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

X-ray Phase-Contrast Imaging and Metrology through Unified Modulated Pattern Analysis

We present a method for x-ray phase-contrast imaging and metrology applications based on the sample-induced modulation and subsequent computational demodulation of a random or periodic reference interference pattern. The proposed unified modulated pattern analysis (UMPA) technique is a versatile approach and allows tuning of signal sensitivity, spatial resolution, and scan time. We characterize the method and demonstrate its potential for high-sensitivity, quantitative phase imaging, and metrology to overcome the limitations of existing methods

RALGAPA1 Deletion in Belgian Shepherd Dogs with Cerebellar Ataxia.

Several genetically distinct forms of cerebellar ataxia exist in Belgian shepherd dogs. We investigated a litter in which two puppies developed cerebellar ataxia. The clinical signs stabilized at around six weeks of age, but remained visible into adulthood. Combined linkage and homozygosity mapping delineated a 5.5 Mb critical interval. The comparison of whole-genome sequence data of one affected dog to 929 control genomes revealed a private homozygous ~4.8 kb deletion in the critical interval, Chr8:14,468,376_14,473,136del4761. The deletion comprises exon 35 of the RALGAPA1 gene, XM_038544497.1:c.6080-2893_6944+1003del. It is predicted to introduce a premature stop codon into the transcript, truncating ~23% of the wild-type open reading frame of the encoded Ral GTPase-activating protein catalytic subunit α 1, XP_038400425.1:(p.Val2027Glnfs*7). Genotypes at the deletion showed the expected co-segregation with the phenotype in the family. Genotyping additional ataxic Belgian shepherd dogs revealed three additional homozygous mutant dogs from a single litter, which had been euthanized at five weeks of age due to their severe clinical phenotype. Histopathology revealed cytoplasmic accumulation of granular material within cerebellar Purkinje cells. Genotyping a cohort of almost 900 Belgian shepherd dogs showed the expected genotype-phenotype association and a carrier frequency of 5% in the population. Human patients with loss-of-function variants in RALGAPA1 develop psychomotor disability and early-onset epilepsy. The available clinical and histopathological data, together with current knowledge about RALGAPA1 variants and their functional impact in other species, suggest the RALGAPA1 deletion is the likely causative defect for the observed phenotype in the affected dogs

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

Comparison of laboratory grating-based and speckle-tracking x-ray phase-contrast imaging

Phase-contrast imaging with x-rays is a developing field for imaging weakly absorbing materials. In this work, two phase-contrast imaging methods, grating- and speckle-based imaging, that measure the derivative of the phase shift, have been implemented with a laboratory source and compared experimentally. It was found that for the same dose conditions, the speckle-tracking differential phase-contrast images have considerably higher contrast-to-noise ratio than the grating-based images, but at the cost of lower resolution. Grating-based imaging performs better in terms of resolution, but would require longer exposure times, mainly due to absorption in the grating interferometer

Hard X-ray stereographic microscopy for single-shot differential phase imaging

The characterisation of fast phenomena at the microscopic scale is required for the understanding of catastrophic responses of materials to loads and shocks, the processing of materials by optical or mechanical means, the processes involved in many key technologies such as additive manufacturing and microfluidics, and the mixing of fuels in combustion. Such processes are usually stochastic in nature and occur within the opaque interior volumes of materials or samples, with complex dynamics that evolve in all three dimensions at speeds exceeding many meters per second. There is therefore a need for the ability to record three-dimensional X-ray movies of irreversible processes with resolutions of micrometers and frame rates of microseconds. Here we demonstrate a method to achieve this by recording a stereo phase-contrast image pair in a single exposure. The two images are combined computationally to reconstruct a 3D model of the object. The method is extendable to more than two simultaneous views. When combined with megahertz pulse trains of X-ray free-electron lasers (XFELs) it will be possible to create movies able to resolve 3D trajectories with velocities of kilometers per second

