysis of thousands of particles using x-ray phase contrast holotomography in a LiNi0.8Mn0.1Co0.1O2-based cathode, we found that the local network heterogeneity results in asynchronous activities in the early cycles, and subsequently the particle assemblies move toward a synchronous behavior. Our study pinpoints the chemomechanical behavior of individual particles and enables better designs of the conductive network to optimize the utility of all the particles during operation.

Targeted Positioning of Quantum Dots Inside 3D Silicon Photonic Crystals Revealed by Synchrotron X-ray Fluorescence Tomography.

It is a major outstanding goal in nanotechnology to precisely position functional nanoparticles, such as quantum dots, inside a three-dimensional (3D) nanostructure in order to realize innovative functions. Once the 3D positioning is performed, the challenge arises how to nondestructively verify where the nanoparticles reside in the 3D nanostructure. Here, we study 3D photonic band gap crystals made of Si that are infiltrated with PbS nanocrystal quantum dots. The nanocrystals are covalently bonded to polymer brush layers that are grafted to the Si-air interfaces inside the 3D nanostructure using surface-initiated atom transfer radical polymerization (SI-ATRP). The functionalized 3D nanostructures are probed by synchrotron X-ray fluorescence (SXRF) tomography that is performed at 17 keV photon energy to obtain large penetration depths and efficient excitation of the elements of interest. Spatial projection maps were obtained followed by tomographic reconstruction to obtain the 3D atom density distribution with 50 nm voxel size for all chemical elements probed: Cl, Cr, Cu, Ga, Br, and Pb. The quantum dots are found to be positioned inside the 3D nanostructure, and their positions correlate with the positions of elements characteristic of the polymer brush layer and the ATRP initiator. We conclude that X-ray fluorescence tomography is very well suited to nondestructively characterize 3D nanomaterials with photonic and other functionalities.

An Internal Promoter Drives the Expression of a Truncated Form of CCC1 Capable of Protecting Yeast from Iron Toxicity.

In yeast, iron storage and detoxification depend on the Ccc1 transporter that mediates iron accumulation in vacuoles. While deletion of the CCC1 gene renders cells unable to survive under iron overload conditions, the deletion of its previously identified regulators only partially affects survival, indicating that the mechanisms controlling iron storage and detoxification in yeast are still far from well understood. This work reveals that CCC1 is equipped with a complex transcriptional structure comprising several regulatory regions. One of these is located inside the coding sequence of the gene and drives the expression of a short transcript encoding an N-terminally truncated protein, designated as s-Ccc1. s-Ccc1, though less efficiently than Ccc1, is able to promote metal accumulation in the vacuole, protecting cells against iron toxicity. While the expression of the s-Ccc1 appears to be repressed in the normal genomic context, our current data clearly demonstrates that it is functional and has the capacity to play a role under iron overload conditions.

Multiscale X-ray phase contrast imaging of human cartilage for investigating osteoarthritis formation.

BACKGROUND: The evolution of cartilage degeneration is still not fully understood, partly due to its thinness, low radio-opacity and therefore lack of adequately resolving imaging techniques. X-ray phase-contrast imaging (X-PCI) offers increased sensitivity with respect to standard radiography and CT allowing an enhanced visibility of adjoining, low density structures with an almost histological image resolution. This study examined the feasibility of X-PCI for high-resolution (sub-) micrometer analysis of different stages in tissue degeneration of human cartilage samples and compare it to histology and transmission electron microscopy. METHODS: Ten 10%-formalin preserved healthy and moderately degenerated osteochondral samples, post-mortem extracted from human knee joints, were examined using four different X-PCI tomographic set-ups using synchrotron radiation the European Synchrotron Radiation Facility (France) and the Swiss Light Source (Switzerland). Volumetric datasets were acquired with voxel sizes between 0.7 × 0.7 × 0.7 and 0.1 × 0.1 × 0.1 µm3. Data were reconstructed by a filtered back-projection algorithm, post-processed by ImageJ, the WEKA machine learning pixel classification tool and VGStudio max. For correlation, osteochondral samples were processed for histology and transmission electron microscopy. RESULTS: X-PCI provides a three-dimensional visualization of healthy and moderately degenerated cartilage samples down to a (sub-)cellular level with good correlation to histologic and transmission electron microscopy images. X-PCI is able to resolve the three layers and the architectural organization of cartilage including changes in chondrocyte cell morphology, chondrocyte subgroup distribution and (re-)organization as well as its subtle matrix structures. CONCLUSIONS: X-PCI captures comprehensive cartilage tissue transformation in its environment and might serve as a tissue-preserving, staining-free and volumetric virtual histology tool for examining and chronicling cartilage behavior in basic research/laboratory experiments of cartilage disease evolution.

