[en] The use of soft X-ray imaging is considered for the determination of the re macromolecular structure of biological fibers, with available image resolution, and subject to the effects of radiation damage. A comparison is made between the structure in sarcomere (2μ to 3 μ long repeating unit) of striated muscle as seen directly by X-ray microscopy and as derived from published interpretations of X-ray diffraction data from whole muscle. The comparison shows that the loss by radiation damage of the ability of a muscle myofibril to contract is related to the loss of fine structure. Ways to minimize the effects of beam damage are discussed, including the use of images taken in phase, rather than amplitude contrast, and with photon energies above the water window

[en] Myofibrils, the contractile organelles from striated muscles, have been examined in the X-ray microscope to determine the effect of radiation on their function and structure. Using X-rays of energy 350-385 eV in the water window we find that after an exposure to 7.5 x 10⁵ photons/μm² (calculated to give an absorbed dose of 20,000 Gy) the myofibrils will no longer contract. The use of the free radical scavenging agent, DMSO, gives some protection to the fibrils. It has also been found that after this much irradiation the fibrils lose up to 20% of their mass. Further substantial mass loss occurs on subsequent irradiation. After 25 times the loss-of-function exposure only 30% of the mass remains. Analysis of a series of images of the same myofibril covering this range of exposures shows that the mass is preferentially lost in some areas of the structure and consequently significant structural changes occur. (author)

[en] The future of X-ray microscopy lies mainly in its potential for imaging fresh, hydrated biological material at a resolution superior to that of light microscopy. For the image to be accepted as representing the cellular organization of the living cell, it is essential that artifacts are not introduced as a result of the image collection system. One possible source of artifacts is cellular damage resulting from the irradiation of the material with soft X-rays. Cells of the unicellular alga Chlorella have been examined by transmission electron microscopy (TEM) following exposure to different doses of monochromatic (380eV) soft X-rays. Extreme ultrastructural damage has been detected following doses of 10³ -10⁴ Gy, in particular loss of cellular membranes such as the internal thylakoid membranes of the chloroplast. This is discussed in relation to dosage commonly used for imaging by soft X-ray microscopy

[en] This article reports the continuation of a series of experiments investigating the effects of soft x-ray radiation damage on the contractile elements of mammalian striated muscle (myofibrils), using their ability to contract as a functional assay. The myofibrils were exposed to 385 eV x rays. This energy is within the ''water window'' between the oxygen and carbon K edges, where the x-ray absorption coefficient of biological materials, such as protein, is about an order of magnitude greater than that for water. An exposure of 8x10⁵ photons μm^-1 was found to prevent contraction in the majority of myofibrils. Preliminary results indicate that it is possible to increase this exposure level by approximately 25% by adding the radioprotective dimethyl sulphoxide (DMSO), an OH radical scavenger to the myofibril buffer during irradiation. This suggests that OH radicals are important in the inactivation of myofibrils through irradiation

[en] Previously high-resolution soft x-ray microscopy has only been possible with synchrotron sources. Here, the first successful attempts at using a scanning transmission x-ray microscope with a laser-plasma source are reported. Spatial resolutions were limited to about 650 nm by electrical noise in the detector, but single shot per pixel images were obtained of test and real specimens. The microscope was not optimized to the source since it was designed for use on the undulator beam line of a synchrotron. With an improved system, it is demonstrated that single shot per pixel imaging at high resolution (better than 50 nm) will routinely be possible

[en] The benefits of microscopy using x-rays stem from the nature of the x-ray interaction with materials. Unlike electron beam interaction, x-rays exhibit, large variations in their absorption and scattering by elements and molecules with changes in the incident energy. It is thus possible to choose an x-ray energy where the element or chemical species of interest absorbs the incident x-rays to a much greater extent than the surrounding material, thus giving a high level of contrast. In brief, the advantages offered by tunable x-ray energy microscopy are: 1. Spatial resolution of better than 0.05μm is available on whole, wet state cell cultures and thin sections. 2. Elemental and chemical state mapping is available via absorption difference imaging in the time-scale of minutes. 3. Elemental imaging via x-ray fluorescence can be done on both thick and thin specimens with very low background, giving high sensitivity. 4. Tuning the x-ray energy to give the optimum contrast in the specimen minimizes the dose to the specimen. 5. Good contrast may obtained on thick specimens, and 3D imaging is an option. These attributes make the new x-ray microscopy techniques well suited to imaging mineralized tissues. In this paper, the authors report work on the imaging of crystal deposits in normal and arthritic articular cartilage by three types of scanning x-ray microscope, and indicate where x-ray microscopy could be used to observe the distribution of labelled molecules (such as human growth hormone) on cells in bone-like cultures

[en] Elemental imaging via scanning transmission x-ray microscopy (STXM) and scanning fluorescence x-ray microscopy (SFXM) has been used to image calcium deposits in cartilage. In the case of STXM, 0.1 μm thick sections were imaged to investigate the proximity of calcium deposits in relation to chondrocyte cells. The resolution available was 0.5 μm, and field widths of up to 25 μm were used at this resolution. The resolution available in SFXM was 10 μm, and field widths of up to 2 mm were used at this resolution on 5-μm thick specimens. Together these techniques were used to map calcium deposits at the cellular level, and at the full tissue size level