3D Magnetic Resonance Microscopy of Dehydrated Biological Specimens

Abstract

Objectives: The major theme of this thesis is the evaluation of the potential of Magnetic Resonance Microscopy (MRM) for branches of the life sciences that have previously seen few or no applications of this non-invasive methodology, particularly for cell biology, palaeontology and cryobiotechnology. An emphasis will be put on the role of liquid water whose multiple biological functions - as a solvent, structural element and metabolite - render it essential for life as we know it. Background: Dehydration beyond a critical threshold poses a serious threat to most organisms in their active state, and mechanisms helping to cope with draught stress present an evolutionary advantage in environments lacking permanent access to liquid water. From a biotechnological point of view, mastering the reversible transition between hydrated and dehydrated states of biological material would allow their long-term storage and facilitate a continuous supply, especially in cases where cell and tissue culture are impossible or not desirable. One of the ways to achieve this is cryopreservation, an arsenal of methods designed to store biological materials for long terms at biologically low temperatures to minimise degradation. In recent years, a trend has developed towards freezing of small cell clusters or even single cells, as opposed to large chunks of tissue. This creates an increased demand for miniaturised cryopreservation systems, and the reduced amount of material raises the need for high-resolution non-invasive supervision of the cryoprocessing. Method: Magnetic Resonance (MR) techniques have become famous precisely for their non-invasiveness and their sensitivity to liquid water, yet microscopic MR applications to dehydrated samples have been scarce, mainly because (i) the signal-to-noise ratio decreases upon dehydration - even dramatically so upon freezing - and (ii) high spatial resolutions translate into a considerable reduction of the already low signal intensity. The primary goals of this study were, therefore, to determine whether these two major barriers can be overcome individually as well as in combination and to evaluate whether the strong magnetic fields necessary for such experiments could interfer with cellular physiology. Results: Microscopic MR image series allowed the non-invasive assessment of the morphology within a well-hydrated cell biological model system (oocytes and embryos of the frog Xenopus laevis), in extreme examples of long-term preservation and dehydration (fossil remains of invertebrate, vertebrate and plant species) and in cryobiological samples ranging from tumor cell spheroids to larvae of cold-hardy insects. Cell division and embryogenesis could be observed in MRM images of Xenopus embryos, and spatially localised MR spectra from subcellular compartments delivered biochemical information about Xenopus oocytes in their normal state and upon uptake of an externally applied drug. A previously described apparent magnetic field effect on Xenopus embryos could be shown not to depend on the magnetic field, as opposed to a new effect found in oocytes artificially deprived of their jelly coat. MRM data allowed the diagnosis of pathological alterations in fossils and the monitoring of cryoprotectant effects in frozen or supercooled insects. Conclusions: These experiments demonstrate that, from a technical perspective, MRM indeed has the potential to become a tool for cell biology, palaeontology as well as cryobiotechnology and that side effects of the methodology, though detectable under unphysiological conditions, do not prevent that.