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

Abstract

Transmission soft x-ray microscopy (SXM) is an important new technique for visualizing and quantifying biological specimens, especially individual cells. In SXM the specimen is illuminated with "water-window" soft x-rays (i.e., x-ray photons with energies in the range 284-543eV). As the illuminating light passes through the specimen, it is attenuated more strongly by carbon- and nitrogencontaining biomolecules than by water. Consequently, even subtle differences in molecular composition produce SXM images with quantitative contrast. In this chapter, we will discuss the basic concepts behind SXM and describe how 2D SXM data is used to calculate high fidelity 3D soft x-ray tomographic (SXT) reconstructions of the specimen. SXT offers many advantages over other high-resolution 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 standalone use, SXT data can be integrated with data from other imaging modalities. The recent development of cryogenic confocal fluorescence tomography (CFT) has permitted 3D SXT reconstructions to be combined directly with 3D molecular localization data measured from the same cryopreserved specimen. With the advent of correlated CFT-SXT imaging, cell biologists can now accurately locate the position of specific molecules in the context of a high-resolution reconstruction of a near-native state cell.