Chemo-physical properties of asbestos bodies in human lung tissues studied at the nano-scale by non-invasive, label free x-ray imaging and spectroscopic techniques.

In the lungs, asbestos develops an Fe-rich coating (Asbestos Body, AB) that becomes the actual interface between the foreign fibers and the host organism. Conventional approaches to study ABs require an invasive sample preparation that can alter them. In this work, a novel combination of x-ray tomography and spectroscopy allowed studying unaltered lung tissue samples with chrysotile and crocidolite asbestos. The thickness and mass density maps of the ABs obtained by x-ray tomography were used to derive a truly quantitative elemental analysis from scanning x-ray fluorescence spectroscopy data. The average mass density of the ABs is compatible with that of highly loaded ferritin, or hemosiderin. The composition of all ABs analyzed was similar, with only minor differences in the relative elemental fractions. Silicon concentration decreased in the core-to-rim direction, indicating a possible partial dissolution of the inner fiber. The Fe content in the ABs was higher than that possibly contained in chrysotile and crocidolite. This finding opens two opposite scenarios, the first with Fe coming from the fiber bulk and concentrating on the surface as long as the fiber dissolves, the second where the Fe that takes part to the formation of the AB originates from the host organism Fe-pool.

Effect of nanomolar concentrations of lanthanum on Desmodesmus quadricauda cultivated under environmentally relevant conditions.

Toxicity of lanthanides is generally regarded as low, and they even have been suggested to be beneficial at low concentrations. This research was conducted to investigate effects of Lanthanum (La) on Desmodesmus quadricauda, a freshwater green microalga. The algal cultures were treated with nanomolar La concentrations under controlled environmentally relevant conditions. Intracellular localization of La was analyzed with µXRF tomography in frozen-hydrated samples. At sublethal concentration (128 nM) La was in hotspots inside the cells, while at lethal 1387 nM that led to release of other ions (K, Zn) from the cells, La filled most of the cells. La had no clear positive effects on growth or photosynthetic parameters, but increasing concentrations led to a dramatic decrease in cell counts. Chlorophyll fluorescence kinetic measurements showed that La led to the inhibition of photosynthesis. Maximal photochemical quantum yield of the PSII reaction center in dark-adapted state (Fv/Fm) decreased at > 4.3 nM La during the 2nd week of treatment. Minimum dark-adapted fluorescence quantum yield (F0) increased at > 13.5 nM La during the 2nd week of treatment except for control (0.2 nM La, baseline from chemicals) and 0.3 nM La. NPQ at the beginning of the actinic light phase showed significant increase for all the treatments. Metalloproteomics by HPLC-ICPMS showed that La binds to a >500 kDa soluble protein complex already in the sub-nM range of La treatments, in the low nM range to a small-sized (3 kDa) soluble peptide, and at >100 nM La additionally binds to a 1.5 kDa ligand.

Three-dimensional virtual histology of the cerebral cortex based on phase-contrast X-ray tomography.

In this work, we optimize the setups and experimental parameters of X-ray phase-contrast computed-tomography for the three-dimensional imaging of the cyto- and myeloarchitecture of cerebral cortex, including both human and murine tissue. We present examples for different optical configurations using state-of-the art synchrotron instruments for holographic tomography, as well as compact laboratory setups for phase-contrast tomography in the direct contrast (edge-enhancement) regime. Apart from unstained and paraffin-embedded tissue, we tested hydrated tissue, as well as heavy metal stained and resin-embedded tissue using two different protocols. Further, we show that the image quality achieved allows to assess the neuropathology of multiple sclerosis in a biopsy sample collected during surgery.

Quantification of the bone lacunocanalicular network from 3D X-ray phase nanotomography images.

There is a growing interest in developing 3D microscopy for the exploration of thick biological tissues. Recently, 3D X-ray nanocomputerised tomography has proven to be a suitable technique for imaging the bone lacunocanalicular network. This interconnected structure is hosting the osteocytes which play a major role in maintaining bone quality through remodelling processes. 3D images have the potential to reveal the architecture of cellular networks, but their quantitative analysis remains a challenge due to the density and complexity of nanometre sized structures and the need to handle and process large datasets, for example, 20483 voxels corresponding to 32 GB per individual image in our case. In this work, we propose an efficient image processing approach for the segmentation of the network and the extraction of characteristic parameters describing the 3D structure. These parameters include the density of lacunae, the porosity of lacunae and canaliculi, and morphological features of lacunae (volume, surface area, lengths, anisotropy etc.). We also introduce additional parameters describing the local environment of each lacuna and its canaliculi. The method is applied to analyse eight human femoral cortical bone samples imaged by magnified X-ray phase nanotomography with a voxel size of 120 nm, which was found to be a good compromise to resolve canaliculi while keeping a sufficiently large field of view of 246 µm in 3D. The analysis was performed on a total of 2077 lacunae showing an average length, width and depth of 17.1 µm × 9.2 µm × 4.4 µm, with an average number of 58.2 canaliculi per lacuna and a total lacuno-canalicular porosity of 1.12%. The reported descriptive parameters provide information on the 3D organisation of the lacuno-canalicular network in human bones.

