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Biomedical applications of X-ray tomography
Francesco Brun
CNR Nanotec, Rome, Italy
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The letter X
F. Brun | Biomedical applications of X-ray tomography 2 Warsaw – November, 7th 2016
The letter X will always have the meaning of:
Unknown things…
“Mysterious” / Hard to explain things…
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The History of X-rays: 1901
F. Brun | Biomedical applications of X-ray tomography 3 Warsaw – November, 7th 2016
At the same time he discovered one
of the most interesting applications
W. Röntgen discovered
“A new kind of radiation”
(Eine neue art von Strahlen)
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Absorption of X-rays
F. Brun | Biomedical applications of X-ray tomography 4 Warsaw – November, 7th 2016
The traditional way to get contrast is the absorption of X-rays
The same principle used by Röntgen is still used in the medical field
Hard tissues (e.g. bones) result well defined
Soft tissues are poorly identified, i.e. “transparent” to X-rays
Röntgen original radiography Modern radiography
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The incident beam is attenuated by the absorbing sample
The sample attenuation is recorded in the A2 area of a projection image
The background signal is recorded in the A1 areas
Absorption X-Ray radiography
(image courtesy of Alberto Astolfo, UCL London)
F. Brun | Biomedical applications of X-ray tomography 5 Warsaw – November, 7th 2016
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Absorption X-Ray CT
F. Brun | Biomedical applications of X-ray tomography 6 Warsaw – November, 7th 2016
The physics behind modern clinical CT is still the absorption of X-rays
To image soft tissues contrast agents or staining procedures might be used
When considering:
Digital detectors
Radiographs at different angles
Some math and digital image processing
X-ray Computed Tomography (CT) is possible
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The History of X-rays: 1927
F. Brun | Biomedical applications of X-ray tomography 7 Warsaw – November, 7th 2016
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Refraction of light
F. Brun | Biomedical applications of X-ray tomography 8 Warsaw – November, 7th 2016
The Dark Side of the Moon (Pink Floyd, 1973)
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Absorption (shadow) and refraction
F. Brun | Biomedical applications of X-ray tomography 9 Warsaw – November, 7th 2016
Looking at the shadow
(absorption) is hard to identify
the three transparent media
(air, glass, water)
Looking at the “broken” straw
(refraction) we can better identify
the presence of air, glass, water
Can we exploit refraction also with X-rays?
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The dark side of X-rays…
F. Brun | Biomedical applications of X-ray tomography 10 Warsaw – November, 7th 2016
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The dark side of X-rays…
F. Brun | Biomedical applications of X-ray tomography 11 Warsaw – November, 7th 2016
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Refraction of X-rays…
F. Brun | Biomedical applications of X-ray tomography 12 Warsaw – November, 7th 2016
The refraction (or phase variation) of X-rays is in the range of microradians
There’s the need to “play” with long relative distances
(among the elements, i.e. X-ray source, sample, detector)
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Synchrotron
F. Brun | Biomedical applications of X-ray tomography 13 Warsaw – November, 7th 2016
A “special” X-ray source called synchrotron is needed
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Conventional source:
Incoherent and polychromatic
(white) light
Cone beam geometry
Limited photon flux
Synchrotron source:
Coherent (and monochromatic) light
(Nearly) parallel beam geometry
High photon flux very fast CT
A synchrotron in a nutshell…
F. Brun | Biomedical applications of X-ray tomography 14 Warsaw – November, 7th 2016
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Parallel beam:
Mathematically and computationally “friendly”
Whole 3D reconstruction decomposed in 2D “slice-by-slice” reconstruction
High photon flux:
Ultra-fast tomography can be performed
4D in situ studies can be considered at synchrotrons
Coherence and monochromaticity
No beam hardening artifact thanks to monochromaticity
Phase contrast CT can be easily performed
Advantages for X-ray CT
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• It is the simplest method as no specific X-ray optics is needed.
