High resolution X-ray micrography of live Candida albicans using laser plasma pulsed point X-ray sources

Abstract

Electron microscopy is still the most frequently used method for visualization of subcellular structures in spite of limitations due to the preparation required to visualize the specimen. High resolution X-ray microscopy is a relatively new technique, still under development and restricted to a f e w large synchrotron X-ray sources. We utilized a single-shot laser (nanosecond) plasma to generate X-rays similar to synchrotron facilities to image live cells of Candida albicans.The emission spectrum was tuned for optimal absorption by carbon-rich material. The photoresist was then scanned by an atomic force microscope to give a differential X-ray absorption pattern. Using this technique, with a sample image time of 90 min, w e have visualized a distinct 152.24 nm thick consistent ring structure around cells of C. albicans representing the cell wall, and distinct 'craters' inside, one of 57090 nm diameter and three smaller ones, each 400 nm in diameter. This technique deserves further exploration concerning its application in the ultrastructural study of live, hydrated microbiological samples and of macromolecules.Keywords : Candida albicans, X-ray micrography, high resolution, ultrastructure, laser plasmas INTRODUCTIONThe detection and study of microbial clells is performed by low magnification optical microscopy, and direct and indirect labelling techniques. In bacteriology, cationic (methylene blue, crystal violet, safranin) or anionic (eosin, acid fuchsin, congo red) colour dyes are used for resolution at the 1-100 pm scale and radioactively labelled substrate uptake is measured for very slow growing mycobacteria (Chapin-Robertson & Edberg, 1991). In virology, for nm-scale detection, secondaryconjugated fluorescent antibodies are applied. Visual ultrastructural studies on subcellular organelles are possible with variations of electron microscopy (thin section, scanning and freeze fracture) a1 though specimen preparation steps such as fixation, dehydration, resin embedding, ultra-thin sectioning, coating and staining are very technical, extensive and may introduce artefacts in the original sample. Although electron microscopy can be used at the nm resolution level, the sample Abbreviations: AFM, atomic force microscopy; PMMA, poly(methy1 methacrylate).preparation steps involved limit its use for routine studies of microbial cells (Kay, 1976 ; Lichfeld, 1976).X-ray microscopy is a relatively new technique that has not been applied to any significant extent for biological specimens (DeMeis, 1996). It eliminates specimen preparatipn which may alter the target, has resolution at the 100 A level (Ohnesorge & Binning, 1993) and is able to probe the internal structures of in vivo assemblies, enabling the observation of complex features in their natural, live state (Feder et al., 1985;Hoh et al., 1992).Most X-ray microscope development has been made so far using large synchrotron sources (Neiman, 1992), thus limiting X-ray microscopy as a research tool to where such facilities are available. The use of a laser plasma X-r...

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.…”

Direct Ultrastructural Imaging of Macrophages Using a Novel X-Ray Contact Microscopy

“…Figure 2 shows a representative x-ray image of a hydrated macrophage that has been fixed with glutaraldehyde, the same as for electron microscopy. However, we could observe viable microorganisms by x-ray microscopy (17) and have obtained preliminary data regarding viable macrophages without fixing cells. 3), the x-ray images showed some structural components of the macrophages, but these seemed not rigid, which means not clear as compared with TEM images.…”

Direct Ultrastructural Imaging of Macrophages Using a Novel X‐Ray Contact Microscopy

“…Since, in the present study, we used fixed cells with glutaraldehyde for the initial study of x-ray microscopy applied to macrophages, it was not possible to examine the physiological state of the macrophages. However, we could observe viable microorganisms by x-ray microscopy (17) and have obtained preliminary data regarding viable macrophages without fixing cells. Thus, x-ray microscopy may be useful for analysis of the physiological state of cells.…”