25 mm

5 µm 2.4 mm

An Overview of 3D
X-ray Microscopy

Essential
Knowledge
Briefings Second Edition, 2020
2 An Overview of 3D X-ray Microscopy

Cover: Selection of images produced by X-ray microscopes

of different materials, including geological, electrical and
advanced materials (clockwise from top).
Segmentation showing the lithological classification of a
100 mm carbonate rock core. Imaging was performed using the
FPX detector on a ZEISS Xradia 520 Versa X-ray microscope. This
rendering was created with ORS Visual SI Advanced.
Mobile phone camera lens assembly imaged by ZEISS Xradia
520 Versa. Brown section is overlay of an interior tomography scan.
A portion of a solid oxide fuel cell (SOFC) was imaged
using ZEISS Xradia 810 Ultra. There are three layers of the
SOFC visible. The porous top section is the cathode, which is
a lanthanum-strontium-manganite (LSM) composition. The
LSM network has been color labeled according to its local
thickness. Blue is thin and red is thick. The center of the sample
is the electrolyte, which is made of yttria-stabilized zirconia
(YSZ). In this portion of the sample, the image does not show
the solid YSZ, but actually the voids that exist within the YSZ.
One void is labeled orange because it also connects to the pore
network in the lower portion of the cell. The bottom layer is the
anode, which is a porous composite of nickel and YSZ. YSZ is
blue, nickel is red.

© 2020 John Wiley & Sons Ltd, The Atrium, Southern Gate,
Chichester, West Sussex PO19 8SQ, UK
Microscopy EKB Series Editor: Dr Julian Heath
Spectroscopy and Separations EKB Series Editor:
Dr Christina Poggel
CONTENTS
4 INTRODUCTION
6 INTRODUCING X-RAY MICROSCOPY
14 FLEXIBLE X-RAY MICROSCOPES
WITH SUB-MICRON RESOLUTION
20 ADVANCED X-RAY MICROSCOPES
WITH NANOSCALE IMAGING
23 HIGH-RESOLUTION IN SITU
EXPERIMENTS
26 THE MANY APPLICATIONS OF XRM
28 EXAMPLES

About Essential Knowledge Briefings

Essential Knowledge Briefings, published by John Wiley &
Sons, comprise a series of short guides to the latest techniques,
appli­cations and equipment used in analytical science. Revised
and updated annually, EKBs are an essential resource for scien-
tists working in both academia and industry looking to update
their understanding of key developments within each specialty.
Free to download in a range of electronic formats, the EKB
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4 An Overview of 3D X-ray Microscopy

INTRODUCTION
Scientists have long used X-rays to peer within solid
objects, but the recent rise of X-ray microscopy (XRM) means
they are now able to probe the secrets of matter in ever greater
detail. Unlike most forms of microscopy, XRM can deliver
high resolution and contrast in three dimensions, and can do
so without destroying samples.
Recent advances in XRM technology have come from
pioneering work in synchrotron facilities across the world.
In these facilities, researchers use brilliant beams of X-rays
produced by particle accelerators to achieve unprecedented
levels of resolution and contrast. While synchrotron scientists
continue to look ever deeper into materials, commercially
available, lab-based XRM systems are now benefiting
from their developments in detector and focusing optic
technologies.
These commercial lab-based XRM systems have a
key role to play in three-dimensional (3D) imaging and
tomography, offering resolutions well beyond that of classical
X-ray tomography or micro-CT. They can provide non-
destructive 3D imaging of samples across a wide range of
length scales, revealing features from nanometers to
millimeters. They also offer a unique opportunity to study
samples in situ to examine how the microstructure changes
over time, known as four-dimensional (4D) imaging. With
these strengths, it should come as no surprise that XRM is
being used to study an ever-increasing range of materials, from
biological samples to batteries to advanced alloys to geological
material, for both research and industrial applications.
 An Overview of 3D X-ray Microscopy 5

The amount of data being produced by the systems

around the world is impressive, both in its breadth and scope.

42 6
Countries publishing XRM Continents with
materials science papers co-authors
1374*
Zeiss XRM
5.7 publications
On average, new since 2015 400+
ZEISS XRM Unique institutions listed
publications every week as authors or co-authors

*as of August 2019

6 An Overview of 3D X-ray Microscopy

INTRODUCING X-RAY MICROSCOPY

With micro-CT architecture, you can image the whole
object (e.g. an apple), but if you want to see small things inside
(e.g. a seed), you need to cut it open. Cutting an apple might
be OK, but what if:
• It is a precious sample you can’t destroy?
• It is an intact device (battery, electronics component)?
• Cutting your sample risks damaging the structure?
• You need to preserve your sample for future studies?
• You have sparse features and don’t know where to cut?
• You are working inside an in situ chamber or rig?
There are frequent cases where working with larger or intact
samples is beneficial. Only an X-ray microscope can scan an
apple seed at high resolution without cutting the apple open
(Figure 1).
A

B
 An Overview of 3D X-ray Microscopy 7

Figure 1. With micro-CT architecture you can image the whole apple, but if you want to
see the seed, you need to cut it open (A–C). Only an X-ray microscope can scan an apple
seed at high resolution without cutting the apple open (D)

X-ray microscopes use a powerful X-ray source and

advanced optics to create compelling 3D images of samples.
Because X-rays can penetrate through solid objects, there
is no need for sectioning thick or opaque samples. Instead,
by taking multiple X-ray projections from different angles
as the sample is rotated, X-ray microscopes can build up a
detailed 3D representation of a sample’s internal structures.
At their simplest, X-ray microscopes comprise an
X-ray source, usually an X-ray tube, and a detector, usually
a combination of a scintillator to convert the X-rays into
visible light and a charge-coupled device (CCD) to detect
this light. The sample is placed between the source and the
detector and illuminated with X-rays. Although X-rays can
8 An Overview of 3D X-ray Microscopy

pass through solid objects, they are absorbed to different

extents by different materials based on the local density, and
it is this contrast that produces the image on the detector.
With the combination of the latest scintillators carefully
tuned to enhance sensitivity, glass lenses to induce varying
degrees of optical magnification, and the latest CCDs
containing small detector pixel sizes, it becomes possible to
achieve imaging resolutions of 500 nm and voxel sizes as
small as 40 nm, as seen within the ZEISS Xradia Versa family
of X-ray microscopes (Figures 2 and 3). But even higher
resolutions, defined as the smallest individual features that can
be resolved in the magnified image, can be achieved by taking
advantage of the latest advanced X-ray optics (Figure 4).
In the ZEISS Xradia Ultra family of X-ray microscopes,
X-rays emitted from a source initially pass through a capillary

Scintillators

CCD detector
(not visible)

Optical magnification
Figure 2. ZEISS Xradia Versa: multiple scintillator-coupled optics for different
magnifications
 An Overview of 3D X-ray Microscopy 9

Resolution at a distance (RaaDTM)

0
High

2 Xradia Versa XRM

4
Resolution (µm)

8 Geometric mag-based micro-CTs

10

12
Low

14
0 10 20 30 40 50
Working distance (mm) – similar to sample radius
Source to center of sample rotation

Figure 3. A comparison of X-ray microscopy and traditional micro-CT architectures

Sample
Capillary on rotation Objective Phase X-ray
condenser axis zone plate ring detector
X-ray
source

Figure 4. Schematic diagram of the optics for ZEISS Xradia Ultra

condenser (Figure 4) to produce even illumination upon the

sample. After passing through the object, the transmitted
X-rays are then focused by a Fresnel zone plate objective lens
to produce a magnified image. Optionally, the X-rays can
also pass through a Zernike phase ring to produce an edge-
enhancing phase contrast effect before hitting the detector.
With the addition of these unique X-ray optical elements, this
set-up can achieve resolutions as low as 50 nm.
10 An Overview of 3D X-ray Microscopy

BOX 1. A century of X-rays

X-rays are a form of electromagnetic radiation, with
wavelengths much shorter than visible light, between 0.01 and
10 nm. Their short wavelengths mean that, unlike visible light,
X-rays can penetrate visually opaque objects, making them ideal for
medical and security scanning, and various research applications.
German physicist Wilhelm Röntgen was the first to
discover X-rays, in 1895, and to create an X-ray image of the
human body – his wife’s hand. His discovery was quickly leapt
upon: within six months, his mysterious ‘X’ rays were being used
by battlefield medics to locate bullets inside injured soldiers.
The development of XRM as an advanced microscopy
technique took a century longer. On the one hand the short
wavelengths of X-rays mean they penetrate opaque objects and
interrogate internal structures, offering new information beyond
what can be obtained from light microscopy. But conversely, this

Figure 5. SEM image of

Fresnel zone plate
 An Overview of 3D X-ray Microscopy 11

characteristic also gives rise to the challenge that X-rays can’t be

focused using the familiar glass lenses that produce magnification
in light microscopes.
So producing magnified X-ray images at high resolutions
has required the development, over the past few decades, of
appropriate scintillators, advanced optics, and detectors with
tiny pixel sizes. These optics include capillary condensers for
evenly illuminating samples and Fresnel zone plates, which
comprise concentric rings of opaque and transparent zones that
can focus X-rays via diffraction.
Originally developed for use in synchrotrons, which
produce powerful X-ray beams by accelerating electrons around
a circular track at high speeds, these technologies have since
found their way into commercially available laboratory-scale
X-ray microscopes, such as the Versa and Ultra families of
X-ray microscopes.
12 An Overview of 3D X-ray Microscopy

BOX 2. Focus on magnification

There are two ways to produce a magnified image:
geometric magnification and optical magnification. Geometric
magnification is generated purely by the relative distance between
the light source, the object and the detector. A huge shadow of a
small object projected by a light source onto a wall behind it is
produced by geometric magnification – like a shadow puppet.
To make the shadow larger, and thus increase the
magnification, either the light source needs to be moved
closer to the object or the object needs to be moved further
away from the wall, or both. As the magnification increases,
the sharpness of the image necessarily decreases, a phenomenon
referred to as penumbral blur. This blur, and the practical
distances between light source and detector, set boundaries on
the magnification that can be realized in a system relying solely
on this geometric effect.

Figure 6. Image of
resolution measure
 An Overview of 3D X-ray Microscopy 13

Optical magnification is conventionally produced by glass

lenses. It allows for a much greater degree of magnification than
geometric magnification, while retaining high levels of resolution.
By focusing and refracting the light interacting with an object,
the lenses in a modern light microscope can magnify the object
by a factor of hundreds.
But because the glass lenses that focus and refract visible
light don’t work with X-rays, magnification of X-ray images has
traditionally been limited by the geometric effect alone. This is
fine for generating X-ray images of broken limbs or cancerous
tumours, which don’t require much magnification, but it
has prevented X-rays from being used to study materials at
microscopic or even sub-micrometer scales.
This all changed with the development of dual-stage
magnification systems, combining moderate amounts of
geometric magnification with user-selected optical magnification.
This is enabled by means of scintillator-coupled optics, whereby
an X-ray image is first formed on a scintillator and then further
magnified by an optical lens before reaching the CCD detector
plane. Combining these magnification schemes produces a level of
resolution and flexibility not previously accessible in laboratory
tomography.
Furthermore, additional technological advances have
produced focusing optics that act upon X-rays directly, namely
capillary condensers and Fresnel zone plates, further pushing the
limits of the total magnification achievable by XRM.
14 An Overview of 3D X-ray Microscopy

FLEXIBLE X-RAY MICROSCOPES WITH

SUB-MICRON RESOLUTION
A powerful range of X-ray microscopes that uses
advanced optics and detector technology to resolve features
as small as 500 nm, comprising four different models – 410,
510, 610 and 620 – that differ in their precise mix of features,
the ZEISS Xradia Versa family employs standard geometric
magnification as well as novel optical magnification
implemented between the scintillator and the CCD. With
this two-stage magnification, the level of detail that can be
resolved is nearly independent of the relative positions of the
X-ray source, sample and detector. We call this ‘resolution
at a distance’ (RaaD) – capable of maintaining 1 µm spatial
resolution even at 50 mm source-sample distance – ideal for
larger samples or samples contained within in situ chambers.
The Xradia Versa family benefits from many other
advanced features. These include several methods for
improving image contrast in instances where X-ray
absorption by different materials in a sample is very similar

Figure 7. ZEISS Xradia 620 Versa

 An Overview of 3D X-ray Microscopy 15

(see Box 3) and the ability to zoom in on interesting features,

just like with a light microscope. It also boasts proprietary
stabilization mechanisms, a sample stage with multiple
degrees of freedom and automated workflows for high
volume, repetitive scanning.
For ZEISS Xradia 510/610/620 Versa models, there is
an optional flat panel detector, which extends the application
space to larger volumes, allowing entire large samples – such
as an intact smartphone – to be imaged rapidly in 3D. The
technically most advanced model of the family, ZEISS Xradia
620 Versa also offers optional modes for studying the crystal
structure of samples (see Box 4) and for manipulating X-ray
images taken at two different wavelengths to discern subtle
details, such as mineral phases in a geological sample.
With these advanced features, the Xradia Versa
instruments allow scientists to conduct studies that would
be impossible with conventional X-ray imaging systems.
Take, for example, an investigation of how tiny silicon
particles are distributed in a chunk of aluminium-silicon
alloy treated with heat and friction to make it less brittle.
Using propagation phase contrast, scientists can detect
the silicon particles in the processed part of the sample
and then use optional grain structure analysis to examine
their orientation. Other applications include mapping the
location of individual neurons in brain tissue in 3D, or high-
resolution inspection of defects and failures within large
electronics packages.
16 An Overview of 3D X-ray Microscopy

BOX 3. Enhancing contrast

Absorption contrast imaging is the standard imaging
mode used in hospital X-rays. It produces a shadow picture where
materials that absorb the most X-rays – such as bone – produce
the most contrast. Utilizing optimal scintillator and detector
designs, the Versa and Ultra X-ray microscopes maximize the
sensitivity to these small absorption variations. In the case of
polychromatic X-rays as used in Versa, this demands enhanced
detection of low-energy photons and decreased detection of
contrast-reducing high-energy photons. However, for samples
containing materials that absorb X-rays to very similar extents,
absorption contrast imaging on its own may not be sufficient
for producing a detailed image. Examples of such low-contrast
samples include soft tissue, polymers and fossilized organisms
encased in amber.
So the Xradia Versa and Ultra families of X-ray microscopes
employ alternative methodologies to enhance the contrast. These

200 µm

Figure 8. Propagation phase contrast image of aluminium silicon (right) alloy

discerns whisker interfaces not present in absorption image (left)
 An Overview of 3D X-ray Microscopy 17

include phase contrast imaging, which takes advantage of the

fact that all light, including X-rays, will pass through materials
at different speeds, altering the phase of the light wave. Using
appropriate detectors and acquisition geometry, this phase
difference can be used to enhance contrast, especially at the
interfaces between materials. In ZEISS Xradia Ultra, a Zernike
phase ring is inserted into the beam path and used to amplify this
effect, leveraging a variation of a technology originally developed
for light microscopy.
18 An Overview of 3D X-ray Microscopy

