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Stephen J. Blackband

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11 papers

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YNIMG Journal 2020 Journal Article

Visualization of live, mammalian neurons during Kainate-infusion using magnetic resonance microscopy

Jeremy J. Flint
Kannan Menon
Brian Hansen
John Forder
Stephen J. Blackband

Since its first description and development in the late 20th century, diffusion magnetic resonance imaging (dMRI) has proven useful in describing the microstructural details of biological tissues. Signal generated from the protons of water molecules undergoing Brownian motion produces contrast based on the varied diffusivity of tissue types. Images employing diffusion contrast were first used to describe the diffusion characteristics of tissues, later used to describe the fiber orientations of white matter through tractography, and most recently proposed as a functional contrast method capable of delineating neuronal firing in the active brain. Thanks to the molecular origins of its signal source, diffusion contrast is inherently useful at describing features of the microenvironment; however, limitations in achievable resolution in magnetic resonance imaging (MRI) scans precluded direct visualization of tissue microstructure for decades following MRI’s inception as an imaging modality. Even after advancements in MRI hardware had permitted the visualization of mammalian cells, these specialized systems could only accommodate fixed specimens that prohibited the observation and characterization of physiological processes. The goal of the current study was to visualize cellular structure and investigate the subcellular origins of the functional diffusion contrast mechanism (DfMRI) in living, mammalian tissue explants. Using a combination of ultra-high field spectrometers, micro radio frequency (RF) coils, and an MRI-compatible superfusion device, we are able to report the first live, mammalian cells—α-motor neurons—visualized with magnetic resonance microscopy (MRM). We are also able to report changes in the apparent diffusion of the stratum oriens within the hippocampus—a layer comprised primarily of pyramidal cell axons and basal dendrites—and the spinal cord’s ventral horn following exposure to kainate.

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YNIMG Journal 2012 Journal Article

Magnetic resonance microscopy of human and porcine neurons and cellular processes

Jeremy J. Flint
Brian Hansen
Sharon Portnoy
Choong-Heon Lee
Michael A. King
Michael Fey
Franck Vincent
Greg J. Stanisz

With its unparalleled ability to safely generate high-contrast images of soft tissues, magnetic resonance imaging (MRI) has remained at the forefront of diagnostic clinical medicine. Unfortunately due to resolution limitations, clinical scans are most useful for detecting macroscopic structural changes associated with a small number of pathologies. Moreover, due to a longstanding inability to directly observe magnetic resonance (MR) signal behavior at the cellular level, such information is poorly characterized and generally must be inferred. With the advent of the MR microscope in 1986 came the ability to measure MR signal properties of theretofore unobservable tissue structures. Recently, further improvements in hardware technology have made possible the ability to visualize mammalian cellular structure. In the current study, we expand upon previous work by imaging the neuronal cell bodies and processes of human and porcine α-motor neurons. Complimentary imaging studies are conducted in pig tissue in order to demonstrate qualitative similarities to human samples. Also, apparent diffusion coefficient (ADC) maps were generated inside porcine α-motor neuron cell bodies and portions of their largest processes (mean=1. 7±0. 5μm2/ms based on 53pixels) as well as in areas containing a mixture of extracellular space, microvasculature, and neuropil (0. 59±0. 37μm2/ms based on 33pixels). Three-dimensional reconstruction of MR images containing α-motor neurons shows the spatial arrangement of neuronal projections between adjacent cells. Such advancements in imaging portend the ability to construct accurate models of MR signal behavior based on direct observation and measurement of the components which comprise functional tissues. These tools would not only be useful for improving our interpretation of macroscopic MRI performed in the clinic, but they could potentially be used to develop new methods of differential diagnosis to aid in the early detection of a multitude of neuropathologies.