Correlating STED and synchrotron XRF nano-imaging unveils cosegregation of metals and cytoskeleton proteins in dendrites.

Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecular mechanisms behind these processes are still elusive due in part to the difficulty of imaging trace metals together with proteins at the synaptic level. We correlate stimulated-emission-depletion microscopy of proteins and synchrotron X-ray fluorescence imaging of trace metals, both performed with 40 nm spatial resolution, on primary rat hippocampal neurons. We reveal the co-localization at the nanoscale of zinc and tubulin in dendrites with a molecular ratio of about one zinc atom per tubulin-αß dimer. We observe the co-segregation of copper and F-actin within the nano-architecture of dendritic protrusions. In addition, zinc chelation causes a decrease in the expression of cytoskeleton proteins in dendrites and spines. Overall, these results indicate new functions for zinc and copper in the modulation of the cytoskeleton morphology in dendrites, a mechanism associated to neuronal plasticity and memory formation.

Dense neuronal reconstruction through X-ray holographic nano-tomography.

Imaging neuronal networks provides a foundation for understanding the nervous system, but resolving dense nanometer-scale structures over large volumes remains challenging for light microscopy (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.

Nano-imaging trace elements at organelle levels in substantia nigra overexpressing α-synuclein to model Parkinson's disease.

Sub-cellular trace element quantifications of nano-heterogeneities in brain tissues offer unprecedented ways to explore at elemental level the interplay between cellular compartments in neurodegenerative pathologies. We designed a quasi-correlative method for analytical nanoimaging of the substantia nigra, based on transmission electron microscopy and synchrotron X-ray fluorescence. It combines ultrastructural identifications of cellular compartments and trace element nanoimaging near detection limits, for increased signal-to-noise ratios. Elemental composition of different organelles is compared to cytoplasmic and nuclear compartments in dopaminergic neurons of rat substantia nigra. They exhibit 150-460 ppm of Fe, with P/Zn/Fe-rich nucleoli in a P/S-depleted nuclear matrix and Ca-rich rough endoplasmic reticula. Cytoplasm analysis displays sub-micron Fe/S-rich granules, including lipofuscin. Following AAV-mediated overexpression of α-synuclein protein associated with Parkinson's disease, these granules shift towards higher Fe concentrations. This effect advocates for metal (Fe) dyshomeostasis in discrete cytoplasmic regions, illustrating the use of this method to explore neuronal dysfunction in brain diseases.

Three-dimensional architecture of human diabetic peripheral nerves revealed by X-ray phase contrast holographic nanotomography.

A deeper knowledge of the architecture of the peripheral nerve with three-dimensional (3D) imaging of the nerve tissue at the sub-cellular scale may contribute to unravel the pathophysiology of neuropathy. Here we demonstrate the feasibility of X-ray phase contrast holographic nanotomography to enable 3D imaging of nerves at high resolution, while covering a relatively large tissue volume. We show various subcomponents of human peripheral nerves in biopsies from patients with type 1 and 2 diabetes and in a healthy subject. Together with well-organized, parallel myelinated nerve fibres we show regenerative clusters with twisted nerve fibres, a sprouted axon from a node of Ranvier and other specific details. A novel 3D construction (with movie created) of a node of Ranvier with end segment of a degenerated axon and sprout of a regenerated one is captured. Many of these architectural elements are not described in the literature. Thus, X-ray phase contrast holographic nanotomography enables identifying specific morphological structures in 3D in peripheral nerve biopsies from a healthy subject and from patients with type 1 and 2 diabetes.

Machine-learning-revealed statistics of the particle-carbon/binder detachment in lithium-ion battery cathodes.

