Macura, Slobodan I

While zebrafish embryos in the first five days after fertilization are clear and amenable to optical analysis, older juveniles and adults are not, due to pigmentation development and tissue growth. Thus other imaging methods are needed to image adult specimens. NMR is a versatile tool for studies of biological systems and has been successfully used for in vivo zebrafish microscopy. In this work we use NMR microscopy (MRM) for assessment of zebrafish specimens, which includes imaging of formalin fixed (FF), formalin fixed and paraffin embedded (FFPE), fresh (unfixed), and FF gadolinium doped specimens. To delineate the size and shape of various organs we concentrated on 3D MRM. We have shown that at 7 T a 3D NMR image can be obtained with isotropic resolution of 50 μm/pxl within 10 min and 25 μm/pxl within 4 h. Also, we have analyzed sources of contrast and have found that in FF specimens the best contrast is obtained by T 1 weighting (3D FLASH, 3D FISP), whereas in FFPE specimens T 2 weighting (3D RARE) is the best. We highlight an approach to perform segmentation of the organs in order to study morphological changes associated with mutations. The broader implication of this work is development of NMR methodology for high contrast and high resolution serial imaging and automated analysis of morphology of various zebrafish mutants.

The magnetization exchange between two groups of equivalent spins each having different populations and overall relaxation rates has been analyzed. The results suggest that either the spin population difference or the overall relaxation rate difference in an exchange spectrum can produce cross-peaks with volumes larger than that of the corresponding diagonal line. This is important for interpretation of the magnetization exchange between water and macromolecular protons where both the population differences and auto-relaxation rate differences can be very large. Theoretical predictions of peak volume evolution were experimentally verified in the intermolecular magnetization exchange between the water and labile amide proton in a model system N-acetylglycine/water.

We propose to use organic and mixed solvents for nuclear magnetic resonance microscopy of fixed tissue as a means for improving image information content. NMR properties of some standard solvents (methanol, acetone, DMSO) and solvents in use for tissue processing in pathology (xylenes, paraffin, ?Clearify?) have been measured, reviewed, and analyzed. It was found that DMSO and paraffin are very useful solvents that provide images of better quality than those obtained in water (neutralized formalin buffer). This is illustrated on the formalin fixed mouse brain sections imaged at 16.4 teslas (700 MHz).