X-ray microscopy and imaging of Escherichia coli, LPS and DNA.
Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs. X-ray microscopy can take instant images of specimens but is mostly restricted to a few synchrotron X-ray sources. We have utilized a bench-top nanosecond laser-plasma to produce a single-shot source of nanosecond X-rays tuned for maximum contrast with carbon-rich material. To examine the ultrastructure by absorption profiles, we utilized a laser-produced plasma generated by a single-shot laser (1.06 microns wavelength, 5 x 10(12) W cm-2 intensity) focused on to a silicon target as an X-ray source for high-resolution X-ray microscopy. This approach eliminates the specimen preparation steps. Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)-coated grids (resist). This resist was exposed to X-rays under vacuum at a distance of 2.5 cm from the target disc. The silicon plasma produced by a 10-ns burst of laser energy (at 20J) radiates strong emission lines in the region of 300 eV. The X-rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image. By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E.coli, darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules. This technique has resolution at the 100 A level, produces images similar to the space-filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens.
Rajyaguru
J M
JM
Department of Pediatric Infectious Diseases Research, Arnold Palmer Hospital for Children and Women, Orlando, FL 32806, USA. JAYSHREE@orhs.org
Kado
M
M
Torres
D
D
Richardson
M
M
Muszynski
M J
MJ
D004269:DNA, Bacterial; D004926:Escherichia coli / Q000737:chemistry*; D008070:Lipopolysaccharides; D008853:Microscopy / Q000379:methods*; D014965:X-Rays
D004269:DNA, Bacterial; D008070:Lipopolysaccharides