An X-ray microscopy image of a living 10-days-old canola plant.
An X-ray microscope uses electromagnetic radiation in the soft X-ray
band to produce magnified images of objects. Since X-rays penetrate
most objects, there is no need to specially prepare them for X-ray
microscopy observations.
Unlike visible light,
X-rays do not reflect or refract easily, and they are invisible to the
human eye. Therefore, an X-ray microscope exposes film or uses a charge-coupled device
(CCD) detector to detect X-rays that pass through the specimen. It is a
contrast imaging technology using the difference in absorption of soft
X-rays in the water window
region (wavelengths: 2.34-4.4 nm, energies: 280-530 eV) by the carbon
atom (main element composing the living cell) and the oxygen atom (main
element for water).
Microfocus X-ray
also achieves high magnification by projection. A microfocus X-ray tube
produces X-rays from an extremely small focal spot (5 µm down to
0.1 µm). The X-rays are in the more conventional X-ray range (20 to 300
kV), and they are not re-focused.
Invention and Development
The history of X-ray microscopy can be traced back to the early 20th century. After the German physicist Rontgen
discovered X-rays in 1895, scientists soon illuminated an object using
an X-ray point source and captured the shadow images of the object with a
resolution of several microns. In 1918, Einstein pointed out that the refractive index for X rays in most mediums should be just slightly less than 1, which means refractive optical parts would be difficult to use for X-ray applications.
Early X-ray microscopes by Paul Kirkpatrick and Albert Baez used grazing incidence reflective X-ray optics to focus the X-rays, which grazed X-rays off parabolic curved mirrors at a very high angle of incidence. An alternative method of focusing X-rays is to use a tiny Fresnel zone plate of concentric gold or nickel rings on a silicon dioxide substrate. Sir Lawrence Bragg produced some of the first usable X-ray images with his apparatus in the late 1940s.
Indirect drive laser inertial confinement fusion
uses a "hohlraum" which is irradiated with laser beam cones from either
side on its inner surface to bathe a fusion microcapsule inside with
smooth high intensity X-rays. The highest energy X-rays which penetrate
the hohlraum can be visualized using an X-ray microscope such as here,
where X-radiation is represented in orange/red.
In the 1950s Sterling Newberry
produced a shadow X-ray microscope which placed the specimen between
the source and a target plate, this became the basis for the first
commercial X-ray microscopes from the General Electric Company.
After a silent period in the 1960s, X-ray microscopy regained people's attention in the 1970s. In 1972, Horowitz and Howell built the first synchrotron-based X-ray microscope at the Cambridge Electron Accelerator.
This microscope scanned samples using synchrotron radiation from a tiny
pinhole and showed the abilities of both transmission and fluorescence
microscopy. Other developments in this period include the first
holographic demonstration by Sadao Aoki and Seishi Kikuta in Japan, the first TXMs using zone plates by Schmahl et al., and Stony Brook’s experiments in STXM.
The uses of synchrotron light sources brought new possibilities
for X-ray microscopy in the 1980s. However, as new synchrotron
source-based microscopes were built in many groups, people realized that
it was difficult to perform such experiments due to insufficient
technological capabilities at that time, such as poor coherent
illuminations, poor quality x-ray optical elements, and user-unfriendly
light sources.
Entering the 1990s, new instruments and new light-sources greatly
fueled the improvement of X-ray microscopy. Microscopy methods
including tomography, cryo, and cryo-tomography were successfully
demonstrated. With rapid development, X-ray microscopy found itsnew
applications in soil science, geochemistry, polymer sciences, and
magnetism. The hardware was also miniaturized so that researchers could
perform experiments in their own laboratories.
Extremely high intensity sources of 9.25 keV X-rays for X-ray
phase-contrast microscopy, from a focal spot about 10 um x 10 um, may be
obtained with a non-syncrotron X-ray source which uses a focused
electron beam and a liquid metal anode. This was demonstratred in 2003,
and in 2017 was used to image mouse brain at a voxel size of about one
cubic micrometer (see below).
With the applications continuing to grow, X-ray microscopy has
become a routine, proven technique used in environmental and soil
sciences, geo- and cosmo-chemistry, polymer sciences, biology,
magnetism, material sciences. With this increasing demand for X-ray
microscopy in these fields, microscopes based on synchrotron, liquid
metal anode, and other laboratory light sources are being built around
the world. X-ray optics and components are also being commercialized
rapidly.
Instrumentation
X-ray optics
Synchrotron Light Sources
Advanced Light Source
The
Advanced Light Source (ALS) in Berkeley, California, is home to XM-1, a
full-field soft X-ray microscope operated by the Center for X-ray
Optics and dedicated to various applications in modern nanoscience, such
as nanomagnetic materials, environmental and materials sciences and
biology. XM-1 uses an X-ray lens to focus X-rays on a CCD, in a manner
similar to an optical microscope. XM-1 held the world record in spatial
resolution with Fresnel zone plates down to 15 nm and is able to combine
high spatial resolution with a sub-100ps time resolution to study e.g.
ultrafast spin dynamics. In July 2012, a group at DESY claimed a record spatial resolution of 10 nm, by using the hard X-ray scanning microscope at PETRA III.
