Imaging has long been one of the principal techniques used in biological and biomedical research. Indeed, the field of cell biology grew out of the first electron microscopy images of organelles in a cell. Since this landmark event, much work has been carried out to image and classify the organelles in eukaryotic cells using electron microscopy. Fluorescently labeled organelles can now be tracked in live cells, and recently, powerful light microscope techniques have pushed the limit of optical resolution to image single molecules. In this paper we describe the use of soft x-ray tomography, a new tool for quantitative imaging of organelle structure and distribution in whole, fully-hydrated eukaryotic Schizosaccharomyces pombe cells. In addition to imaging intact cells, soft x-ray tomography has the advantage of not requiring the use of any staining or fixation protocols-cells are simply transferred from their growth environment to a sample holder and immediately cryofixed. In this way the cells can be imaged in a near native state. Soft x-ray tomography is also capable of imaging relatively large numbers of cells in a short period of time, and is therefore a technique that has the potential to produce information on organelle morphology from statistically significant numbers of cells.

Silver diamine fluoride (SDF) is a brush-on treatment for tooth decay that stops 81% of cavitated caries lesions (dental cavities). Before this innovation, caries was treatable only with operative approaches (dental fillings). SDF-treated lesions harden and become resistant to further decay. We hypothesized that the hardening is due to reaction with silver, rather than classic fluoride-mediated remineralization, because infected dentin is not amenable to remineralization. Using synchrotron microCT with 1.3 μm resolution, we observe filamentous densities up to 500 μm in length and 0.25-7.0 μm in diameter, formed in situ by applying SDF to caries lesions. We show that these “microwires” fill voids in the lesion caused by disease, and permeate through surrounding dentinal tubules. Using spectroscopy, we confirm that the chemical composition of the observed microwires is predominantly silver. To our knowledge, this represents the first structural microscale observations resulting from clinical SDF treatment. These novel observations hint at mechanistic explanations for the first clinical method to harden carious dentin besides remineralization. We hypothesize that SDF may not only achieve its antimicrobial functions by biochemical interactions, but also through its inherent ability to integrate into dentin.

Soft X-ray microscopy can image whole, hydrated, biological specimens up to 10 microns thick with a spatial resolution better than 50 nm [1-4]. Soft X-ray microscopy uses photons with energies between the K shell absorption edges of carbon (284 eV, =4.4 nm) and oxygen (543 eV, =2.3 nm). These photons readily penetrate the aqueous environment while encountering significant absorption from carbon-and nitrogen-containing organic material. In this energy range, referred to as the 'water window,' organic material absorbs approximately an order of magnitude more strongly than water, producing a quantifiable natural contrast and eliminating the need for contrast enhancement procedures to visualize cellular structures.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008