BOX 4. Transforming crystallography

X-ray crystallography is a technique for studying the
regular arrangement of atoms in crystalline materials, such as
ceramics and metals. When a crystal is illuminated by an X-ray
source, the atoms cause the X-rays to spread out – or diffract –
and then interfere with each other to create a pattern specific to
that material.
Traditionally, the X-ray diffraction (XRD) technique
has been used as an analytical approach for identifying and
describing crystalline phases, but without the power to produce
spatially resolved images. Concurrently, electron backscatter
diffraction (EBSD) in electron microscopes has developed to
provide 2D maps of the crystal structure of surfaces, but lacks
the power to generate 3D information. But, since the early 2000s,
scientists have been developing a technique known as diffraction
contrast tomography (DCT), using the powerful X-ray beams

Figure 9. Non-destructive
LabDCT 3D grain
structure of iron. Internal
crystallography (colour)
revealed by diffraction
information (black and white)
 An Overview of 3D X-ray Microscopy 19

produced by synchrotrons to visualize cracks, impurities,

locations, shape, and orientation of grains in 3D. Unfortunately,
only a handful of facilities worldwide offer this technology,
despite a growing demand from material scientists.
ZEISS Xradia 620 Versa offers DCT in a commercially
available X-ray microscope, bringing 3D non-destructive grain
mapping into the lab. With the LabDCT module, grains in
the sample act as tiny lenses to focus diffracted X-rays onto a
detector. The GrainMapper3D software then reconstructs the
positions, shapes and orientations of the grains in 3D from a
combination of these diffraction patterns and ordinary X-ray
absorption images.
20 An Overview of 3D X-ray Microscopy

ADVANCED X-RAY MICROSCOPES WITH

NANOSCALE IMAGING
The ZEISS Xradia Ultra family are the first X-ray
microscopes to bring non-destructive 3D imaging at
nanometer-scale resolutions into the laboratory. Comprising
two different models – 800 and 810 – that differ in their X-ray
energies and precise mix of features, the Xradia Ultra family
uses advanced optics and detector technologies adopted
from synchrotron-based designs to achieve resolutions
down to 50 nm, intermediate between light microscopy and
electron microscopy.
Models with two different X-ray energies to enhance
contrast are available. ZEISS Xradia 800 Ultra uses a higher-
energy X-ray source (8.0 keV) for imaging thicker, denser
samples, because more powerful X-rays are required to
penetrate them, while Xradia 810 Ultra uses a lower-energy
X-ray source (5.4 keV) for thinner, less dense samples. The
advantage of using lower energy X-rays where possible is that
they are absorbed more strongly by the sample and therefore
provide a higher contrast. So whereas Xradia 810 Ultra is more
suitable for analyzing polymers, energy storage materials and
soft tissue, Xradia 800 Ultra is more suitable for analyzing
electronic components and metals.
With the Xradia Ultra family, the option of adding a
phase ring behind the Fresnel zone plate is also offered to
further improve contrast, which can be useful for studying
low density samples as well as tiny features like cracks and
defects. In addition, an optional Load Stage can be integrated
to perform in situ 3D nanomechanical testing (see Box 5). The
 An Overview of 3D X-ray Microscopy 21

Xradia Ultra family also benefits from a low vibration platform

and precise positioning stages for holding the microscope
steady during several hours of unattended imaging.
With its unprecedented resolution and advanced
features, the Xradia Ultra microscopes can take XRM to
places where it’s never been before. For example, scientists are
using Xradia Ultra to determine the internal porosity of shale
at nanoscale resolutions and to explore dendritic structures in
advanced metal alloys. With the optional Load Stage, they are
able to study crack formation in dentin, a major component
of teeth that protects them from fracture, by observing how
cracks initiate, propagate and ultimately lead to failure.
22 An Overview of 3D X-ray Microscopy

BOX 5. Imaging under pressure

Scientists have long
used microscopes to study
materials under strain, but
these techniques have generally
required very thin samples
(e.g. TEM) or were limited to
surface deformation (e.g. SEM
10 µm
or most light microscopy).
The Ultra Load Stage Figure 10. A rendering showing the 3D
morphology of a selected crack (gold) in
is an optional accessory dentin (grey) in relation to neighboring
tubules (blue) and the indenter tip (orange)
for ZEISS Xradia Ultra that
overcomes these limitations. It allows, for the first time, the effects
of compression, tension and indentation on whole samples to be
imaged in 4D at nanoscale resolutions.
The stage consists of a motor, a force sensor, and two anvils
at the top and bottom that exert force on the sample while being
rotated and imaged by XRM. In indentation mode, for example,
the top anvil presses down on the surface of a sample to reveal
how micro-cracks appear and propagate through the sample.
Applications of this innovative new technology include
studying how building materials fail when compressed or
stretched, and how this relates to their microstructural features.
For example, scientists can observe whether damage initiation
is localized or widespread, what are the related structural
features, and how does it propagate through the sample with
increasing load.
 An Overview of 3D X-ray Microscopy 23