The microstructure of a composite electrode determines how individual battery particles are charged and discharged in a lithium-ion battery. It is a frontier challenge to experimentally visualize and, subsequently, to understand the electrochemical consequences of battery particles' evolving (de)attachment with the conductive matrix. Herein, we tackle this issue with a unique combination of multiscale experimental approaches, machine-learning-assisted statistical analysis, and experiment-informed mathematical modeling. Our results suggest that the degree of particle detachment is positively correlated with the charging rate and that smaller particles exhibit a higher degree of uncertainty in their detachment from the carbon/binder matrix. We further explore the feasibility and limitation of utilizing the reconstructed electron density as a proxy for the state-of-charge. Our findings highlight the importance of precisely quantifying the evolving nature of the battery electrode's microstructure with statistical confidence, which is a key to maximize the utility of active particles towards higher battery capacity.

Nanofocused Scanning X-ray Fluorescence Microscopy Revealing an Effect of Heterozygous Hemoglobin S and C on Biochemical Activities in Plasmodium falciparum-Infected Erythrocytes.

While there is ample evidence suggesting that carriers of heterozygous hemoglobin S and C are protected from life-threatening malaria, little is known about the underlying biochemical mechanisms at the single cell level. Using nanofocused scanning X-ray fluorescence microscopy, we quantify the spatial distribution of individual elements in subcellular compartments, including Fe, S, P, Zn, and Cu, in Plasmodium falciparum-infected (P. falciparum-infected) erythrocytes carrying the wild type or variant hemoglobins. Our data indicate that heterozygous hemoglobin S and C significantly modulate biochemical reactions in parasitized erythrocytes, such as aberrant hemozoin mineralization and a delay in hemoglobin degradation. The label-free scanning X-ray fluorescence imaging has great potential to quantify the spatial distribution of elements in subcellular compartments of P. falciparum-infected erythrocytes and unravel the biochemical mechanisms underpinning disease and protective traits.

Effect of sample preparation techniques upon single cell chemical imaging: A practical comparison between synchrotron radiation based X-ray fluorescence (SR-XRF) and Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS).

Analytical capabilities of Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS) and Synchrotron Radiation based X-ray Fluorescence (SR nano-XRF) techniques were compared for nanochemical imaging of polymorphonuclear human neutrophils (PMNs). PMNs were high pressure frozen (HPF), cryo-substituted, embedded in Spurr's resin and cut in thin sections (500 nm and 2 µm for both techniques resp.) Nano-SIMS enabled nanoscale mapping of isotopes of C, N, O, P and S, while SR based nano-XRF enabled trace level imaging of metals like Ca, Mn, Fe, Ni, Cu and Zn at a resolution of approx. 50 nm. The obtained elemental distributions were compared with those of whole, cryofrozen PMNs measured at the newly developed ID16A nano-imaging beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Similarities were observed for elements more tightly bound to the cell structure such as phosphorus and sulphur, while differences for mobile ions such as chlorine and potassium were more pronounced. Due to the observed elemental redistribution of mobile ions such as potassium and chlorine, elemental analysis of high pressure frozen (HPF), cryo-substituted and imbedded cells should be interpreted critically. Although decreasing analytical sensitivity occurs due to the presence of ice, analysis of cryofrozen cells - close to their native state - remains the golden standard. In general, we found nanoscale secondary ion mass spectrometry (nano-SIMS) and synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) to be two supplementary alternatives for nanochemical imaging of single cells at the nanoscale.

Assessment of the human bone lacuno-canalicular network at the nanoscale and impact of spatial resolution.

Recently, increasing attention has been given to the study of osteocytes, the cells that are thought to play an important role in bone remodeling and in the mechanisms of bone fragility. The interconnected osteocyte system is deeply embedded inside the mineralized bone matrix and lies within a closely fitted porosity known as the lacuno-canalicular network. However, quantitative data on human samples remain scarce, mostly measured in 2D, and there are gaps to be filled in terms of spatial resolution. In this work, we present data on femoral samples from female donors imaged with isotropic 3D spatial resolution by magnified X-ray phase nano computerized-tomography. We report quantitative results on the 3D structure of canaliculi in human femoral bone imaged with a voxel size of 30 nm. We found that the lacuno-canalicular porosity occupies on average 1.45% of the total tissue volume, the ratio of the canalicular versus lacunar porosity is about 37.7%, and the primary number of canaliculi stemming from each lacuna is 79 on average. The examination of this number at different distances from the surface of the lacunae demonstrates branching in the canaliculi network. We analyzed the impact of spatial resolution on quantification by comparing parameters extracted from the same samples imaged with 120 nm and 30 nm voxel sizes. To avoid any bias related to the analysis region, the volumes at 120 nm and 30 nm were registered and cropped to the same field of view. Our results show that the measurements at 120 and 30 nm are strongly correlated in our data set but that the highest spatial resolution provides more accurate information on the canaliculi network and its branching properties.