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YNIMG Journal 2012 Journal Article

Temporal scaling characteristics of diffusion as a new MRI contrast: Findings in rat hippocampus

Evren Özarslan
Timothy M. Shepherd
Cheng Guan Koay
Stephen J. Blackband
Peter J. Basser

Features of the diffusion-time dependence of the diffusion-weighted magnetic resonance imaging (MRI) signal provide a new contrast that could be altered by numerous biological processes and pathologies in tissue at microscopic length scales. An anomalous diffusion model, based on the theory of Brownian motion in fractal and disordered media, is used to characterize the temporal scaling (TS) characteristics of diffusion-related quantities, such as moments of the displacement and zero-displacement probabilities, in excised rat hippocampus specimens. To reduce the effect of noise in magnitude-valued MRI data, a novel numerical procedure was employed to yield accurate estimation of these quantities even when the signal falls below the noise floor. The power-law dependencies characterize the TS behavior in all regions of the rat hippocampus, providing unique information about its microscopic architecture. The relationship between the TS characteristics and diffusion anisotropy is investigated by examining the anisotropy of TS, and conversely, the TS of anisotropy. The findings suggest the robustness of the technique as well as the reproducibility of estimates. TS characteristics of the diffusion-weighted signals could be used as a new and useful marker of tissue microstructure.

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YNIMG Journal 2011 Journal Article

Diffusion tensor microscopy in human nervous tissue with quantitative correlation based on direct histological comparison

Brian Hansen
Jeremy J. Flint
Choong Heon-Lee
Michael Fey
Franck Vincent
Michael A. King
Peter Vestergaard-Poulsen
Stephen J. Blackband

Thanks to its proven utility in both clinical and research applications, diffusion tensor tractography (DTT) is regularly employed as a means of delineating white-matter tracts. While successful efforts have been made to validate tractographic predictions, comparative methods which would permit the validation of such predictions at microscopic resolutions in complex biological tissues have remained elusive. In a previous study, we attempted to validate for the first time such predictions at microscopic resolutions in rat and pig spinal cords using a semi-quantitative analysis method. In the current study, we report improved quantitative analysis methods that can be used to determine the accuracy of DTT through comparative histology and apply these techniques for the first time to human tissue (spinal cord) samples. Histological images are down-sampled to resolutions equivalent to our magnetic resonance microscopy (MRM) and converted to binary maps using an automated thresholding tool. These maps (n=3) are co-registered to the MRM allowing us to quantify the agreement based on the number of pixels which contain tracts common to both imaging datasets. In our experiments, we find that—on average—89% of imaging pixels predicted by DTT to contain in-plane white-matter tract structure correspond to physical tracts identified by histology. In addition, angular analysis comparing the orientation of fiber tracts measured in histology to their corresponding in-plane primary eigenvector components is presented. Thus, as well as demonstrating feasibility in human tissue, we report a robust agreement between imaging datasets taken at microscopic resolution and confirm the primary eigenvector's role as a fundamental parameter with clear physical correlates in the microscopic regime.

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YNIMG Journal 2010 Journal Article

Cellular-level diffusion tensor microscopy and fiber tracking in mammalian nervous tissue with direct histological correlation

Jeremy J. Flint
Brian Hansen
Michael Fey
Daniel Schmidig
Michael A. King
Peter Vestergaard-Poulsen
Stephen J. Blackband

Magnetic resonance imaging techniques have literally revolutionized neuroimaging with an unprecedented ability to explore tissue structure and function. Over the last three decades, the sensitivity and array of imaging techniques available have improved providing ever finer structural information and more sensitive functional techniques. Among these methods, diffusion imaging techniques have facilitated the generation of fiber-tract maps of the brain enabling an examination of issues related to brain structure and neural connectivity. Despite the potential utility of the techniques described, validation has not yet been achieved on biological samples. Recently, using newly developed surface microcoils on small samples at high magnetic fields, we demonstrated the ability of MR microscopy to image individual neurons in mammalian brain tissue. In the present work, we combine MR microscopy with the highest resolution (15μm) fiber tracking yet reported and demonstrate the accuracy of the fiber tract maps with direct histological validation. Thus it becomes possible to delineate fiber structure in tissues at the cellular level. A semi-quantitative approach was used to estimate the cell overlap fraction (cOF) and fiber tract overlap fraction (tOF), with cOFs of 94%, 92% and 100%, and tOFs of 84%, 86% and 100%, in rat cervical, rat lumbar, and pig spinal cord tissue, respectively. These methods provide a way to directly validate fiber tracking techniques with histology so that contemporary tracking techniques may be compared and refined using the microstructural details of a biological template as a ground truth.

