Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging Gerd Schneider a,⇑ , Peter Guttmann a , Stefan Rehbein a , Stephan Werner a , Rolf Follath b a Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Institute for Soft Matter and Functional Materials, Albert-Einstein-Str. 15, 12489 Berlin, Germany b Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Institute for Nanometre Optics and Technology, Albert-Einstein-Str. 15, 12489 Berlin, Germany article info Article history: Available online 18 January 2012 Keywords: X-ray microscopy X-ray tomography Correlative microscopy Cryo microscopy abstract X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ide- ally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating mono- chromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spec- tral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochroma- tor close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10 nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Modern microscopic techniques are important tools for struc- tural biology. Structural information about biological cells can be obtained with light microscopes operating in phase-contrast mode, fluorescence detection mode, confocal scanning mode for three- dimensional (3-D) imaging, and spatially resolving Raman spectros- copy mode. Independent of the imaging mode, the spatial resolution obtainable with these far-field microscopes is limited by the wave- length of the visible light or UV radiation used for imaging. Advanced fluorescence microscopes overcome the Abbe resolution limit, as for example STED or PALM (Betzig et al., 2006; Hell and Wichmann, 1994). However, these techniques rely on the location of fluorescent dyes to visualize indirectly the cell structures. The approach to selectively visualize labeled structures has many advantages but does not give a complete picture of the whole cell. Recent instrumental developments are the scanning near-field optical microscope (SNOM) and the atomic force microscope (AFM). These techniques overcome the diffraction limitation of the far-field light microscopes, but deliver only information of structures near the surface of an object. Much higher resolution can be obtained by using electrons with short de Broglie wave- lengths. Transmission electron microscopes (TEM’s) can resolve single atoms in radiation stable materials like crystals. Despite its excellent resolution, electron microscopy applied to biological cells has some limitations. Living biological cells are in an aqueous envi- ronment. However, cells are commonly dried for standard TEM studies. Caused by the surface tension during the drying process, the cells can structurally be altered. The cryo TEM overcomes this problem; however, the strong inelastic scattering of the electrons in the samples restricts the maximum sample thickness for TEM investigations to the sub-lm range, i.e. only thin sections of cells can be imaged with high spatial resolution in the sub-10 nm range (Baumeister, 2002; Dubochet et al., 1988). To extend the microscopy techniques for the examination of matter and to overcome the diffraction limit of visible light micro- scopes, it is obvious to image objects with electromagnetic radia- tion of shorter wavelengths, e.g., with X-rays. For biological applications already Wolter (1952) found that organic structures possess a natural elemental contrast against water in the so-called 1047-8477/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2011.12.023 ⇑ Corresponding author. Fax: +49 (0)30 806212114. E-mail address: gerd.schneider@helmholtz-berlin.de (G. Schneider). Journal of Structural Biology 177 (2012) 212–223 Contents lists available at SciVerse ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi