Phase-contrast x-ray imaging and tomography of the nematodeCaenorhabditis elegans

Abstract

We have analyzed the model organism Caenorhabditis elegans with the help of phase-contrast x-ray tomography. This work combines techniques from x-ray imaging studies of single biological cells by in-line holography with three-dimensional reconstruction and furthermore extends these studies to the multicellular level. To preserve the sub-cellular ultrastructure of the nematodes, we used the near-native sample preparation of high-pressure freezing as commonly used in the field of electron microscopy. For the presented samples, a standard, non-magnifying parallel-beam setting, as well as a magnifying, divergent-beam setting using nanofocusing optics is evaluated based on their tomographic reconstruction potential. In this paper, we address difficulties in sample preparation and issues of image processing. By experimental refinement and through optimized reconstruction procedures, we were able to perform x-ray imaging studies on a living specimen.

Efficient focusing of 8 keV X-rays with multilayer Fresnel zone plates fabricated by atomic layer deposition and focused ion beam milling

“…The utilization of focused X-rays has spread continuously over recent decades and includes applications such as imaging, chemical analysis (Sakdinawat & Attwood, 2010) and time-resolved investigation by exploiting pulses of synchrotron radiation (Kammerer et al, 2011;Van Waeyenberge et al, 2006;Saes et al, 2003). In contrast to electron microscopy, X-ray microscopy provides high penetration depth and enables three-dimensional imaging of complete cells (Vogt et al, 2000), large alive biological samples (Olendrowitz et al, 2012) and in situ investigation under extreme conditions (Fife et al, 2012). As the theoretical resolution is determined by the wavelength, hard X-ray microscopy is a very promising technique for highresolution imaging with chemical sensitivity (Schroer et al, 2004).…”

Phase retrieval problems in x-ray physics

“…Here the sample is given by a C. elegans nematode, prepared by high-pressure freezing and epon-embedding, placed on a 1 mm thick glass [59]. The experiment was carried out at the Göttingen Instrument for Nano Imaging with coherent X-rays (GINIX), which is installed at the undulator beamline P10 of the PETRA III storage ring of the Deutsches Elektronen SYnchrotron (DESY).…”

“…On the other hand, [59] provides examples for the empty beam correction performing well. Besides cleaner windows and optimization of parameters like the photon energy or the sample distance R the main reason for the successive application there is probably the use of a clean-up pinhole which compactifies the source size.…”

X-ray phase contrast tomography from whole organ down to single cells

“…Directly before the measurements one slice was mounted in PBS between two polypropylene foils on aluminum rings forming a liquid chamber. 23 The rings were glued together and mounted on a sample holder. Figure 5 (b) shows a photograph of a final preparation with the brain slice kept in PBS between the two rings.…”