Assessing Myocardial Microstructure with Biophysical Models of Diffusion MRI

Biophysical models are a promising means for interpreting diffusion weighted magnetic resonance imaging (DW-MRI) data, as they can provide estimates of physiologically relevant parameters of microstructure including cell size, volume fraction, or dispersion. However, their application in cardiac microstructure mapping (CMM) has been limited. This study proposes seven new two-compartment models with combination of restricted cylinder models and a diffusion tensor to represent intra-and extracellular spaces, respectively. Three extended versions of the cylinder model are studied here: cylinder with elliptical cross section (ECS), cylinder with Gamma distributed radii (GDR), and cylinder with Bingham distributed axes (BDA). The proposed models were applied to data in two fixed mouse hearts, acquired with multiple diffusion times, q-shells and diffusion encoding directions. The cylinderGDR-pancake model provided the best performance in terms of root mean squared error (RMSE) reducing it by 25% compared to diffusion tensor imaging (DTI). The cylinderBDA-pancake model represented anatomical findings closest as it also allows for modelling dispersion. High-resolution 3D synchrotron X-ray imaging (SRI) data from the same specimen was utilized to evaluate the biophysical models. A novel tensor-based registration method is proposed to align SRI structure tensors to the MR diffusion tensors. The consistency between SRI and DW-MRI parameters demonstrates the potential of compartment models in assessing physiologically relevant parameters

Increased dose efficiency of breast CT with grating interferometry

Refraction-based x-ray imaging can overcome the fundamental contrast limit of computed tomography (CT), particularly in soft tissue, but so far has been constrained to high-dose ex vivo applications or required highly coherent x-ray sources, such as synchrotrons. Here we demonstrate that grating interferometry (GI) is more dose efficient than conventional CT in imaging of human breast under close-to-clinical conditions. Our system, based on a conventional source and commercial gratings, outperformed conventional CT for spatial resolutions better than 263 µm and absorbed dose of 16 mGy. The sensitivity of GI is constrained by grating fabrication, and further progress will lead to significant improvements of clinical CT

Measuring cardiomyocyte cellular characteristics in cardiac hypertrophy using diffusion‐weighted MRI

Purpose: This paper presents a hierarchical modeling approach for estimating cardiomyocyte major and minor diameters and intracellular volume fraction (ICV) using diffusion‐weighted MRI (DWI) data in ex vivo mouse hearts. Methods: DWI data were acquired on two healthy controls and two hearts 3 weeks post transverse aortic constriction (TAC) using a bespoke diffusion scheme with multiple diffusion times ( Δ $$\Delta$$ ), q‐shells and diffusion encoding directions. Firstly, a bi‐exponential tensor model was fitted separately at each diffusion time to disentangle the dependence on diffusion times from diffusion weightings, that is, b‐values. The slow‐diffusing component was attributed to the restricted diffusion inside cardiomyocytes. ICV was then extrapolated at Δ = 0 $$\Delta =0$$ using linear regression. Secondly, given the secondary and the tertiary diffusion eigenvalue measurements for the slow‐diffusing component obtained at different diffusion times, major and minor diameters were estimated assuming a cylinder model with an elliptical cross‐section (ECS). High‐resolution three‐dimensional synchrotron X‐ray imaging (SRI) data from the same specimen was utilized to evaluate the biophysical parameters. Results: Estimated parameters using DWI data were (control 1/control 2 vs. TAC 1/TAC 2): major diameter—17.4 μ $$\mu$$ m/18.0 μ $$\mu$$ m versus 19.2 μ $$\mu$$ m/19.0 μ $$\mu$$ m; minor diameter—10.2 μ $$\mu$$ m/9.4 μ $$\mu$$ m versus 12.8 μ $$\mu$$ m/13.4 μ $$\mu$$ m; and ICV—62%/62% versus 68%/47%. These findings were consistent with SRI measurements. Conclusion: The proposed method allowed for accurate estimation of biophysical parameters suggesting cardiomyocyte diameters as sensitive biomarkers of hypertrophy in the heart

Fast Multi-scale imaging using the Beamline I13L at the Diamond Light Source

The DIAMOND beamline I13L is dedicated to multi-scale and multi-modal imaging in real and reciprocal space. The beamline consists of two independently operating experimental stations, located at a distance of more than 200 m from the source. The Imaging Branch performs micro-tomography with in-line phase contrast in the 6-30 keV energy range. In addition, a grating interferometry setup and a full-field microscope for nano-tomography are currently implemented. Other techniques providing high-resolution three-dimensional information, in particular coherent X-ray diffraction, are hosted on the Coherence Branch. All imaging methods are tested to operate with large energy bandwidths and therefore shorter exposure times. To this end, two options are currently used: the so-called ‘pink-beam’ mode using a reflecting mirror and X-ray filters and monochromatic mode using a multilayer monochromator. The operation mode enables science for in-situ and operando studies across a wide range of scientific areas