X-ray Microscopes

Why use X-rays for Microscopy?

Microscopes using visible light are convenient, and ubiquitous. They have limitations: The resolution is generally limited by the wavelength of visible light to some 100-s of nanometers. Furthermore many materials are opaque to visible light, so there is need for better penetration and/or higher resolution.

Transmission electron microscopes (TEM) offer unsurpassed resolution, but samples to be examined must be ultra-thin (generally less than a fraction of a micrometer). Scanning electron microscopes (SEM) can handle any specimen with a conducting surface, but the stunning images it creates represent only the surface contour, and has no information about the bulk.

There are other options as well: Scanning Probe Microscopes offer very high resolution, but once again the information comes from the surface, not from what lies below it.

X-ray microscopes are particularly powerful in providing tomographic information on the 3D structure of samples too large for electron microscopy, where the resolution required is better than what visible light microscopes can deliver, or where the sample is opaque to visible light.

Microscope Type	Detect	Resolution	Contrast
visible light	transmitted light	500 nm	bright field
		500 nm	phase contrast
	scattered light	500 nm	dark field
	fluorescence	50 – 500 nm	label
electron microscope (TEM)	scattered electrons	0.1 – 1 nm	heavy metal stain
scanning electron micr. (SEM)	secondary electrons	3 – 10 nm	surface relief
scanning tunneling micr. (STM)	tunneling current	0.1 nm	surface atoms
scanning force micr. (AFM)	force on probe tip	0.5 nm	surface relief
X-ray projection microscope	transmitted X-rays	>1000 nm	absorption
X-ray microscope (TXM)	transmitted X-rays	25 nm	absorption
		25 nm	phase contrast
scanning X-ray microscope (STXM)	transmitted X-rays	25 nm	absorption
			XANES (chemical)
nanoprobe	fluorescence	30 nm	elements
	diffraction	30 nm	strain

Xradia is a pioneer in the field of X-ray optical components and their application to ultra-high resolution imaging and tomography. The development of advanced X-ray lenses, so-called zone plates, makes it possible to focus an X-ray beam down to a spot as small as 30nm or about 150 atoms across. This has enabled the development of extremely high resolution X-ray microscope systems having wide application in fields from semiconductor development and inspection, to materials science, environmental science, nanotechnology and life sciences.

Penetrating Power

A very important property of X-rays is that by selecting the X-ray wavelength, the penetrating power can be adjusted from the size of a bacterium to the size of an ox.

Penetrating power of X-rays through an organic material.

Contrast

Most X-ray microscopes form the image based on the X-rays transmitted by the sample. Where the sample absorbs more, the image is darker, where it transmits more the image is brighter. Absorption increases with density and with thickness, and it is also generally higher for elements with a higher atomic number in the periodic table. The absorption maxima above absorption edges for a given element can also be used to isolate elemental and chemical constituents (see the section on elemental and chemical contrast).

If the sample has largely uniform density and thickness, it is often desirable to make use of phase contrast. The phase depends on the optical properties of the sample, and can provide contrast even if absorption is minimal or has little spatial variation. Zernike phase contrast is implemented in X-ray microscopes much as in the light microscope, using a strategically placed "phase ring".

While most samples show good contrast in their unmodified, natural state, in some cases it is desirable to highlight certain features, and for that purpose it is often possible to use specific labels. Heavy metals, such as gold, in the form of tiny gold particle attached to molecules that bind to the structure in question, are used in such cases.

Elemental and Chemical Contrast

When X-rays are absorbed, an electron bound in the absorbing atom is ejected in what is known as the photoelectric effect. The hole this electron leaves behind is filled by a less strongly bound electron, and the surplus energy is commonly released by the emission of another X-ray. This phenomenon, X-ray fluorescence, results in X-rays with energy that is characteristic of the element that emitted it. By detecting and analyzing the energy of these X-rays, the elemental make-up of the irradiated sample can be determined with high sensitivity. For the incident X-ray to be able to remove a bound electron, it must have an energy that is larger than the energy with which the electron is bound. The X-ray absorption spectrum as a function of energy shows sharp increases wherever one of these binding energy levels is exceeded. These "absorption edges" are therefore also characteristic of the absorbing atom. A detailed study of the position of the edge and shape of the absorption spectrum reveals that it is influenced by the chemical environment of the absorbing atom. As a result, high resolution X-ray spectroscopy (NEXAFS – Near Edge X-ray Absorption Fine Structure) can provide information not only about the elements present in the sample, but about the chemistry as well.

Resolution

X-ray microscopes are capable of very high resolution – to 50 nm or even better. To achieve such high resolution, zone plate lenses are used. These high-end microscopes reveal very fine detail in small samples (less than 50 micron field of view, with the possibility to tile together much larger images). At the highest resolutions radiation-sensitive samples (especially biological samples) may suffer radiation damage, unless imaged at liquid nitrogen temperature.

At more moderate resolution the operation of the microscope, as well as sample mounting become very simple. Radiation damage becomes much less of a problem, image acquisition becomes faster, and larger samples are imaged in a single-shot. For example, at 5 micron resolution the field of view is several millimeters, and one can make physiological observations (seed sprouting, plant growth, metabolic processes), as well as watch phenomena in the inanimate world (percolation in porous rocks and mixing in viscous fluids for example).

The reasons are easy to see: with a 1 Megapixel (1000x1000 pixel) detector, if one pixel corresponds to 50 nm on the sample, the entire array covers 50 microns. If one pixel corresponds to 5 microns on the sample, the array covers a 5 mm field. To obtain information from any given volume element (voxel) in a sample, you have to have a minimum number of incident X-ray quanta interact with that voxel. This would suggest that the required exposure time and the radiation dose should scale as the volume, or the resolution cubed. In fact, detailed analysis shows that the situation is a bit worse, and the minimum exposure and radiation dose depends on the fourth power of the resolution. Going from 5 micron resolution to 50 nm resolution would then involve 10⁸ times more radiation dose, and a comparably longer exposure time. In practice the exposure time (but not the radiation dose) is reduced for the higher resolution instruments compared to these predictions using a proprietary illumination scheme.

The Sample Environment

In X-ray microscopes (unlike electron microscopes) the sample is generally at normal atmospheric pressure. It can be heated or cooled if desired. Wet samples are mounted between X-ray transparent windows or in thin-walled capillaries.

X-rays are not affected by electric or magnetic fields, hence computer chips may be imaged under real operating conditions, or while subject to mechanical forces. Time-varying phenomena (curing of paint, concrete, adhesive, etc.) may be observed by repeated exposures and combined into movies.