Stephen J. Blackband - US grants

The goal of this proposal is to develop nuclear magnetic resonance (NMR) microscopy and spatially resolved spectroscopy at 360 MHz (8.5 T/8.9cm bore) and 200MHz (4.7T/33cm bore) on single cells, extracted tissues, and small animals with and without tumors and thus assess their feasibility and applicability. The cytoplasmic characteristics and cell dynamics and metabolism of large single cells (more than 100 microns), specifically frogs eggs, will be examined by 1H studies before and after fertilization in vivo. Imaging and spectrosocopic studies conducted on lenses under normal and high glucose media conditions will improve understanding of diabetes and cataractogenesis, since the effects of diabetes on the lens are reflective of its effect on other tissues. Tumors on small animals will be examined by microscopy and localized spectroscopy and the significance of tumor heterogeneity thus addressed. From these studies the morphologic and spectroscopic characteristics of tumors implanted in animals will be obtained and their implications with respect to cancer diagnosis investigated. The resolution limits defined on the 200MHz instrument on rats and rabbits (with and without implanted tumors) will determine the applicability of microscopy to larger systems, specifically humans. The biological systems in these studies are selected because they are well defined, are currently being investigated in this laboratory, and are areas in which application of this technology will have great potential in their understanding and clinical application. The specific objectives of this proposal are (1) to obtain the best possible resolution and define the limits of spatial resolution with NMR microscopy at both fields on different sized biological systems; (2) to perform NMR microscopy on single cells and study cell metabolism, dynamics (division) and compartmentalization; (3) to determine the capability of resolving the structure of cell clusters in intact living tissues (lenses) and small animals (with tumors); (4) to implement 31P and 1H spatially resolved spectroscopy on imaged regions of small animals, lenses and cells (1H only) at both fields and define the resolution limits; and (5) to obtain spatially resolved relaxation times (T1 and T2) and self-diffusion coefficients in selected studies.

High magnetic field strengths are essential for optimized signal to noise and spectral resolution in NMR. To this end, a 4.7T/40cm bore GE instrument was purchased three years ago and is used for basic research on a variety of animal models. Since that time, however, NMR techniques and technology have radically improved. The present instrument is outdated, and restricts the scope and quality of the research that can be performed. Several techniques, especially spatially localized spectroscopy, are not feasible with the present system. Consequently this proposal requests an upgrade of this instrument to keep it at the cutting edge of NMR technology. Two major upgrades are requested. The first is for two sets of shielded gradients of differing diameters. This will greatly improve our ability to perform spatially localized spectroscopy, NMR microscopy and high speed imaging on large and small animal models. The second upgrade is for a new computer console based on a SUN computer. The present system is outdated and very restrictive with regards to pulse sequence programming, graphic prescription, real time parameter optimization, data processing, and data storage. These improvements are essential for the continued development of spatially localized spectroscopy and imaging for a variety of studies in oncology, cardiology, radiology and neurology at JHMI.

EXCEED THE SPACE PROVIDED. The overall objective of this Resource is to develop technology and techniques for high magnetic field MR imaging and spectroscopy to support the study of significant biomedical problems. We are in a prime position to do this through the acquisition of two of the world's highest field instruments; a 750MHz/9cm and a 11.7T/40cm imaging spectrometer. The push towards higher field strengths is driven by increases in signal strength and spectral resolution available at higher fields. These improvements will be used to develop biomedical applications of MR through collaborative projects locally, nationally and internationally. These objectives are supported by a strong synergy between the Universityof Florida (UF) and Florida State University (FSU) as partners in the National High Magnetic Field Laboratory (NHMFL), with support from the McKnight Brain Institute at UF. There is little point in stronger magnets if the associated technology andtechniques are not developed - indeed, the potential gains would otherwise be lost. These developments form the basis of this proposal, and will be carried out through three research cores plus an administrative core, in support of 19 collaborative projects (15 funded, 4 pending) and 7 service projects (6 funded). The three cores focus on sensitivity improvements for (1) high field small animal imaging and spectroscopy (novel large volume and phased array coils), (2) microimaging and spatially localized microspectroscopy (microcoils, microphased arrays, strong gradient coils, and high field imaging techniques) and (3) high sensitivity and high throughput solution-state NMR spectroscopy (microcoils, multicoil arrays, high temperature superconducting probes). The collaborative projects range from studies of cancer, stroke, hydrocephalus, ischemia, and brain and spinal cord trauma, HIV, diabetes, epilepsy, Alzheimers, DCA toxicity, bone dosimetry, through protein folding,structure-function studies and drug design, as well as basic studies on the origins of MR signals in tissues. The Resource will provide training and disseminate knowledge resulting from these studies.

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DESCRIPTION (provided by applicant): Over the last two decades MR microscopy has evolved into a subset of MR Imaging with a wide range of applications, with its greatest benefit still the ability to image live tissue non-invasively. Still, the resolution is limited compared to other microscopies and until recently cellular and subcellular resolutions on mammalian tissue were not possible. Additionally, the cellular origins of MR signals in tissues are still unknown, and mathematical models attempting to elucidate this issue are the subject of great debate. Recently, using microsurface coils at high fields, we have obtained the first direct MR images of mammalian cells, and further, fiber tract maps at the cellular level with direct histological correlation. Still, these studies were of fixed tissue and the data took several hours to acquire. This proposal will demonstrate that the combination of smaller microsurface coils, higher magnetic fields and smaller, faster and stronger planar gradient coils can, conservatively, improve the SNR by an order of magnitude or more. Then, coupled with new microperfusion chambers, mammalian sub-cellular resolution MR microscopy of live mammalian tissue can be achieved in physiologically acceptable imaging times. Additionally new microvolume coils will be developed for accurate quantitative studies. Aims 1-5 will implement MR microscopy at successively higher magnetic field strengths (14.1, 17.6 and 21 Tesla) using new microsurface and volume microcoils, new planar microgradients and optimized sequences, and testing the system for stability and accuracy of quantitation. We will explore the utility of these developments primarily on neural tissue (single Aplysia neurons and rat brain slices, both on fixed tissue and then live perfused tissue) and similarly in cardiac tissue. When successful, a wide range of tissues will be possible to study. Through quantitation of intra and extracellular signals and how they change with physiological perturbations (for example, ischemia), we will be able to develop working realistic mathematical models of MR signals in tissues. Additionally, we will be able to accurately validate fiber tracking techniques at the cellular level. Thus, MR microscopy will provide a complementary microscopy technique for imaging live tissue at the sub-cellular level. Relevance: The development of the MR microscope capable of imaging live mammalian tissue at the sub-cellular level in physiologically acceptable imaging times will for the first time facilitate a quantitative understanding of the signal origins in MRI. This in turn will impact the sensitivity and specificity of MRI, improving its clinical potential. For example, a quantitative understanding of the signal changes in brain and cardiac ischemia may be able to resolve the difference between reversible and irreversible damage in stroke and heart attack, and have a major impact in improving the utility of MRI in a wide variety of tissues and diseases. PUBLIC HEALTH RELEVANCE: An MR microscope will be developed capable of obtaining cellular and sub-cellular resolution in live mammalian tissue in physiologically relevant acquisition times using new microcoils, microgradients and a micro-perfusion system at high magnetic fields. Quantitative studies will be undertaken on live brain and cardiac tissue. Consequently an understanding of the origins in MR signals will be developed, impacting on the sensitivity and specificity of clinical MRI.