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X-rays, which are similar to visible light, are beneficial for creating an image of the internal structures of an object. This is because X-rays have higher energy.
X-ray tomography is perhaps the most well-known imaging technique that employs X-rays. However, this form of electromagnetic radiation isn’t exclusive to CT scanners and the medical imaging world. X-rays are also indispensable in the life sciences industry.
First, let’s review the basics. X-rays are defined as having wavelengths of 0.01 to 10 nanometers (nm). Depending on their wavelengths, they are further subdivided into soft and hard X-rays. Hard X-rays have high energy and relatively longer wavelengths of around 0.2 to 0.1 nm, while soft X-rays have lower energy and longer wavelengths of approximately 10 nm.
The shorter wavelengths and higher energy properties hard X-rays exhibit mean they are excellent at penetrating deep inside of solid objects, making them useful for applications such as medical radiography.
On the other hand, soft X-rays are more suitable for applications such as X-ray crystallography, which requires minimal radiation exposure. Soft X-rays are excellent for gathering the molecular and atomic structure of a crystal (i.e., a biological specimen or protein that’s been crystallized).
Furthermore, soft X-ray microscopy has been used to bridge the gap between optical microscopy and electron microscopy. It is often performed at cryogenic temperatures.
X-ray microscopy, specifically, uses electromagnetic radiation found in soft X-rays to produce magnified images of objects that would otherwise be invisible to the human eye.
First, the object is shot with an X-ray beam, and the soft X-ray photons strike the sample. Because X-rays do not reflect or refract, a charged coupled device detector (CCD) or exposed film must be used to pick up the X-rays as they pass through the sample. Then, the collected X-rays are analyzed, and a magnified image is produced.
A significant advantage that X-ray microscopes have over conventional microscopes is that, due to the penetrative properties of X-rays, biological samples can be imaged with minimal preparation and in their natural state.
Additionally, due to their wavelengths being shorter than visible light, X-ray microscopes have higher spatial resolution compared to normal optical microscopes. Magnification is also comparable.
Microscopic techniques, such as optical, confocal, and electron microscopy, provide important information regarding the surface or near-surface of a sample. However, internal information from the sample is often required as well. X-ray microscopy is capable of doing just that. It’s what makes it such an invaluable technique and tool in the lab.
While researchers have conventionally had to rely on invasive procedures to obtain internal structural information, X-ray microscopy provides a non-invasive alternative. This feature makes X-ray microscopy critical in several fields of study, including cell biology, medicine, and materials science.
Below we cover the main components, types, and techniques of X-ray microscopes and microscopy.
X-ray microscope components can vary significantly based on design; however, each model typically has three main parts:
X-ray optics are specifically designed to manipulate X-rays rather than visible light because X-rays behave pretty differently than visible light. Visible light is easily redirected using lenses and mirrors. That said, X-rays change direction much less, as they are prone to penetrating and being absorbed by an object.
Instead, various techniques are used to redirect X-rays where they need to go. This includes diffraction and interference, typically in the form of various compound refractive lenses and zone plates and certain types of crystals and crystal planes.
Initially, a tungsten filament inside a vacuum tube was used as the X-ray source in these microscopes. However, the development of synchrotron radiation as an X-ray source opened up new possibilities in X-ray microscopy. The synchrotron light source was brighter and more brilliant and tunable and coherent as well.
Furthermore, synchrotron radiation-producing imaging systems can be coupled with spectroscopy to map chemical composition with sub-micron resolution. This includes X-ray fluorescence and X-ray spectroscopy.
There are different types of X-ray detectors for other applications. However, one of the most common detection devices in X-ray microscopes tends to be the charge-coupled device (CCDs) detector, a highly sensitive photon detector essential to digital imaging today. These detectors are excellent for measuring spatial intensity distributions.
Other detectors include X-ray photographic film, image plates, scanning point detectors, converter screens, and more.
X-ray microscopy is generally categorized as either full-field or scanning. However, full-field X-ray microscopy can be divided into two distinct versions. The first involves lensless imaging with parallel and conical beams. The second version is most commonly seen in transmission X-ray microscopes, which use the same optical configuration as conventional light microscopes and transmission electron microscopes.
Full-field microscopes use optical elements like Fresnel zone plates or refractive optics as objective lenses for high-resolution imaging and typically image the whole field of view to a detector plane simultaneously.
In a scanning X-ray microscope, a small but intensive X-ray beam is used to scan the sample, creating a finely focused spot or microprobe through which the sample is rasterized, or converted as an image into pixels. The extremely small X-ray beam is typically generated using focusing optics. In this case, an X-ray optic.
Each sample position produces an X-ray signal during the scan, which is then recorded. These signals include X-ray fluorescence, absorption spectroscopy, or diffraction. In this way, the sample’s elemental, chemical, and structural information can be determined.
Using high-energy X-rays or gamma rays, XRF microscopes can analyze the secondary, or fluorescent, X-rays that are excited by the sample. These secondary emissions can then be analyzed to produce an image.
When the gamma rays strike molecules, they fluoresce at specific energies, allowing for elemental image analysis. This technique also gives the user depth control and horizontal and vertical aiming control. Advanced models can analyze and image multiple elements at once.
Besides electron microscopes, most conventional microscopy could not provide fully 3D, high-resolution images without destructive sectioning. That said, three-dimensional (3D) X-ray microscopy is an exception; it’s a non-destructive 3D imaging method able to image submicron to the nanometer scale.
X-rays’ inherent penetrative qualities and their ability to not harm organic material allow this to happen.
By employing a zone plate to focus the X-ray beam onto a specific and small area, STXM allows for analyzing wet samples. Specifically, STXM uses near-edge X-ray absorption spectroscopy as the contrast mechanism to produce an image.
Samples in water pose issues when being imaged due to how various forms of light interact with water. X-rays can penetrate water and only interact with the sample.
Another significant advantage of STXM, as opposed to other similar transmission microscopy techniques, is that it inflicts relatively minimal damage to the actual material.
Three-dimensional X-ray diffraction microscopy, which has led to the implementation of high-energy X-ray diffraction microscopy (HEDM), is a somewhat young technique. However, it is pretty powerful (and, of course, nondestructive).
X-ray diffraction microscopy uses the far-field scattering of coherent X-rays to form the 2D or 3D image of a scattering object in a way that resembles crystallography.
HEDM generates a map of the internal crystalline structure of a sample and allows researchers to obtain specific information regarding the object’s internal measurements on a tiny scale.
Thanks to its non-destructive qualities in hard materials like ceramics and metals, it is an invaluable technique in materials science.
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