X-ray microscopy and biomedical specimens

Application of X-ray microscopy to biological specimens has been very limited to date, and is restricted to a few large synchrotron X-ray sources which need specimen exposure for a few seconds before an image is obtained. In diagnosis of infectious diseases, detection of microbial pathogens is performed using low resolution optical microscopy, Gram-stain, growth on selective media and polymerase chain reaction (PCR). These procedures require fixation-staining for optical microscopy, overnight incubation for growth of the pathogen on selective liquid or plated media, and complete cell lysis for PCR. The authors utilized a bench top source of single shot laser (nanosecond) plasma to generate X-rays in the "water window" similar to synchrotron facilities to image live hydrated cells (in 0.9% phosphate buffered saline). A 5 /spl mu/l aliquot suspension was placed on a small (5 mm/sup 2/) polymethylmethyacrylate (PMMA) coated photoresist and covered with a thin (100 nm) SiN window. This sealed specimen was placed in vacuum close (2 cm) to the laser-plasma point X-ray source. The X-ray emission spectrum was tuned for optical absorption by carbon-rich material to etch an image on the PMMA coated photoresist, The photoresist was then developed by immersion in solvent to remove the broken polymeric PMMA material and then scanned by an atomic force microscope to obtain a topographical image of differential X-ray absorption. The authors have applied high resolution X-ray microscopy to image various types of biomedical specimens to assess its usefulness in this area. The images that they obtained are similar to those observed by other direct microscopy techniques, such as optical, fluorescence or variations of electron microscopy. However, these images represent the status of the cell in its live hydrated and therefore natural dynamic state.