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YNIMG Journal 2009 Journal Article

Diffusion weighted magnetic resonance imaging of neuronal activity in the hippocampal slice model

Jeremy Flint
Brian Hansen
Peter Vestergaard-Poulsen
Stephen J. Blackband

Functional magnetic resonance imaging (fMRI) has become the leading modality for studying the working brain. Being based on measuring the haemodynamic changes after enhanced mass neuronal activity the spatiotemporal resolution of the method is somewhat limited. Alternative MR-based methods for detection of brain activity have been proposed and investigated and studies have reported functional imaging based on diffusion weighted (DW) MRI. The basis for such DW fMRI is believed to be the sensitivity of diffusion weighted MRI to changes in tissue micro-structure. However, it remains unclear whether signal changes observed with these methods reflect cell swelling related to neural activation, residual vascular effects, or a combination of both. Here we present evidence of a detectable, activity-related change in the diffusion weighted MR-signal from the cellular level in live hippocampal slices in the absence of vasculature. Slices are exposed to substances which evoke or inhibit neural activity and the effects are evaluated and compared. The results are also compared to earlier DW fMRI studies in humans.

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YNIMG Journal 2009 Journal Article

Magnetic resonance microscopy of mammalian neurons

Jeremy J. Flint
Choong H. Lee
Brian Hansen
Michael Fey
Daniel Schmidig
Jonathan D. Bui
Michael A. King
Peter Vestergaard-Poulsen

Magnetic resonance imaging (MRI) is now a leading diagnostic technique. As technology has improved, so has the spatial resolution achievable. In 1986 MR microscopy (MRM) was demonstrated with resolutions in the tens of micrometers, and is now an established subset of MRI with broad utility in biological and non-biological applications. To date, only large cells from plants or aquatic animals have been imaged with MRM limiting its applicability. Using newly developed microsurface coils and an improved slice preparation technique for correlative histology, we report here for the first time direct visualization of single neurons in the mammalian central nervous system (CNS) using native MR signal at a resolution of 4–8 μm. Thus MRM has matured into a viable complementary cellular imaging technique in mammalian tissues.

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YNIMG Journal 2009 Journal Article

Postmortem interval alters the water relaxation and diffusion properties of rat nervous tissue — Implications for MRI studies of human autopsy samples

Timothy M. Shepherd
Jeremy J. Flint
Peter E. Thelwall
Greg J. Stanisz
Thomas H. Mareci
Anthony T. Yachnis
Stephen J. Blackband

High-resolution imaging of human autopsy tissues may improve our understanding of in vivo MRI findings, but interpretation is complicated because samples are obtained by immersion fixation following a postmortem interval (PMI). This study tested the hypotheses that immersion fixation and PMI's from 0–24 h would alter the water relaxation and diffusion properties in rat cortical slice and spinal cord models of human nervous tissue. Diffusion data collected from rat cortical slices at multiple diffusion times (10–60 ms) and b-values (7–15, 000 s/mm2) were analyzed using a two-compartment model with exchange. Rat spinal cords were characterized with standard diffusion tensor imaging (21 directions, b =1250 s/mm2). Switching from perfusion- to immersion-fixation at 0 h PMI altered most MRI properties of rat cortical slices and spinal cords, including a 22% decrease in fractional anisotropy (P <0. 001). After 4 h PMI, cortical slice T1 and T2 increased 22% and 65% respectively (P <0. 001), transmembrane water exchange decreased 23% (P <0. 001) and intracellular proton fraction increased 25% (P =0. 002). After 6 h PMI, spinal cord white matter fractional anisotropy had decreased 38% (P <0. 001). MRI property changes were observed for PMIs up to 24 h. The MRI changes correlated with protease activity and histopathological signs of autolysis. Thus, immersion fixation and/or even short PMIs (4–6 h) altered the MRI properties of rat nervous tissue. This suggests comparisons between in vivo clinical MRI and MRI data from human autopsy tissues should be interpreted with caution.

