Visualization of 30 nm structures in frozen-hydrated biological samples by cryo transmission X-ray microscopy

Abstract

A new object stage with extremely low thermal drift at −170 °C was developed for the cryo transmission X-ray microscope (cryo-TXM) at the electron storage ring BESSYI (Berlin). The new set-up enables high resolution studies of frozen-hydrated cells and was applied in investigations of cryogenic Kupffer cells from a rat liver. The ultrastructure and numerous X-ray dense vacuoles are resolved allowing a more comprehensive interpretation of data obtained by TEM studies. Furthermore, the cryo-TXM has been recently used for non-destructive computed tomography of intact frozen-hydrated objects. The resolution obtainable in TXM micrographs is limited significantly by the photon density applied to illuminate an object. The contrast transfer of the TXM was evaluated including the real X-ray optical elements with the help of a so-called multiple plane-wave model which is based on Fourier optics. It allowed to optimize the X-ray optical set-up for best contrast transfer and to minimize the photon density required to detect ice-embedded protein structures. However, the results show that details in biological objects smaller than 30 nm in size, e.g. single chromatin fibers in cell nuclei, can only be visualized if a drastically increased photon flux of the X-ray source is available from undulator insertion devices of electron storage rings. Furthermore, for this purpose new condenser concepts like a rotating condenser and highly efficient X-ray objectives with smallest zone structures of 20 nm have to be employed. This progress in the instrumentation will enable new applications ultimately resulting in artifact–free high-resolution images of radiation sensitive biological samples.