• Digital imaging microscopy;
  • exocrine pancreas;
  • glycol methacrylate tissue embedding and sectioning;
  • microscopic protein mass measurement;
  • nitrogen-based contrast;
  • quantitative morphometry;
  • radiation tolerance;
  • sample preparation;
  • synchrotron;
  • transmission soft X-ray microscopy;
  • XANES;
  • zymogen granules

We describe the preparation of a biological tissue for imaging in a transmission soft X-ray microscope. Sections of exocrine pancreas embedded in glycol methacrylate polymer, an embedding medium widely used in visible light and electron microscopy, were examined. Contrast was based primarily on the nitrogen content of the tissue, and dual-wavelength imaging at the nitrogen K-shell absorption edge was used to map the distribution and provide quantitative densitometry of both the protein and embedding matrix components of the sample. The measurements were calibrated by obtaining the absorption spectrum of protein near the nitrogen edge. The contrast was consistent and reproducible, making possible the first large-scale X-ray microscopic study on sections of plastic-embedded soft tissue. At radiation doses of up to 108 Gray, much more than required for routine imaging, no distortion and little mass loss were observed. This sample preparation method should permit routine imaging of tissues in X-ray microscopes, previously a difficult task, as well as multimodal imaging (using visible light, X-ray, electron, and scanned probe microscopies) on the same sample.