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YNIMG Journal 2006 Journal Article

MR microscopy of rat hippocampal slice cultures: A novel model for studying cellular processes and chronic perturbations to tissue microstructure

Timothy M. Shepherd
Bjorn Scheffler
Michael A. King
Greg J. Stanisz
Dennis A. Steindler
Stephen J. Blackband

Brain slices provide a useful nervous tissue model to investigate the relationships between magnetic resonance imaging (MRI) contrast mechanisms and tissue microstructure; yet, these acutely isolated tissues remain viable for only 10–12 h. To study slower biological processes, this work describes the first MRI microscopy characterization of organotypic rat hippocampal slice cultures that can be maintained for several weeks. Diffusion-weighted images of slice cultures acquired with a 14. 1-T magnet demonstrated the laminar anatomy of the hippocampus with relatively high signal-to-noise ratios. Diffusion data analyzed using a two-compartment model with exchange indicated that cultured slices had a comparable microstructure to acute brain slices and to in vivo brain. Immunohistochemistry indicated that slice cultures tolerated the conditions required for MRI study well. MRI of cultured tissue slices is highly amenable to correlative microscopy techniques and offers great promise for future MRI investigations of pathological tissue reorganization, molecular imaging and stem cell therapies.

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YNIMG Journal 2006 Journal Article

Resolution of complex tissue microarchitecture using the diffusion orientation transform (DOT)

Evren Özarslan
Timothy M. Shepherd
Baba C. Vemuri
Stephen J. Blackband
Thomas H. Mareci

This article describes an accurate and fast method for fiber orientation mapping using multidirectional diffusion-weighted magnetic resonance (MR) data. This novel approach utilizes the Fourier transform relationship between the water displacement probabilities and diffusion-attenuated MR signal expressed in spherical coordinates. The radial part of the Fourier integral is evaluated analytically under the assumption that MR signal attenuates exponentially. The values of the resulting functions are evaluated at a fixed distance away from the origin. The spherical harmonic transform of these functions yields the Laplace series coefficients of the probabilities on a sphere of fixed radius. Alternatively, probability values can be computed nonparametrically using Legendre polynomials. Orientation maps calculated from excised rat nervous tissue data demonstrate this technique's ability to accurately resolve crossing fibers in anatomical regions such as the optic chiasm. This proposed methodology has a trivial extension to multiexponential diffusion-weighted signal decay. The developed methods will improve the reliability of tractography schemes and may make it possible to correctly identify the neural connections between functionally connected regions of the nervous system.

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YNIMG Journal 2006 Journal Article

Structural insights from high-resolution diffusion tensor imaging and tractography of the isolated rat hippocampus

Timothy M. Shepherd
Evren Özarslan
Michael A. King
Thomas H. Mareci
Stephen J. Blackband

The hippocampus is a critical structure for learning and memory formation injured by diverse neuropathologies such as epilepsy or Alzheimer's disease. Recently, clinical investigations have attempted to use diffusion tensor MRI as a more specific surrogate marker for hippocampal damage. To first better understand the tissue architecture of healthy hippocampal regions, this study characterized 10 rat hippocampi with diffusion tensor imaging (DTI) at 50-μm in-plane image resolution using a 14. 1-T magnet. Chemical fixation of the dissected and straightened rat hippocampus provided a simple, effective way to reduce partial volume effects when segmenting hippocampal regions and improved mean signal-to-noise per unit time (e. g. 50. 6 ± 4. 4 at b = 1250 s/mm2 in 27 min). Contrary to previous reports that water diffusion is homogeneous throughout the nervous system, statistically different mean diffusivities were observed (e. g. 0. 238 ± 0. 054 and 0. 318 ± 0. 084 μm2/ms for the molecular and granule cell layers respectively) (ANOVA, P < 0. 05). Different hippocampal subregions had lower fractional anisotropy than uniformly fibrous structures like corpus callosum because of their complex architecture. DTI-derived color fiber orientation maps and tractography demonstrated most components of the trisynaptic intrahippocampal pathway (e. g. orientations in stratum lacunosum–moleculare were dominated by perforant and Schaffer fibers) and also permitted some assessment of connectivity in the rat hippocampus.

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