X‐ray microscopy and imaging of Escherichia coli, LPS and DNA

Abstract

SummaryUltrastructural 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 laserplasma 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 mm wavelength, 5 × 10 12 W cm ¹2 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 20 J) 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 Å 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.

Nanoconstruction of Microspheres and Microcapsules Using Proton-Induced Phase Transitions: Molecular Self-Recognition by Diamide Diacids in Water

“…At the border between the UV and X-ray regions of the electromagnetic spectrum (also known as the “water window”), organic molecules absorb radiation (λ < 20 Å) more strongly than water. The absorptivity differences (between organic molecules and water) are large enough that the water matrix is relatively transparent to these wavelengths . The “soft” X-rays pass through a thin film (20 μm thick) of the aqueous suspension, are partially absorbed by the suspended organic assemblies, and impact a poly(methyl methacrylate) (PMMA) photoresist with reduced intensity.…”

“…There are several advantages to this technique. XRM provides the ability to image the assembly process as it takes place in an aqueous environment, with no further sample manipulation and, unlike SEM, without the use of high vacuum . Moreover, the drying step required with SEM sample preparation may actually preclude the detection of supramolecular intermediates (rods), which likely coalesce upon concentration of the suspension.…”

“…The sample was then placed in a sample holder which was located 1 cm away from a gold foil target and 45° from the target normal in a vacuum chamber. The chamber was evacuated, and a plasma was produced by irradiating the gold target with a 5 ns pulse of 1064 nm light at 20 J from a Nd:glass laser, allowing the sample to be irradiated by “soft” X-rays (λ = 2−4 nm) …”

“…Remarkably, this minor platform alteration changed the assembly outcome from microcapsules (with 3 ) to microspheres (with 5 ). To better understand this observation, a detailed study of 5 was initiated using Fourier transform infrared (FTIR), variable-temperature NMR (VT-NMR), transmission electron microscopy (TEM), X-ray crystallography, Raman microscopy, and a novel imaging technique known as “soft” X-ray microscopy (XRM) . These diverse techniques have now provided the evidence necessary to formulate a mechanism by which these systems generate microspheres in aqueous environments.…”

Application of X‐ray microscopy in analysis of living hydrated cells

“…That is, well‐maintained fragile outer structures of macrophages are observed by X‐ray microscopy, providing acceptable resolution for the study of cell–cell interactions. The application of X‐ray microscopy to biological specimens, such as chromosome fibers, protozoa, bacteria, sperm, and cultured cells, has also been studied with some success (Tomie et al, 1991; Kinjo et al, 1994; Kirz et al, 1995; Methe et al, 1997; Rajyaguru et al, 1997a, b; Kado et al, 1999; Scharf and Schneider, 1999). In particular, current equipment supported by advanced technologies provides practical X‐ray microscopes, such as those used in this study and for similar cell biology studies by biological scientists.…”