HIGH-RESOLUTION IN SITU EXPERIMENTS

Science is increasingly moving towards looking at
samples in situ in order to fully understand how different
materials perform under real-life working conditions. As
previously stated, one of the advantages of RaaD is that it
allows you to perform high-resolution scans at source-to-
sample distances that a traditional micro-CT architecture
cannot match. While a sample may be able to be reduced
in size, this is not the case if you want to perform an in situ
experiment: your effective sample size is no longer the sample,
but the size of the in situ chamber.
Taking advantage of this unique feature of XRM, you can
look at your samples in a wide variety of in situ experiments,
using either commercially available in situ chambers or
building your own. These can range from micromechanical
studies on tension, compression or indentation, to temperature
or atmosphere control.
3-layer angle interlocked CMC notched
1200

1000

800
Load (N)

600

400

200
Interior high-resolution
0 tomography
0 0.2 0.4 0.6 0.8 1 1.2 1.4 under tension 450 N
Displacement (mm)

Figure 11. An in situ experiment looking at how a woven ceramic matrix composite
performs under tension
24 An Overview of 3D X-ray Microscopy

BOX 6. Correlative microscopy

Correlative microscopy, in which several different
microscopy techniques are combined into a common workflow,
is becoming an increasingly popular way to study a range of
different samples, including biological specimens, geological
samples and advanced functional materials such as battery
electrodes. The advantage of using several different microscopy
techniques is that it allows scientists to cover a broad range
of length scales and dimensions consistent with the often-
hierarchical nature of their samples, therefore providing an
improved level of contextual understanding for their data as well
as a more complete representation of its complex structure.
With its ability to non-destructively produce 3D and 4D
images of the interior of samples at sub-micrometer resolutions,
intermediate between light microscopy and electron microscopy,

Xradia 520 Versa Xradia 810 Ultra

ZEISS FIB-SEM

20 µm

ZEISS Atlas 5
Correlative Workspace

500 µm 10 µm

Figure 12. Experimental overview. This multiscale correlation study was designed
to better understand corrosion damage in a Mg AZ31B alloy sample. The graphical
user interface of ZEISS Atlas 5, designed as a correlative workspace enabling the
user to combine data from different modalities, was used to link and navigate
between imaging performed on ZEISS Xradia 620 Versa, ZEISS Xradia 810 Ultra
and ZEISS FIB-SEM microscopes
 An Overview of 3D X-ray Microscopy 25

XRM is becoming an integral part of such studies. In one

recent example, a group of scientists used ZEISS Xradia Versa
to monitor corrosion in a magnesium alloy over the course of
a few hours, and subsequently identify a particularly corroded
region within the sample for further study at higher resolution.
They then extracted this region using focused ion beam milling
(on a ZEISS FIB-SEM), and performed more detailed 3D analysis
first with ZEISS Xradia Ultra and then FIB-SEM tomography
(which destroyed the sample in the final step). Such a ‘correlative
tomography’ workflow has the power to provide 3D information
at targeted regions of interest spanning from the mm to nm scale.
ZEISS Atlas 5 is a solution designed to aid such correlative
microscopy studies, by automatically integrating images
produced by different microscopy techniques (even 3D) so that
users can seamlessly identify and navigate to specific features for
further interrogation.
26 An Overview of 3D X-ray Microscopy

THE MANY APPLICATIONS OF XRM

XRM uses X-rays to penetrate deep inside solid objects,
revealing their internal structures, down to the nanoscale. It is
of invaluable use across a wide range of scientific disciplines,
from life sciences to geology to advanced materials.
The microscopic structures of almost all materials can
change over their lifetime, as a result of physical stresses or
exposure to environmental conditions such as moisture and
heat. XRM offers a unique opportunity to understand the
mechanisms and dynamics of these changes inside a sample
in 3D. Examples include improving our understanding of
corrosion inside lightweight magnesium alloys, and studying
the formation of micro-fractures within human bones.
The electronic devices essential to modern life demand
powerful rechargeable batteries, with long battery life and
fast charging times. XRM can image whole batteries in 3D,

Figure 13. 4D non-destructive XRM study tracking the change in microstructure of

lithium ion battery materials as a result of charge cycles. Field of view: 2 cm
 An Overview of 3D X-ray Microscopy 27

without disturbing the packaging, to reveal the micro-cracks

and structural defects that can cause them to fail.
XRM is also at the forefront of cutting-edge geological
investigations. In industrial oil and gas extraction, XRM is
regularly used to provide estimates of yield from potential
drilling sites, especially from today’s less conventional oil
sources, such as shale. XRM can also produce 3D images of
the pores and fractures in the rocks that dictate the likely flow
and accessibility of its oil or gas.
Visible light microscopy including laser scanning
confocal microscopy has transformed the life sciences, but is
limited to thin or translucent samples, with relatively small
volumes. XRM can complement these techniques by creating
3D images of large specimens such as entire zebrafish or other
organisms without damaging them, providing a tantalizing
glimpse of their tissues and organs at the resolution of
individual cells.
28 An Overview of 3D X-ray Microscopy

EXAMPLE: ADDITIVE MANUFACTURING

As the development of additive manufacturing (AM)
for mainstream production continues to grow, so too will the
need for linking the impact of process conditions to material
performance, from raw stock material to final part. Defect
detection and characterization within additive manufactured
parts is a key to efficient and effective process development.
However, the process parameters for fabrication can vary
dramatically for different materials and shapes, resulting in
unwanted defects in the final part.
The unique architecture of ZEISS Xradia Versa, most
notably the variable resolution detector coupled with a
flexible sample stage, allows easy collection of high-resolution
tomography information for detailed analysis of complex AM
parts. In the case that higher resolution is required, ZEISS
Xradia Ultra offers a 50 nm resolution.
For AM using feedstock powder, the powder needs to
be uniform in morphology, shape and size. Satellite particles

Figure 14. Ti-6Al-4V powder for PBF, DED.