• A sufficient sample-to-detector distance d is needed to let the signal propagate
• The detector collects the interference of refracted X-rays with non-refracted rays
• Without additional image processing, the result is an edge enhanced image
Propagation Based Imaging (PBI)
(image courtesy of Alberto Astolfo, UCL London)
F. Brun | Biomedical applications of X-ray tomography 16 Warsaw – November, 7th 2016
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Mimosa flower
F. Brun | Biomedical applications of X-ray tomography 17 Warsaw – November, 7th 2016
Conventional (absorption)
Phase contrast
The first phase contrast image collected at SYRMEP (Elettra) in 1997
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Phase retrieval for PBI
F. Brun | Biomedical applications of X-ray tomography 18 Warsaw – November, 7th 2016
With digital detectors and digital image processing the “edge contrast” in a PBI
image can be “converted” into an “area contrast” image
The operation is called phase retrieval
The most widely used (single distance) phase retrieval algorithm is Paganin et al., 2002
edge enhanced phase retrieved image
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Five main imaging approaches:
the propagation-based (PBI) with single or multiple distances
the analyzer-based (ABI) or diffraction-enhanced (DEI)
the interferometric methods based on the use of crystals
the grating interferometry (GI)
grating non-interferometric methods (coded apertures, edge illumination)
These methods differ for their experimental set-up (gratings, crystals or
nothing) and requirements in terms of the X-ray beam coherence
They differ also for the nature and amplitude of the provided image signal
and for the amount of radiation dose delivered to the sample
Techniques for phase contrast CT
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Applications
F. Brun | Biomedical applications of X-ray tomography 20 Warsaw – November, 7th 2016
Two applications of (single distance) PBI X-ray CT (with phase retrieval):
Vascularization in bone tissue engineering
Neurodegenerative diseases
considering pre-clinical (mice) models (ex vivo)
ToMA (Tomography for Medical Applications) Lab in Rome
with support from synchrotrons (ESRF, SLS, Elettra) and “bio-partners”
Alessia Cedola Michela Fratini Inna Bukreeva Fabrizio Bardelli Lorenzo Massimi Francesco Brun
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I - Tissue Engineering (TE)
F. Brun | Biomedical applications of X-ray tomography 21 Warsaw – November, 7th 2016
Alternative approach to grafts
Cells and biofactors are placed onto a biodegradable and bioresorbable scaffold
In a bioreactor, cells grow forming a new tissue ready to be implanted
The biomaterials and microarchitecture of the scaffold play a crucial role
µ-CT is one of the most attractive techniques to characterize the engineered tissue
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I - Tissue Engineering (TE)
F. Brun | Biomedical applications of X-ray tomography 22 Warsaw – November, 7th 2016
The aim:
Visualize and analyze the 3D micro-vascular networks in an ectopic
bone formation mouse-model
Motivations:
Bone grafts are the most common transplants after blood transfusions
Bone-tissue regeneration research has scientific and social impact
Bone is a complex hierarchical structure:
o the interplay of organic and inorganic mineral phases at different
length scale affects its functionality and health
The understanding of bone tissue regeneration requires:
o high spatial resolution
o Imaging at different length scale
The control of the angiogenesis of the microvascular network with
proper spatial organization is a key step
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I – SR PBI CT for TE
F. Brun | Biomedical applications of X-ray tomography 23 Warsaw – November, 7th 2016
Why SR PBI CT?
2D imaging, such as histology, yields incomplete spatial coverage with
possible data misinterpretation
Conventional micro-CT does not achieve sufficient resolution
Hard to observe blood vessels without using contrast agents
Materials and methods
Scaffolds seeded with BMSC implanted for 4 weeks into mice
Three groups:
o with MICROFIL® right before mouse sacrifice
o with phosphotungstic acid (PTA)
o without staining techniques or contrast agents
SR PBI CT at TOMCAT beamline (SLS) with pixel size of 0.64 µm
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I – Results: µ-CT slice
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Three different tissues:
newly formed bone (B)
collagenous soft tissue (ST)
Skelite TM scaffold (SC)
Relevant information about the “soft” elements of TE can be extracted
Bukreeva I. et al. High-resolution X-ray techniques as new tool to investigate the 3D vascularization of
engineered bone tissue. (2015) Frontiers in Bioengineering and Biotechnolgy 3:133.
Campi, G. et al. Imaging collagen packing dynamics during mineralization of engineered bone tissue. (2015)
Acta Biomaterialia, 23, pp. 309-316.
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I - 3D rendering
F. Brun | Biomedical applications of X-ray tomography 25 Warsaw – November, 7th 2016
After image segmentation the
network of vessels was
quantitatively characterized
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II – Neurodegenerative diseases
F. Brun | Biomedical applications of X-ray tomography 26 Warsaw – November, 7th 2016
The aim:
Simultaneous 3D visualization of the vascular network (VN) and neuronal
network (NN) of ex-vivo mouse spinal cord
No contrast agents, no sectioning, no destructive sample-preparation
Image both the 3D distribution of micro-capillary network and the
micrometric nerve fibers, axon-bundles and neurons
Motivations:
Pre-clinical investigation of neurodegenerative pathologies
Resolve the entangled relationship between VN and NN
The specific case of multiple sclerosis (MS) was considered
(its animal model experimental autoimmune encephalomyelitis - EAE)
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F. Brun | Biomedical applications of X-ray tomography 27 Warsaw – November, 7th 2016
Fratini, M. et al. Simultaneous submicrometric 3D imaging of the micro-vascular network and the neuronal
system in a mouse spinal cord. (2014) Scientific Reports, 5, art. no. 8514.
II – PBI CT of spinal cord
Feasibility study at TOMCAT, SLS
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F. Brun | Biomedical applications of X-ray tomography 28 Warsaw – November, 7th 2016
Massimi, L. et al., Characterization of mouse spinal cord vascular network by means of synchrotron radiation X-
ray phase contrast tomography. (2016) Physica Medica: European Journal of Medical Physics.
A decreased vascularization is detected in the EAE case
After segmentation significant parameters for the vessels are quantified
The specific case of the animal model for multiple sclerosis, called EAE was studied
SR PBI CT at ID17, ESRF
II – Neurodegenerative diseases: EAE
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Conclusion
F. Brun | Biomedical applications of X-ray tomography 29 Warsaw – November, 7th 2016
Two biomedical applications were presented
Ex vivo pre-clinical models were considered
SR (single distance) PBI CT with Paganin’s PR and FBP reconstruction
Relevant biomedical information about “soft” components (usually
transparent with conventional CT) can be derived
About the future:
HR ex vivo phase contrast imaging also with non-synchrotron sources
In vivo and longitudinal studies
Radiation dose is a concern
Benefits are coming from hardware improvements (e.g. photon counting detectors) as well as from research in tomographic reconstruction (e.g. ROI-CT, discrete CT) and image processing (e.g. artifacts compensation)