NMR Microscopy of Formalin Fixed Tissue in Organic Solvents

We propose to use organic solvents as imaging medium in MRM to either improve image quality or shorten the imaging time, or both.
Standard procedure for long term preservation of human and animal tissue is fixation with neutralized formalin buffer (NFB). MRM on ex vivo tissue specimens is mostly performed on the fixed tissue when water in NFB is used as an imaging medium. Water protons in NFB are in fast chemical exchange with protons from various formalin derivatives, which creates a problem. At higher magnetic fields most suitable for MRM this leads to a strong water line broadening or a significant shortening of T2. This reduces the parameter space for image contrast optimization. For example, at echo times longer than 20 ms the NFB tissue signal becomes very small at 16.4 T.
Water is routinely replaced by organic solvents in the course of tissue processing for histology analysis. The tissue specimens are treated by a series of organic solvents and finally are embedded in paraffin to produce formalin fixed paraffin embedded (FFPE) tissue blocks. We used the same processing method (by replacing water by ethanol and ethanol by selected solvent) to prepare specimens for MRM of fixed tissue in organic solvent. We have found that dimethyl sulfoxide (DMSO) is particularly suitable for MRM: the DMSO methyl protons have T1 comparable to T1 of water in NFB, but much longer T2 (due to the absence of chemical exchange) and smaller diffusion coefficient (D). In addition, in the DMSO tissue swells to increase the volume up to 50%. All these changes contribute to an enhanced quality of the images compared to those obtained in NFB.
Melted paraffin appears to be the most interesting imaging medium. At room temperature, T2 of paraffin is so short that no echo could be observed under standard imaging conditions. However, upon melting, the paraffin T2 becomes very long (>200 ms at 60 C and 16.4 T) enabling the use of all MRM methods. Also, D of paraffin is almost ten times smaller than D of water. Interestingly, T1 of paraffin is not affected by the presence of tissue and the contrast mostly depends on the T2 and T2*.
There are millions of FFPE tissue blocks in biomedical community maintained in tissue banks. They are invaluable source of research and clinical information but they are consumed during standard histological analysis. MRM of FFPE blocks could be an invaluable tool for nondestructive imaging even if MRM is no match for optical microscopy analysis. Three dimensional MRM images of tissue block could be useful for global analysis of heterogeneous tissue and could guide pathologist toward the regions of interest.
MRM imaging of formalin fixed tissues in organic solvents at 16.4 T (700 MHz) is illustrated by images of mouse and human neurological tissue.