Defects in the raw powder material for
additive manufacturing can be analyzed
using XRM
 An Overview of 3D X-ray Microscopy 29

Figure 15. An Inconel 3D-printed

lattice structure with the porosity
shown by the blue dots. Sample
courtesy of Kavan Hazeli, Mechanical
and Aerospace Engineering, The
University of Alabama, Huntsville, USA

and internal porosity also must be avoided in order to avoid

the appearance of defects in the final part. In Figures 14 and
15, we are able to see a number of defects in the raw material,
which may lead to an inferior final product, depending on
their quantity and distribution.
We are also able to get statistical analysis of final parts,
such as porosity. Ideally, metal AM parts, such as the Inconel
sample in Figure 15, have a density greater than 99.5% to
match physical properties and the reliability of traditionally
machined/formed parts. An unwanted concentration of
porosity can cause failures in your final product.
30 An Overview of 3D X-ray Microscopy

EXAMPLE: BATTERIES
Analysis of batteries is challenging – many critical
efficiency and safety effects only become apparent with aging,
and thus studies of microstructure over time are critical to
understand lifetime dynamics. This makes non-destructive
measurement techniques particularly appealing. However,
this is complicated by the fact that we would prefer to measure
intact devices to see their microstructure. Many X-ray CT
systems claim sub-micron resolution, but this is only possible

Figure 16. Top: An 18650 Li-ion battery measured with ZEISS Xradia 620 Versa.
Bottom: A single cathode particle of NMC 111 imaged in High Resolution Zernike
Phase Contrast mode on ZEISS Xradia 810 Ultra, voxel size 32 nm
 An Overview of 3D X-ray Microscopy 31

when the sample is prepared very carefully – usually cut down

to a small piece, and often requires a special filament exchange
and adjustment of the X-ray source. Luckily, with RaaD we
are able to scan the entire device to identify areas of interest
and then zoom in for high-resolution imaging.
In Figure 16, we see an example of XRM’s ability to
measure intact devices with a measurement of a commercial
18650 Li-ion battery measured with ZEISS Xradia 620 Versa.
We are clearly able to distinguish between the different layers
in the battery and can see how charge cycling, for example,
will affect each of them.
If nanometer-scale resolution is required, we can
correlatively turn to ZEISS Xradia Ultra to measure battery
materials. In the example shown in Figure 16, we see a single
cathode particle of NMC 111. In this case, the study was
motivated by understanding how morphological changes in
the particle will affect device performance, such as any changes
in the internal crack visible in the scan. Due to the non-
destructive qualities of XRM, this high-resolution imaging can
fit seamlessly and correlatively into your workflow with other
imaging modalities.
32 An Overview of 3D X-ray Microscopy

EXAMPLE: CRYSTALLOGRAPHY
With LabDCT, ZEISS brings you the first-ever
laboratory-based diffraction contrast tomography imaging
module. This unique grain imaging analytical technology
enables non-destructive mapping of orientation and
microstructure in 3D. No longer confined to conventional
2D metallography investigations, direct visualization of 3D
crystallographic grain orientation opens up a new dimension
in the characterization of metal alloys and polycrystalline
materials.
You can combine 3D grain orientation with 3D
microstructural features such as defects or precipitates
that you have observed in tomography, thus seeing new
possibilities for characterizing damage, deformation and
growth mechanisms – or even to couple with modeling.
LabDCT extends metals research to 3D – and on to
4D with routine tool access for longitudinal studies such as

Grain boundary Grain boundary Grain boundary

normal curvature misorientation

330 µm

Figure 17. Abnormal grain growth in Armco iron using LabDCT. Laboratory
diffraction contrast tomography is a contrast method that is uniquely available on the
ZEISS Xradia Versa XRM instruments. Sample courtesy of Prof. Burton R. Patterson,
University of Florida, USA
 An Overview of 3D X-ray Microscopy 33

corrosion. Compared to measurements done in a synchrotron,

being able to expose your samples to environments in the
microscope across days, weeks or even months is a unique
strength of laboratory-based XRM experiments.
The possibilities cover a wide range, from metals and
alloys, such as in the example shown in Figure 17 of abnormal
grain growth in Armco iron, to abrasives, energetic materials,
or semiconductors. By being able to routinely acquire grain
statistics on larger volumes at faster acquisition times, the
crystallographic information provided by LabDCT lets you
supplement other analyses like EBSD or synchrotron methods.
34 An Overview of 3D X-ray Microscopy

