biomedical applications of x-ray tomography · (image courtesy of alberto astolfo, ucl london) f....

Biomedical applications of X-ray tomography

Francesco Brun

CNR Nanotec, Rome, Italy

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…

The History of X-rays: 1901


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)

Absorption of X-rays


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

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)


Absorption X-Ray CT


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

The History of X-rays: 1927


Refraction of light


The Dark Side of the Moon (Pink Floyd, 1973)

Absorption (shadow) and refraction


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?

The dark side of X-rays…


Refraction of X-rays…


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)

Synchrotron


A “special” X-ray source called synchrotron is needed

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…


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


• 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)


Mimosa flower


Conventional (absorption)

Phase contrast

The first phase contrast image collected at SYRMEP (Elettra) in 1997

Phase retrieval for PBI


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

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


Applications


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

I - Tissue Engineering (TE)


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

I - Tissue Engineering (TE)


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

I – SR PBI CT for TE


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

I – Results: µ-CT slice


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.

I - 3D rendering


After image segmentation the

network of vessels was

quantitatively characterized

II – Neurodegenerative diseases


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)


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


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

Conclusion


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)

biomedical applications of x-ray tomography · (image courtesy of alberto astolfo, ucl london) f....

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