Quantitative 3D imaging of cells at 50 nm resolution using soft x-ray tomography

Abstract

Soft X-ray microscopy combines features associated with light and electron microscopy — it is an imaging technique that is both fast and relatively easy to accomplish (like light microscopy) that produces high-resolution, absorption-based images (like electron microscopy). As with light microscopy, one can examine whole, hydrated cells (between 10–15 µm thick), eliminating the need for time-consuming and potentially artifact-inducing embedding and sectioning procedures. Similar to electron microscopy, the image is formed based upon density of cellular components. With soft x-ray microscopy, however, the image is based on the inherent contrast of the organic material in the cell or tissue being examined1. Soft x-ray microscopy utilizes photons in the ‘water window,’ with energies between the K shell absorption edges of carbon (284 eV, λ=4.4 nm) and oxygen (543 eV, λ=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 structures2.