EXAMPLE: BIOMATERIALS
From the smallest features measured by ZEISS Xradia
Ultra microscopes to larger samples, XRM has a lot to offer
for biomaterial samples. With the Scout and Zoom approach,
you can observe interior details of larger samples without
destroying them, which is particularly advantageous for
precious samples such as fossils.
Spinules

Figure 18. Using ZEISS Xradia Ultra XRM, digital copies of natural and copied Gecko‐
lizard nanotipped microspinules are generated with unprecedented levels of accuracy
to provide design instructions for soft lithography replication. Moreover, the X‐ray data
are easily transcribed in silico for fine editing and refinement before eventual real‐life
projection into 3D printouts. https://doi.org/10.1002/admi.201800201

5 mm

5 mm 1 mm

Figure 19. A bear jaw imaged with ZEISS Xradia Versa, using the Scout and Zoom
software that enables the creation of efficient workflows by using a simple control
system to scout a region of interest and specify scanning parameters
 An Overview of 3D X-ray Microscopy 35

EXAMPLE: SEMICONDUCTORS
Over the last decade, ZEISS Xradia Versa 3D X-ray
microscopes have become the standard for non-destructive
failure analysis of semiconductor packages. Xradia 620
Versa RepScan, a sub-micron resolution, 3D non-destructive
imaging solution for inspection and measurement, adds a
new dimension of capabilities to XRM by enabling linear and
volumetric inspection and measurement of critical buried
structures in advanced semiconductor packages.
RepScan enables automatic loading, scanning and
unloading of identical samples without the need for operator
intervention. Scan results may be automatically transferred
to a separate workstation where a variety of measurements
can be executed semi-automatically. This establishes a new
benchmark for non-destructive off-line measurements
that support process optimization, product development
and quality analysis and control of complex fine-pitch 3D

Figure 20. Image taken by manual Figure 21. Image taken by 3D X-ray
cross-section microscope
• Mechanically destructive to sample • Not mechanically destructive
• Linear measurements in only one plane • Linear and volumetric measurements
• Difficult to position/capture fine • Infinite flexibility for all planes/
features features
36 An Overview of 3D X-ray Microscopy

architectures, including 2.5D interposers, high bandwidth

memory stacks with TSVs and microbumps, wafer-level
packages with package-on-package interconnects, and ultra-
thin memory with multiple chips in a stack.
By being able to image and measure buried features
non-destructively with sub-micron resolution, you can base
development decisions on richer, higher-accuracy engineering
data and use acquired data for designs of experiments, process
skews, corner lot or other statistical process analysis.
 An Overview of 3D X-ray Microscopy 37

CASE 1: From advanced materials to insect pests

Since 2017, Ralf Wehrspohn, director of the
Fraunhofer Institute for Microstructure of Materials and
Systems IMWS in Halle and professor of Microstructured
Material Design at the Martin Luther University Halle-
Wittenberg, Germany, has been using ZEISS Xradia 810
Ultra to study advanced materials developed by his group.
But now, he is also beginning to apply the expertise his
group has built up in 3D XRM to other, completely different
scientific fields.
‘In our group, we use XRM to produce 3D virtual
representations of the samples non-destructively,’ explains
Wehrspohn. ‘These representations enable the morphological
characterization of the samples and also the estimation of
structural parameters using software analysis. From those 3D
images, we have already characterized polymer fibers, protein
fleeces, porous materials of diverse compositions, catalysts
and drugs.’
For example, Wehrspohn and his group used ZEISS
Xradia 810 Ultra to study novel electrodes they had
developed for splitting water by electrolysis, which offers an
efficient way to produce hydrogen for energy applications.
The electrodes consist of stainless steel coated in oxidized
carbon nanotubes and then covered in catalytic particles of
platinum or ruthenium dioxide, with the carbon nanotubes
providing an effective way to link the catalytic particles to
the stainless-steel support. With ZEISS Xradia 810 Ultra, the
scientists were able to confirm that the stainless-steel support
38 An Overview of 3D X-ray Microscopy

had become fully coated with carbon nanotubes, increasing

its surface area by almost seven times.1
They also used ZEISS Xradia 810 Ultra to study
polymer yarns that are simultaneously strong and tough,
properties that are usually mutually exclusive in synthetic
materials. These polymer yarns consist of nanofibrils
of polyacrylonitrile aligned in the same direction and
connected together via a polymer called poly(ethylene glycol)
bisazide. The yarns are then heated while being stretched
to enhance their strength and toughness. With ZEISS Xradia
810 Ultra, the scientists were able to confirm that the
preparation process had aligned all the nanofibrils in the
same direction.2
For Wehrspohn, the main advantage of 3D XRM is that
it is non-destructive. ‘It means that we can visualize in detail
the internal parts of the sample without the need to cut or
destroy it, which is usually necessary for other microscopy
techniques,’ he says.

