In this review we propose to address the question: for the life-science researcher, what does X-ray microscopy have to offer that is not otherwise easily available?We will see that the answer depends on a combination of resolution, penetrating power, analytical sensitivity, compatibility with wet specimens, and the ease of image interpretation.

Soft X-ray microscopes and their biological applications

The depth and composition of human airway surface liquid (ASL) may depend on secretion from airway glands, ion transport across the surface epithelium, goblet cell discharge, transepithelial gradients in hydrostatic pressure, and surface tension. Published values for the frequency of airway glands and for the secretory rates of individual glands suggest that total gland secretion in human trachea can amount to approximately 60 microL x cm(-2) x h(-1). Volume absorption directly measured across cultures of surface epithelium from human trachea is approximately 5 microL x cm(-2) x h(-1). These flows should alter the depth of ASL at +10 and -1 microm x min(-1). We have looked for changes in ASL depth of this magnitude using low-temperature scanning electron microscopy (LT-SEM) of rapidly frozen specimens of bovine trachea. Stimulation of gland secretion with methacholine led to an initial increase in depth of approximately 25 microm x min(-1) followed by a decline at approximately 1.5 microm x min(-1). Whereas the initial increase in depth was probably due to transient gland secretion, the later decline reflected active absorption of liquid across the surface epithelium. Finally, we present preliminary data showing that LT-SEM can be combined with X-ray microanalysis to determine the elemental composition of ASL.

https://erj.ersjournals.com/content/erj/10/12/2892.full.pdf

Regulation of depth and composition of airway surface liquid

A scanning soft x‐ray microscope has been built and operated at the National Synchrotron Light Source. It makes use of a mini‐undulator as a bright source of 3.2‐nm photons. An electron beam fabricated Fresnel zone plate focuses the beam onto the specimen, which is scanned under computer control. The scanning stage can be moved by both piezoelectric transducers and stepping motors, and the location is monitored by a high‐speed laser interferometer. X rays transmitted through the specimen are detected using a flow proportional counter. Images of biological specimens and of artificial microstructures have been made with resolution in the 75–100‐nm range. Acquisition time for 256×256‐pixel images is about 5 min.

Scanning x‐ray microscope with 75‐nm resolution

Summary This paper presents the first application of high-resolution X-ray synchrotron tomography to the imaging of large microvascular networks in biological tissue samples. This technique offers the opportunity of analysing the full three-dimensional vascular network from the micrometre to the millimetre scale. This paper presents the specific sample preparation method and the X-ray imaging procedure. Either barium or iron was injected as contrast agent in the vascular network. The impact of the composition and concentration of the injected solution on the X-ray synchrotron tomography images has been studied. Two imaging modes, attenuation and phase contrast, are compared. Synchrotron high-resolution computed tomography offers new prospects in the three-dimensional imaging of in situ biological vascular networks.

/pdf/x-ray-high-resolution-vascular-network-imaging-4kzclafdj2.pdf

X-ray high-resolution vascular network imaging

A modern 1- to 2-billion-electron-volt synchrotron radiation facility (based on high-brightness electron beams and magnetic undulators) would generate coherent (laser-like) soft x-rays of wavelengths as short as 10 angstroms. The radiation would also be broadly tunable and subject to full polarization control. Radiation with these properties could be used for phase- and element-sensitive microprobing of biological assemblies and material interfaces as well as reserch on the production of electronic microstructures with features smaller than 1000 angstroms. These short wavelength capabilities, which extend to the K-absorption edges of carbon, nitrogen, and oxygen, are neither available nor projected for laboratory XUV lasers. Higher energy storage rings (5 to 6 billion electron volts) would generate significantly less coherent radiation and would be further compromised by additional x-ray thermal loading of optical components.

Tunable Coherent X-rays

This article describes the scanning transmission X-ray microscope operated at the National Synchroton Light Source. The application of the instrument to elemental analysis is detailed. In particular, qualitative results on the calcium distribution in human skull tissue are presented.

Absorption microanalysis with a scanning soft X-ray microscope: mapping the distribution of calcium in bone.

A scanning soft X-ray microscope has been developed that uses synchrotron radiation focused by a Fresnel zone plate to form a submicron beamspot on the specimen. Transmitted X-rays are detected and used to form a quantitative map of specimen absorptivity. Applications of the instrument to the imaging of whole wet cells and to the mapping of calcium in sections of bone are presented, with a resolution of 300 nm and an elemental sensitivity of 2 mu g cm-2.

/pdf/quantitative-imaging-and-microanalysis-with-a-scanning-soft-16oartv1cq.pdf

Quantitative imaging and microanalysis with a scanning soft X-ray microscope

X-ray differential absorption microanalysis is presented as a technique for trace element analysis of hydrated biological specimens of about 0.1-5 μm thickness. For the study of the light elements (Z≲20), the absorption technique minimizes the radiation dose and, thus, damage to such specimens when compared with X-ray fluorescence. A Scanning Transmission X-ray Microscope (SXTM) is described, which has been used to map the concentration of calcium in bone with better than 300 nm spatial resolution and a sensitivity to 5% calcium by weight. Future plans are briefly discussed that offer the hope of achieving 0.1% trace element sensitivity and 75 nm spatial resolution.

Elemental analysis using differential absorption techniques.

In order to explore the potential of soft X ray microanalysis, we have, in collaboration with scientists from IBM and the Brookhaven National Synchrotron Light Source (NSLS), constructed a scanning soft X ray microprobe, which is operating on the U15 beamline of the 750 MeV VUV storage ring at the NSLS.' A schematic drawing of the instrument is shown in FIGURE 1. A toroidal grating monochromator, of resolving power 300, focuses a selected wavelength onto a pinhole. A Fresnel zone plate is used to form a demagnified image of this pinhole in the plane of the specimen. The specimen is scanned through this point in a raster fashion while the transmitted X rays are detected using a flow proportional counter. This data is stored by computer and displayed on a television screen. The ultrahigh vacuum requirements of the synchrotron source are far removed from the ideal conditions for biological cells, so the specimen and source are decoupled in two stages. A 150-nm aluminum contamination barrier separates the ultrahigh vacuum region from a relatively low vacuum area, keeping out hydrocarbon contamination, but providing no mechanical strength. A small 120-nm thick silicon nitride window then separates the vacuum from the atmospheric pressure region. Helium is flowed over the specimen, allowing the soft X rays to be transmitted in this region. Having the sample in a helium environment a t atmospheric pressure permits relatively simple handling. The spatial resolution of the instrument is determined by the size of the pinhole image, which depends on the quality of the zone plate. At present, 0.2 pm is the limit, but work underway a t IBM and King's College, London, should enable an order of magnitude improvement within the near future.

Microanalysis with a soft X ray scanning microprobe.

Physiological and morphological changes in bone tissue are poorly understood phenomena associated with the aging process. A decrease in bone volume and a reduction in cellular activity are most commonly noted. For research, for diagnostic purposes, and eventually to find and evaluate specific forms of treatment there is a need to be able to study the distribution of calcium in bone tissue.

F. Cinotti

Papers

Absorption microanalysis with a scanning soft X-ray microscope: mapping the distribution of calcium in bone.

Quantitative imaging and microanalysis with a scanning soft X-ray microscope

Elemental analysis using differential absorption techniques.

Microanalysis with a soft X ray scanning microprobe.

Studies of Calcium Distribution in Bone by Scanning X-ray Microscopy