J.Microscopy 204 (2001) 69

Journal of Microscopy 204 (2001) 69-86.

A new sample preparation method for biological soft X-ray microscopy: nitrogen-based contrast and radiation tolerance properties of glycol methacrylate-embedded and sectioned tissue

B.W.LOO JR*, I. M. SAUERWALD²,A.P.HITCHCOCK³AND S. S. ROTHMAN§

*Bioengineering Graduate Group, University of California, San Francisco and Berkeley, School of Medicine, University of California, Davis, California, U.S.A.

²Department of Growth and Development, University of California, San Francisco, U.S.A.

³Brockhouse Institute for Materials Research, McMaster University, Hamilton, Ontario, L8S 4M1, Canada

§Departments of Physiology and Stomatology, University of California, San Francisco, Bioengineering Graduate Group, University of California, San Francisco and Berkeley, Genteric Corporation, Alameda, California, U.S.A.

Received 18 February 2000; accepted 7 January 2001

Abstract:

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 embed- ding 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 10^8 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 micro- scopes, previously a difficult task, as well as multimodal imaging (using visible light, X-ray, electron, and scanned probe microscopies) on the same sample.

Published on Web 11/22/2001