Putting molecules in the picture: Using correlated lightmicroscopy and soft X-ray tomography to study cells

Abstract

Transmission soft x-ray microscopy (SXM) is well suited to visualizing and quantifying mesoscale biology, that is to say structures ranging from the size of a typical molecular machine up to an intact cell. In SXM, the specimen is illuminated with “water window” soft x-rays (i.e., photons with energies in the range 284 eV to 543 eV). The degree to which the illumination is absorbed by a subcellular feature depends on its chemical composition. Carbon- and nitrogen-containing biomolecules strongly absorb the illumination, while water is relatively transparent. Similarities and differences in subcellular molecular content generate contrast in SXM images of the specimen without the use of stains. In this chapter, we discuss the basic concepts behind SXM and describe how 2D SXM data is used to calculate a 3D soft x-ray tomographic (SXT) reconstruction of the specimen. SXT offers significant advantages over other high-resolution cell imaging methods. A particular strength is the capacity to image intact, fully hydrated cells, including eukaryotes such as yeast or mammalian cells. In addition to stand-alone use, we will show that SXT data can be integrated into other imaging modalities to create a comprehensive view of the specimen. The combination of SXT with fluorescence data measured from the same specimen is particularly important, since this allows molecular localization data to be viewed directly in the context of the cell.