Soft x-ray microscopy in the water window ( ∼ 285 − 535 e V ) is an emerging and unique tool for 2D and 3D imaging of unstained intact cellular samples in their near-native state with few-10-nm detail. However, present microscopes rely on the high x-ray brightness of synchrotron-radiation sources. Having access to water-window microscopy in the home laboratory would increase the impact and extend the applicability of the method. In the present paper, we review three decades of efforts to build laboratory water-window microscopes and conclude that the method is now reaching the maturity to allow biological studies. The instruments as well as their key components are quantitatively and qualitatively compared. We find that the brightness and the reliability of the laboratory source are the most critical parameters, but that the optics as well as the sample preparation also must be optimized to enable high-resolution imaging with adequate exposure times. We then describe the two sister microscopes in Stockholm and Berlin, which allow reliable high-resolution biological imaging with short exposure times of a few tens of seconds in 2D and a few tens of minutes in 3D. They both rely on a liquid-jet laser-plasma source combined with high-reflectivity normal-incidence multilayer condenser optics, high-resolution zone-plate imaging optics, CCD detection, and cryogenic sample handling. Finally, we present several examples of biological imaging demonstrating the unique properties of these instruments.

Scanning transmission X-ray microscopy, especially in combination with X-ray fluorescence detection (STXM-XRF) in the soft X-ray energy range, is becoming an increasingly important tool for life sciences. Using X-ray fluorescence detection, the study of biochemical mechanisms becomes accessible. As biological matrices generally have a low fluorescence yield and thus a low fluorescence signal, high detector efficiency (e.g. large solid angle) is indispensable for avoiding long measurement times and radiation damage. Here, the new AnImaX STXM-XRF microscope equipped with a large solid angle of detection enabling fast scans and the first proof-of-principle measurements on biomedical samples are described. In addition, characterization measurements for future quantitative elemental imaging are presented. research papers J. Synchrotron Rad. (2019). 26, 430-438 Lars Lü hl et al. Biomedical applications of STXM-XRF in the soft/tender energy range research papers J. Synchrotron Rad. (2019). 26, 430-438 Lars Lü hl et al. Biomedical applications of STXM-XRF in the soft/tender energy range research papers 436 Lars Lü hl et al. Biomedical applications of STXM-XRF in the soft/tender energy range J. Synchrotron Rad. (2019). 26, 430-438Figure 7Transmission image (top) and pseudo-colour image (bottom) for C (blue, BAT) and Fe (red, SPIO) for control tissue.

Synchrotron radiation based scanning X-ray spectroscopy (XRS) can determine both elemental concentrations and chemical binding states with high spatial resolution down to the nanoscale. Within the last decade, the need for such high-resolution analytical methods in application fields such as life science, energy storage and conversion as well as climate protection has steadily increased. In order to quantify nanoelectronics, pharmaceuticals, biomolecules and medical imaging contrast agents in biological tissue and cells, the development and adaptation of traceable methodologies are crucial. XRS can be quantitative and recent advances in a novel kind of vibration reduced X-ray fluorescence (XRF) analysis have pushed the spatial resolution towards the 100 nm regime. This could provide the missing link for quantification across the length scales.There are only a few scanning X-ray microscopes that offer XRF analysis in combination with scanning transmission X-ray microscopy (STXM) such as Elettra-TwinMic (STXM and XRF); Diamond -I08 (STXM and XRF); PETRA III -P04 AnImaX (STXM and XRF); Advanced Light Source -11.0.2 (STXM and spectroscopy) and Canadian Light Source -10ID-1 (STXM and spectroscopy).With the advent of UHV-compatible piezo stages with nanometer resolution, it is possible to mount optical elements in a compact manner on a single platform. Relevant parts of any external vibrations might be reduced by such a single platform. The lay-out of its stages and related holders is to ensure amplitudes of relative vibrational movements to be smaller than the absolute ones by avoiding any resonance frequencies. We realized this approach in a compact translational and rotational set-up to align beamstops (or diagnostics), zone plates, order sorting apertures (OSA) and samples on a single mounting plate (see fig.1). This plate was mounted on a sample manipulator of non-microscopy equipment such as an XRF instrument in our case [1]. Furthermore, its overall size offers versatile areas of operation as the compact set-up can be readily inserted in a wide range of experimental (vacuum) chambers [2] through their load-locks. Our nm-XRF and STXM set-up is mounted on a 6-axis manipulator [1] which allows for an alignment of all three linear as well as all three rotational axes. The optics and the transmission sample are mounted on piezo positioning stages with 8 axes providing 1 nm resolution and encoder control on which the zone plate can be aligned in the X-Y plane (perpendicular to the optical axis) and the OSA as well as the transmission sample in X-Y-Z. The fluorescence samples are mounted on a 4-axis manipulator which can be moved in and out of the beam. The optical elements of the scanning X-ray microscope consist of an Au zone plate (with a diameter of 360 µm, an outer zone width of 25 nm and an integrated 50 µm central stop) and a 50 µm OSA. All measurements presented here are carried out with photon energy of 1500 eV at PTB's plane grating monochromator (PGM)

A sub-pixel 16 bit charge coupled device camera featuring superresolution for the soft X-ray regime is presented. Superresolution images (SRIs) are reconstructed from a set of 4 × 4 individual low-resolution images that are recorded for different sub-pixel shifts of the detector. SRIs have a 1.3 times higher resolution than individual low-resolution images which is close to the maximum achievable enhancement factor of about 1.5 in the X-ray regime under ideal conditions. To characterize this camera and demonstrate its potential, an X-ray microscope setup is used to image different objects at different photon energies.

Abstract