We present comprehensive measurements of the C (carbon) K edge near-edge X-ray absorption (NEXAFS) spectra of all 20 amino acids commonly occurring in nature. Qualitative trends among the spectra of amino acids with similar chemical character are identified and spectral features are compared with extensive ab initio calculations. The contributions of individual units and substitutional groups have been determined to explore their fingerprinting character using the building block concept. Several such units are found. Two that give particularly clear features in the C 1s NEXAFS spectra are the carboxyl group (which can be clearly identified by a pronounced structure due to the C 1s→π*C  O transition with maximum at 288.65(5) eV) and modified phenol rings in aromatic amino acids (which give sharp C 1s→π*C  C structures). The latter transitions are located around 285 eV, and their shape is specific for each aromatic amino acid. Other building blocks, such as the CNH n group and the CH, CC, CO, CN pair bonds, are also identified, although their characteristic features are less pronounced in the C K edge spectra than the carboxylic and aromatic structures. This study provides the basis for rigorous assignment of the NEXAFS spectra of the amino acids, and will be helpful in developing X-ray absorption spectroscopy for quantitative analysis of proteins.

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We have developed a cryo scanning transmission X-ray microscope which uses soft X-rays from the National Synchrotron Light Source. The system is capable of imaging frozen hydrated specimens with a thickness of up to 10 microm at temperatures of around 100 K. We show images and spectra from frozen hydrated eukaryotic cells, and a demonstration that biological specimens do not suffer mass loss or morphological changes at radiation doses up to about 1010 Gray. This makes possible studies where multiple images of the same specimen area are needed, such as tomography (Wang et al. (2000) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography. J. Microsc. 197, 80-93) or spectroscopic analysis.

Using a cryo scanning transmission X-ray microscope (Maser, et al. (2000) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy. J. Microsc. 197, 68-79), we have obtained tomographic data-sets of frozen hydrated mouse 3T3 fibroblasts. The ice thickess was several micrometres throughout the reconstruction volume, precluding cryo electron tomography. Projections were acquired within the depth of focus of the focusing optics, and the three-dimensional reconstruction was obtained using an algebraic reconstruction technique. In this first demonstration, 100 nm lateral and 250 nm longitudinal resolution was obtained in images of unlabelled cells, with potential for substantial further gains in resolution. Future efforts towards tomography of spectroscopically highlighted subcellular components in whole cells are discussed.