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Background: Accurate registration between microscopy and MRI data is necessary for validating imaging biomarkers against neuropathology, and to disentangle complex signal dependencies in microstructural MRI. Existing registration methods often rely on serial histological sampling or significant manual input, providing limited scope to work with a large number of stand-alone histology sections. Here we present a customisable pipeline to assist the registration of stand-alone histology sections to whole-brain MRI data. Methods: Our pipeline registers stained histology sections to whole-brain post-mortem MRI in 4 stages, with the help of two photographic intermediaries: a block face image (to undistort histology sections) and coronal brain slab photographs (to insert them into MRI space). Each registration stage is implemented as a configurable stand-alone Python script using our novel platform, Tensor Image Registration Library (TIRL), which provides flexibility for wider adaptation. We report our experience of registering 87 PLP-stained histology sections from 14 subjects and perform various experiments to assess the accuracy and robustness of each stage of the pipeline. Results: All 87 histology sections were successfully registered to MRI. Histology-to-block registration (Stage 1) achieved 0.2–0.4 mm accuracy, better than commonly used existing methods. Block-to-slice matching (Stage 2) showed great robustness in automatically identifying and inserting small tissue blocks into whole brain slices with 0.2 mm accuracy. Simulations demonstrated sub-voxel level accuracy (0.13 mm) of the slice-to-volume registration (Stage 3) algorithm, which was observed in over 200 actual brain slice registrations, compensating 3D slice deformations up to 6.5 mm. Stage 4 combined the previous stages and generated refined pixelwise aligned multi-modal histology-MRI stacks. Conclusions: Our open-source pipeline provides robust automation tools for registering stand-alone histology sections to MRI data with sub-voxel level precision, and the underlying framework makes it readily adaptable to a diverse range of microscopy-MRI studies.

The acquisition of MRI and histology in the same post-mortem tissue sample enables direct correlation between MRI and histologically-derived parameters. However, there still lacks a standardised automated pipeline to process histology data, with most studies relying on manual intervention. Here, we introduce an automated pipeline to extract a quantitative histological measure for staining density (stain area fraction, SAF) from multiple immunohistochemical (IHC) stains. The pipeline is designed to directly address key IHC artefacts related to tissue staining and slide digitisation. Here, the pipeline was applied to post-mortem human brain data from multiple subjects, relating MRI parameters (FA, MD, RD, AD, R2*, R1) to IHC slides stained for myelin, neurofilaments, microglia and activated microglia. Utilising high-quality MRI-histology co-registrations, we then performed whole-slide voxelwise comparisons (simple correlations, partial correlations and multiple regression analyses) between multimodal MRI- and IHC-derived parameters. The pipeline was found to be reproducible, robust to artefacts and generalisable across multiple IHC stains. Our partial correlation results suggest that some simple MRI-SAF correlations should be interpreted with caution, due to the co-localisation of other tissue features (e.g., myelin and neurofilaments). Further, we find activated microglia—a generic biomarker of inflammation—to consistently be the strongest predictor of high DTI FA and low RD, which may suggest sensitivity of diffusion MRI to aspects of neuroinflammation related to microglial activation, even after accounting for other microstructural changes (demyelination, axonal loss and general microglia infiltration). Together, these results show the utility of this approach in carefully curating IHC data and performing multimodal analyses to better understand microstructural relationships with MRI.

Diffusion MRI tractography is the only noninvasive method to measure the structural connectome in humans. However, recent validation studies have revealed limitations of modern tractography approaches, which lead to significant mistracking caused in part by local uncertainties in fiber orientations that accumulate to produce larger errors for longer streamlines. Characterizing the role of this length bias in tractography is complicated by the true underlying contribution of spatial embedding to brain topology. In this work, we compare graphs constructed with ex vivo tractography data in mice and neural tracer data from the Allen Mouse Brain Connectivity Atlas to random geometric surrogate graphs which preserve the low-order distance effects from each modality in order to quantify the role of geometry in various network properties. We find that geometry plays a substantially larger role in determining the topology of graphs produced by tractography than graphs produced by tracers. Tractography underestimates weights at long distances compared to neural tracers, which leads tractography to place network hubs close to the geometric center of the brain, as do corresponding tractography-derived random geometric surrogates, while tracer graphs place hubs further into peripheral areas of the cortex. We also explore the role of spatial embedding in modular structure, network efficiency and other topological measures in both modalities. Throughout, we compare the use of two different tractography streamline node assignment strategies and find that the overall differences between tractography approaches are small relative to the differences between tractography- and tracer-derived graphs. These analyses help quantify geometric biases inherent to tractography and promote the use of geometric benchmarking in future tractography validation efforts.