The ALS is also home to the world's first soft x-ray microscope
designed for biological and biomedical research. This new instrument,
XM-2 was designed and built by scientists from the National Center for
X-ray Tomography. XM-2 is capable of producing 3-dimensional tomograms of cells.
Liquid metal anode X-ray source
Extremely
high intensity sources of 9.25 keV X-rays (gallium K-alpha line) for
X-ray phase-contrast microscopy, from a focal spot about 10 um x 10 um,
may be obtained with an X-ray source which uses a liquid metal galinstan anode. This was demonstratred in 2003.
The metal flows from a nozzle downward at a high rate of speed and the
high intensity electron source is focused upon it. The rapid flow of
metal carries current, but the physical flow prevents a great deal of
anode heating (due to forced-convective heat removal), and the high
boiling point of galinstan inhibits vaporization of the anode. The
technique has been used to image mouse brain in three dimensions at a
voxel size of about one cubic micrometer.
Detection devices
Scanning Transmission
Sources of soft X-rays suitable for microscopy, such as synchrotron
radiation sources, have fairly low brightness of the required
wavelengths, so an alternative method of image formation is scanning
transmission soft X-ray microscopy. Here the X-rays are focused to a
point and the sample is mechanically scanned through the produced focal
spot. At each point the transmitted X-rays are recorded using a detector
such as a proportional counter or an avalanche photodiode.
This type of Scanning Transmission X-ray Microscope (STXM) was first
developed by researchers at Stony Brook University and was employed at
the National Synchrotron Light Source at Brookhaven National Laboratory.
Resolution
The resolution of X-ray microscopy lies between that of the optical microscope and the electron microscope.
It has an advantage over conventional electron microscopy in that it
can view biological samples in their natural state. Electron microscopy
is widely used to obtain images with nanometer to sub-Angstrom level
resolution but the relatively thick living cell cannot be observed as
the sample has to be chemically fixed, dehydrated, embedded in resin,
then sliced ultra thin. However, it should be mentioned that cryo-electron microscopy
allows the observation of biological specimens in their hydrated
natural state, albeit embedded in water ice. Until now, resolutions of
30 nanometer are possible using the Fresnel zone plate lens which forms
the image using the soft x-rays emitted from a synchrotron. Recently,
the use of soft x-rays emitted from laser-produced plasmas rather than
synchrotron radiation is becoming more popular.
Analysis
Additionally, X-rays cause fluorescence in most materials, and these emissions can be analyzed to determine the chemical elements of an imaged object. Another use is to generate diffraction patterns, a process used in X-ray crystallography.
By analyzing the internal reflections of a diffraction pattern (usually
with a computer program), the three-dimensional structure of a crystal
can be determined down to the placement of individual atoms within its
molecules. X-ray microscopes are sometimes used for these analyses
because the samples are too small to be analyzed in any other way.
Biological Applications
One early applications of X-ray microscopy in biology was contact imaging, pioneered by Goby in 1913. In this technique, soft x-rays
irradiate a specimen and expose the x-ray sensitive emulsions beneath
it. Then, magnified tomographic images of the emulsions, which
correspond to the x-ray opacity maps of the specimen, are recorded using
a light microscope or an electron microscope. A unique advantage that
X-ray contact imaging offered over electron microscopy was the ability
to image wet biological materials. Thus, it was used to study the micro
and nanoscale structures of plants, insects, and human cells. However,
several factors, including emulsion distortions, poor illumination
conditions, and low resolutions of ways to examine the emulsions, limit
the resolution of contacting imaging. Electron damage of the emulsions
and diffraction effects can also result in artifacts in the final
images.
X-ray microscopy has its unique advantages in terms of nanoscale
resolution and high penetration ability, both of which are needed in
biological studies. With the recent significant progress in instruments
and focusing, the three classic forms of optics—diffractive, reflective, refractive
optics—have all successfully expanded into the X-ray range and have
been used to investigate the structures and dynamics at cellular and
sub-cellular scales. In 2005, Shapiro et al. reported cellular imaging
of yeasts at a 30 nm resolution using coherent soft X-ray diffraction
microscopy. In 2008, X-ray imaging of an unstained virus was demonstrated.
A year later, X-ray diffraction was further applied to visualize the
three-dimensional structure of an unstained human chromosome.
X-ray microscopy has thus shown its great ability to circumvent the
diffractive limit of classic light microscopes; however, further
enhancement of the resolution is limited by detector pixels, optical
instruments, and source sizes.
A longstanding major concern of X-ray microscopy is radiation damage, as
high energy X-rays produce strong radicals and trigger harmful
reactions in wet specimens. As a result, biological samples are usually
fixated or freeze-dried before being irradiated with high-power X-rays.
Rapid cryo-treatments are also commonly used in order to preserve intact
hydrated structures.
