Three-dimensional NMR microscopy of zebrafish specimens

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 T₁ weighting (3D FLASH, 3D FISP), whereas in FFPE specimens T₂ 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.