Three-dimensional X-ray
image of the polymer yarn,
showing the nanofibrils
aligned in the same direction
 An Overview of 3D X-ray Microscopy 39

He and his group are now bringing this advantage to

biological samples. Recently, in conjunction with colleagues
from the Institute of Biology at the Martin Luther University
Halle-Wittenberg, they used ZEISS Xradia 810 Ultra to study
structures known as setae at the tip of the abdomen of a
species of thrip.3 The thrip uses these setae to hold a drop of
fluid that it can launch at predators as a defense mechanism.
‘In the future, we plan to explore more consistently the
use of XRM to develop new methods for analyzing biological
samples,’ Wehrspohn says.

1. Zhang H, de Souza e Silva JM, Lu X, et al. Novel stable

3D stainless steel-based electrodes for efficient water splitting.
Adv Mater Interfaces 2019;6:1900774. (https://doi.org/10.1002/admi.
201900774)
2. Liao X, Dulle M, de Souza e Silva JM, et al. High strength in
combination with high toughness in robust and sustainable
polymeric materials. Science 2019;366:1376–9. (https://doi.org/
10.1126/science.aay9033)
3. de Souza e Silva JM, Krüger S, Radisch D, et al. Phase-contrast
nano-tomographic imaging in a commercial X-ray microscope
to understand the defensive behavior of Suocerathrips linguis.
Microsc Microanal 2018;24(Suppl S2):398–9. (https://doi.org/
10.1017/S1431927618014277)
40 An Overview of 3D X-ray Microscopy

FURTHER READING
Bachmann F, Bale H, Gueninchault N, et al. 3D grain
reconstruction from laboratory diffraction contrast tomo­
graphy. J Appl Crystallogr 2019;52:643–51. (https://online
library.wiley.com/iucr/doi/10.1107/S1600576719005442)
Carl Zeiss Microscopy. Multi-scale correlative study of
corrosion evolution in a magnesium alloy. Online White Paper,
2015. (https://applications.zeiss.com/C125792900358A3F/0/
39A3BE9DF7D5FAF1C1257F86004A794F/$FILE/EN_44_
013_031_Multi-scale-Correlative-Study-of-Corrosion-
Evolution-in-a-Magnesium-Alloy.pdf)
Carl Zeiss Microscopy. What is 3D X-ray microscopy? High-
resolution non-destructive imaging of internal structures. Online
White Paper, 2015. (https://www.zeiss.com/content/dam/Micro
scopy/us/download/pdf/Products/X-Ray/xraymicroctdigital.pdf)
Carl Zeiss Microscopy. Contrast with a 3D X-ray micro-
scope for difficult-to-image materials. Online White Paper,
2019. (https://applications.zeiss.com/C125792900358A3F/0/
1141F6CD0D40EA01C1258493004F6FE3/$FILE/EN_44_
013_064_contrast_3d_x_ray_microscope.pdf)
Carl Zeiss Microscopy. In situ and 4D science: observing
and quantifying the evolution of 3D microstructure. Online
White Paper, 2019. (https://applications.zeiss.com/C1257929
00358A3F/0/9E4A92E34736BE87C1258493004E9327/
$FILE/EN_44_013_063_insitu_4d_science.pdf)
Green DW, Kelly ST, Lee KK-H, et al. High-definition
X-ray imaging of small gecko skin surface protuberances
for digitization and 3D printing. Adv Mater Interfaces
2018;5:1800201. (https://doi.org/10.1002/admi.201800201)
 An Overview of 3D X-ray Microscopy 41

Holzner C, Lavery L, Bale H, et al. Diffraction contrast

tomography in the laboratory – applications and future
directions. Microsc Today 2016;24:34–43. (https://doi.org/
10.1017/S1551929516000584)
Jespersen KM, Mikkelsen LP. Three dimensional fatigue
damage evolution in non-crimp glass fibre fabric based
composites used for wind turbine blades. Compos Sci Technol
2017;153:261–72. (https://doi.org/10.1016/j.compscitech.2017.
10.004)
Lavery LL, Merkle AP, Gelb J. 3D X-ray microscopy: a
new high resolution tomographic technology for biological
specimens. Microsc Microanal 2015;21(Suppl S3):925–6.
(https://doi.org/10.1017/S1431927615005425)
Merkle AP, Gelb, J. The ascent of 3D X-ray microscopy in
the laboratory. Microsc Today 2013;21:10–5. (https://doi.org/
10.1017/S1551929513000060)
Shashank Kaira C, De Andrade V, Singh SS, et al. Prob-
ing novel microstructural evolution mechanisms in alumi-
num alloys using 4D nanoscale characterization. Adv Mater
2017;29:1703482. (https://doi.org/10.1002/adma.201703482)
ZEISS Xradia 810 Ultra product information. (https://
applications.zeiss.com/C125792900358A3F/0/
D60B411D09D221FDC125835B003B6D57/$FILE/
EN_42_011_080_xradia-810-ultra_rel2-0.pdf)
ZEISS Xradia 610 and 620 Versa product information.
(https://applications.zeiss.com/C125792900358A3F/0/
D 2 0 9 1 8 1 7 B 1 F 1 6 B 9 AC 1 2 5 8 3 8 A 0 0 4 F 1 9 E 2 / $ F I L E /
EN_44_011_068_Product_600-SeriesVersa_rel_1-1.pdf)