Selective signal enhancement in Fourier space as a tool for discovering ultrastructural organization of macromolecules from in situ TEM

Abstract

We present a Fourier transform (FT) based analytical method that allows to obtain of ultrastructural details from TEM images at sub-nanometer scale applying a selective filtering for singular macromolecule electron microscopy density information. It can be applied to high-pressure frozen, frozen hydrated and epoxy freeze substituted and embedded biological species. Both 2D projections and orthoslices from reconstructed tomograms can be used as a source of structural information. The key to the method is to select the macromolecule or organelle of interest with an accuracy of ≥ 7 – 3 nm (depending on pixel size of initial tilt series or singular image acquisition) and explore both the central low frequency FT intensity and diffraction regions to obtain the spatial structural organization and its dimensional characteristics, respectively. We also introduce a structure-specific selective mask FT filtering approach that can significantly improve image information even in poorly contrasted TEM of resin sections without heavy metal been used. The described method elucidates chromatin architecture without the need of averaging. A zigzag symmetry of 30 nm diameter chromatin fibers which in general is a controversial topic of research has been identified for C. elegans cells in vivo with sub-nanometer details being preserved in the images.