X-ray microscopy

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X-ray microscopy is a microscopy method that uses X-rays instead of visible light, i.e. radiation in the wavelength range from 10 nm to 1 pm.

X-rays initially have the advantage of shorter wavelengths, which potentially enables higher resolution. The resolution of a microscope is limited by half the wavelength. In addition, the interaction of X-rays with matter differs from that of visible light (e.g. penetration capacity, immanent element contrast, refractive indices ), which means that additional information about the sample can be obtained. Above all, the depth of information increases. Information can also be obtained from deeper layers than with light microscopes. The problem up to now has been that converging lenses are necessary to build a microscope. In order to be able to build a converging lens, however, one must use a material for the lens whose refractive index is greater than 1. For the frequency range of X-rays, however, the refractive indices for available materials are smaller than 1. Using the Fresnel scheme, so-called zone plates are used to focus the X-ray beam. These act as lenses analogous to the classic microscope; however, they do not use the refraction, but the diffraction of the X-ray waves. Usable lenses for X-ray radiation that work according to the principle of refraction (i.e. "refraction") can only be produced for wavelengths below 1 nm (see refractive X-ray lens ) and for the important spectral range of the "water window" between 2.4 nm and 4.4 nm, in which aqueous samples show good absorption and phase contrast, therefore not feasible. Modern, high-resolution X-ray microscopy achieves a resolution of 20-30 nm and only uses Fresnel zone plates in this spectral range , cf. also X-ray optics .

A distinction is made between imaging and scanning microscopes.

As a rule, imaging microscopes work in transmission. The examined specimen is evenly illuminated from the "front" and the radiation penetrating the specimen is imaged by optics onto a spatially resolving detector (e.g. CCD sensor ).

In scanning microscopes, the X-ray radiation is focused with the help of mirrors under grazing incidence, mirrors with multilayer systems, Fresnel zone plates or refractive X-ray lenses . The sample is moved through the focus and all the light coming from the sample is measured at each sample position and taken as the brightness value for the image. In addition to the reflected light, other particles or radiation originating from the sample can also be used for imaging.

These are for example:

  • Scattered radiation (diffraction analysis)
  • Reflected radiation
  • Transmitted radiation
  • Luminescent light
  • The total electron yield
  • Photoelectrons
  • Photon-stimulated ion desorption

In order to be able to record high-resolution images with an imaging X-ray microscope in a few seconds and with a scanning X-ray microscope in a few minutes, very intense radiation ( brilliance ) is required. Directed synchrotron radiation and recently also plasma sources are suitable as X-ray sources for this.

Compared to electron microscopes, X-ray microscopes have the advantage that much thicker samples - up to typically 10 µm - can be examined, that the dose deposited in the samples is up to a factor of 10,000 lower and that the samples are not expected to be electrically conductive . Biological samples can remain “natural”; d. In other words, they do not have to be colored with heavy metal, dried, embedded in a support material and, after it has hardened, cut into typically 100 nm thin layers - as is necessary for examination in an electron microscope. The expectations of obtaining artifact-free images with X-ray microscopy are correspondingly high. T. already confirmed.

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