Ultrastructural X-ray imaging and molecular modelling of hydrated biological specimen using laser plasma pulsed point X-ray sources

Abstract

Electron microscopy (EM) is stilt the most frequently used method for visualization of subcellular structures inspite of limitations due to the cumbersome preparation required to visualize the specimen. High resolution X-ray microscopy is a relatively new technique still under developement and restricted to a few large synchroton X-ray sources which use specimen exposure to X-rays over a few second interval before obtaining an image. We utilized a bench-top source of single-shot laser (nsec) plasma to generate X-rays similar to those used at synchroton facilities to image live hydrated (in 0.9% saline) cells of several species of bacteria and macromolecules (DNA and lipopolysaccharadie (IPS)) 5|il aliquot suspension was placed on a small (PMMA) photoresist and covered with a thin (100nm) SiN window. This sealed specimen is positioned in a vacuum chamber close to the laser-plasma point X-ray source. The emission spectrum is tuned for optimal absorption by carbon-rich material to etch an image on the resist. The resist is then scanned by an atomic force microscope to give a topographical image of differential X-ray absorption corresponding to specimen properties. By this technique we have visualized distinct consistent layers around whole cells of bacteria reflecting the structure of the envelope, darker stained areas inside the cell corresponding to the chromosomal DNA spread as has been seen by thin sectioned electron micrographs, and 3-dimensional molecular structures of DNA and LPS similar to the space filling models of such molecules.. Show more