http://xrm.phys.northwestern.edu/research/pdf_papers/2005/lerotic_jesrp_2005.pdf

Cluster analysis in soft X-ray spectromicroscopy : Finding the patterns in complex specimens

It is not easy to examine bulky or hydrated biological material with an electron microscope (EM). In contrast, x-ray microscopy (XM) provides great penetration power that electron microscopy can not offer, and high resolution with which light microscopy can not compete. Therefore, x-ray microscopy could occupy a niche in biological research in the form of 3D imaging of thick (in comparison to the sample thickness of standard electron microscopy) and living specimens.

X-Ray Microscopy — Its Application to Biological Sciences

X rays are electromagnetic radiation of the same nature as visible light but with only one-thousandth of the wavelength.(1) X rays behave like light toward photosensitive materials. For example when a dentist takes a radiograph she/he places an unexposed photosensitive film next to the teeth and turns on the X rays. Cavities show up dark on the negative film because they transmit X rays so well. Sound teeth show up less dark because they are denser than air or flesh. Ceramic and plastic fillings are still darker if they are denser than teeth and bone. Silver and gold show up very dark when exposed to X rays because they are very dense (such have high atomic numbers).

X-Ray Microscopy

Scanning Microscopy with Soft X Rays

In scanning soft X‐ray microscopy a quantitative map of specimen absorptivity is obtained. Sharp changes in the transmission spectrum at absorption edges provide contrast and are the basis for elemental analysis by comparison of images taken at differing wavelengths. We have developed an instrument that uses monochromatized synchrotron radiation as its source. A collimated portion of this source is focused with a Fresnel zone plate to a submicron spot, across which the specimen is scanned under computer control. To illustrate the microscope’s capabilities, we have imaged whole cultured cells in a fixed but wet and unstained state, and measured the distribution of calcium in thin sections of human bone tissue.

Scanning soft X-ray microscopy a the national synchrotron light source

We have recently completed our first run [21.1] with the Stony Brook scan­ ning microscope at the National Synchrotron Light Source (NSLS), using a Fresnel zone plate fabricated at IBM as the principal focusing element. The brightness of this synchrotron radiation source coupled with our high- reso­ lution optics have made a substantial improvement in resolution and through­ put compared with our earlier 'work [21.2,3] which lacked these features. The microscope images biological specimens by forming a submicron spot of monochromatic soft x rays which it scans in a raster fashion across the spe­ cimen, located in a one-atmosphere environment. The resolution is presently determined by the size of this spot. The transmitted radiation is detected by a flow proportional counter, the output of which is used to control a computer-generated real-time image. Since the specimen is in air, the ineffi­ cient optics are all located upstream of the specimen, and the detector is over 50% efficient, the radiation dose to unstained wet natural specimens is minimized. We believe [21.4] that the resolution limit for scanning micro­ scopy of unfixed specimens will be set more by damage considerations than by optics; our scheme aims to minimize this damage. This paper considers the components of the microscope in turn, starting from the monochromator and the optics. How we test our optics is considered in some detail as it illustrates the microscope's capabilities as well as the optical performance achieved to date. Some of our first images of biolo­ gical specimens are then presented. We conclude with some remarks about the future of the microscope.

J. Kirz

Papers

Scanning Microscopy with Soft X Rays

Scanning soft X-ray microscopy a the national synchrotron light source

21. Recent Results from the ~tony Brook Scanning Microscope