Abstract
We present a correlative microscopy approach for biology based on holographic X-ray imaging, X-ray scanning diffraction, and stimulated emission depletion (STED) microscopy. All modalities are combined into the same synchrotron endstation. In this way, labeled and unlabeled structures in cells are visualized in a complementary manner. We map out the fluorescently labeled actin cytoskeleton in heart tissue cells and superimpose the data with phase maps from X-ray holography. Furthermore, an array of local far-field diffraction patterns is recorded in the regime of small-angle X-ray scattering (scanning SAXS), which can be interpreted in terms of biomolecular shape and spatial correlations of all contributing scattering constituents. We find that principal directions of anisotropic diffraction patterns coincide to a certain degree with the actin fiber directions and that actin stands out in the phase maps from holographic recordings. In situ STED recordings are proposed to formulate models for diffraction data based on co-localization constraints.
Highlights
Nanoscale imaging links cellular functions to the underlying structure, from organelle down to the molecular level
We present in situ stimulated emission depletion (STED) and X-ray microscopy recorded at the same synchrotron endstation
A designed STED microscope has been installed on the optical table of a nano-focus coherent X-ray imaging endstation, which we have optimized for scanning small-angle X-ray scattering (SAXS), as well as in-line Xray holography
Summary
Nanoscale imaging links cellular functions to the underlying structure, from organelle down to the molecular level. One would like to probe unlabeled biomolecular assemblies in the cell in similar ways as it is possible for in vitro model systems, for example by small-angle X-ray scattering (SAXS) in solution In this way, molecular crowding, short range order or packing effects such as in the problem of DNA compactification could be addressed quantitatively in terms of correlation functions and corresponding structure factors. A designed STED microscope has been installed on the optical table of a nano-focus coherent X-ray imaging endstation, which we have optimized for scanning SAXS, as well as in-line Xray holography This configuration allows for the recording of Xray holograms, diffraction data, and STED images to be gathered from the same cell, and in the same sample environment or mounting. We first introduce the concept of all recording schemes and show how the different contrast mechanisms inform each other for the example of neonatal rat cardiac tissue cells with a labeled actin cytoskeleton
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