Mammalian neurons operate at length scales spanning six orders of magnitude; they project millimeters to centimeters across brain regions, are composed of micrometer-scale-diameter myelinated axons, and ultimately form nanometer scale synapses. Capturing these anatomical features across that breadth of scale has required imaging samples with multiple independent imaging modalities. Translating between the different modalities, however, requires imaging the same brain with each. Here, we imaged the same postmortem mouse brain over five orders of spatial resolution using MRI, whole brain micrometer-scale synchrotron x-ray tomography (μCT), and large volume automated serial electron microscopy. Using this pipeline, we can track individual myelinated axons previously relegated to axon bundles in diffusion tensor MRI or arbitrarily trace neurons and their processes brain-wide and identify individual synapses on them. This pipeline provides both an unprecedented look across a single brain's multi-scaled organization as well as a vehicle for studying the brain's multi-scale pathologies.

Susceptibility weighted magnetic resonance imaging (MRI) is sensitive to the local concentration of iron and myelin. Here, we describe a robust image processing pipeline for quantitative susceptibility mapping (QSM) and R2* mapping of fixed post-mortem, whole-brain data. Using this pipeline, we compare the resulting quantitative maps in brains from patients with amyotrophic lateral sclerosis (ALS) and controls, with validation against iron and myelin histology.Twelve post-mortem brains were scanned with a multi-echo gradient echo sequence at 7T, from which susceptibility and R2* maps were generated. Semi-quantitative histological analysis for ferritin (the principal iron storage protein) and myelin proteolipid protein was performed in the primary motor, anterior cingulate and visual cortices.Magnetic susceptibility and R2* values in primary motor cortex were higher in ALS compared to control brains. Magnetic susceptibility and R2* showed positive correlations with both myelin and ferritin estimates from histology. Four out of nine ALS brains exhibited clearly visible hyperintense susceptibility and R2* values in the primary motor cortex.Our results demonstrate the potential for MRI-histology studies in whole, fixed post-mortem brains to investigate the biophysical source of susceptibility weighted MRI signals in neurodegenerative diseases like ALS.

Diffusion-weighted steady-state free precession (DW-SSFP) is an SNR-efficient diffusion imaging method. The improved SNR and resolution available at ultra-high field has motivated its use at 7T. However, these data tend to have severe B1 inhomogeneity, leading not only to spatially varying SNR, but also to spatially varying diffusivity estimates, confounding comparisons both between and within datasets. This study proposes the acquisition of DW-SSFP data at two-flip angles in combination with explicit modelling of non-Gaussian diffusion to address B1 inhomogeneity at 7T. Data were acquired from five fixed whole human post-mortem brains with a pair of flip angles that jointly optimize the diffusion contrast-to-noise (CNR) across the brain. We compared one- and two-flip angle DW-SSFP data using a tensor model that incorporates the full DW-SSFP Buxton signal, in addition to tractography performed over the cingulum bundle and pre-frontal cortex using a ball & sticks model. The two-flip angle DW-SSFP data produced angular uncertainty and tractography estimates close to the CNR optimal regions in the single-flip angle datasets. The two-flip angle tensor estimates were subsequently fitted using a modified DW-SSFP signal model that incorporates a gamma distribution of diffusivities. This allowed us to generate tensor maps at a single effective b-value yielding more consistent SNR across tissue, in addition to eliminating the B1 dependence on diffusion coefficients and orientation maps. Our proposed approach will allow the use of DW-SSFP at 7T to derive diffusivity estimates that have greater interpretability, both within a single dataset and between experiments.