Hatano Y., Katsumura Y., Mozumder A. (Eds.) Charged Particle and Photon Interactions with Matter - Recent Advances, Applications, and Interfaces
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570 Charged Particle and Photon Interactions with Matter
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575
21
Application of Microbeams
to
the Study of the Biological
Effects
of Low Dose Irradiation
Kevin M. Prise
Queen’s University Belfast
Belfast,
United Kingdom
Giuseppe Schettino
Queen’s University Belfast
Belfast,
United Kingdom
21.1 introduCtion
Humans are continually exposed to ionizing radiation from a range of sources including environ-
mental and occupational sources. The doses from these various sources are very low and generally
of low dose rates. At the level of individual cells within the human body, this can equate to only
Contents
21.1 Introduction .......................................................................................................................... 575
21.2 Low
Dose Radiation Effects and Radiation Risk................................................................. 576
21.3
Introduction
to Microbeams ................................................................................................. 576
21.4
History,
Rationale, and Key Aspects.................................................................................... 577
21.5
Charged-Particle
Microbeams..............................................................................................580
21.5.1
Microbeam Production.............................................................................................580
21.5.2 Beam
Detection ........................................................................................................ 581
21.5.3
Beam
Orientation and Sample Holder...................................................................... 582
21.5.4
Optics
and Sample Preparation ................................................................................ 583
21.5.5
Positioning
Stage ......................................................................................................584
21.6
X-Ray Microbeams...............................................................................................................584
21.6.1 X-Ray
Source............................................................................................................ 585
21.6.2
X-Ray
Optics ............................................................................................................ 587
21.6.3
X-Ray
Detection and Dosimetry ..............................................................................588
21.7
Electron
Microbeams ...........................................................................................................588
21.8
Future Developments............................................................................................................ 589
21.8.1 Beam
Spot Scanning ................................................................................................ 589
21.8.2
Non-Staining
Imaging ..............................................................................................590
21.8.3
Hard
X-Ray Optics ...................................................................................................590
21.8.4
Biological
Advances ................................................................................................. 591
21.9
Summary .............................................................................................................................. 591
Acknowledgments..........................................................................................................................592
References...................................................................................................................................... 592
576 Charged Particle and Photon Interactions with Matter
single tracks of radiation crossing a cell over periods of weeks or years. Our understanding of
the effects of these low doses requires technological approaches that can deliver highly localized
radiation beams into biological models. This chapter reviews the use of novel microbeam technologies
that
allow relevant doses to be tested in biological systems. We outline some of the key technologi-
cal
approaches used to produce microbeams using different types of ionizing radiations and give
examples of some of the biological results that have been obtained with them.
21.2 low dose radiation eFFeCts and radiation risk
Humans are exposed to multiple physical, chemical, and biological agents during their lifetime.
Of these, ionizing radiation(s) has long been known to be deleterious after high dose exposure
(>100mSv), predominantly due to cancer induction; although very high dose exposures cause tissue
damage and ultimately death (see Hall, (2000) for a general textbook). Ionizing radiations are widely
used in society, play a key role in the treatment of cancer, and are an important diagnostic tool.
Despite a century of study, risk estimates for cancer induction in humans to be used for radiation
protection purposes are extrapolated from the Japanese atomic bomb survivors, who were exposed
to both relatively high doses and dose rates. Around 120,000 survivors have been followed up to the
present date; 20,000 of these had received doses of greater than 5 mSv. By 1990, around 6000 deaths
resulted from cancer, of which ∼400 were considered to be deaths due to excess radiation. Other
exposed populations have been studied, including those treated with radiation for various medical
conditions such as ankylosing spondylitis, conditions related to leukemia risk, and other tumors.
Several studies of radiation workers have been undertaken as these populations were exposed to
protracted low-dose exposures (Cardis etal., 1995; Muirhead etal., 1999). From these epidemio-
logical data, a simple extrapolation of risk has been made to low doses generally found in envi-
ronmental and most occupational exposures. This has been called the linear non-threshold model
(LNT), which assumes a linear dose–response relationship between dose and risk. Currently, with
the exception of radiotherapy, the doses that members of the population can be typically exposed to
are lower than the doses received by the bomb survivors and are therefore areas where epidemio-
logical data is scarce. Against a typical background dose of around 3 mSv/year, examples of routine
medical exposures include 3mSv for a breast mammogram and 0.7mSv for a dental x-ray (Brenner
etal., 2003). From this background, a range of experimental approaches have been taken to study
the effects of low dose and low dose rates of radiation exposure and to test the assumptions of the
LNT model. Ionizing radiations are typically compared on the basis of their linear energy transfer
(LET). Sparsely ionizing radiations such as γ-rays and x-rays are classied as low LET and densely
ionizing radiations such as alpha-particles and neutrons are classied as high LET. For low-LET
radiations, a single radiation track will deposit around 1mGy in a human cell. A novel approach has
been the use of microbeams that allow radiation to be precisely targeted to a particular biological
target of interest. They have contributed enormously to our understanding of the effects of low dose
radiation
exposure and this will be the subject of the rest of this chapter.
21.3 introduCtion to miCrobeams
Charged-particle microbeams have been used since the 1960s for quantitative elemental analysis
of geological, historical, and biological samples (Watt and Grime, 1987) where two-dimensional
elemental maps can be obtained by scanning a small ion beam across a sample and monitoring
the x-rays produced by the sample elements (Watt etal., 1982). However, it was only toward the
end of the 1990s that microbeams have attracted the interest of the radiobiology community and
been developed into specic tools to investigate the effect of ionizing radiation on living samples.
Modern “radiobiological microbeams” are instruments capable of delivering accurate predeter-
mined doses of ionizing radiation to individual cells (or parts of cells) and which can assess the
damage induced on a cell-by-cell basis. The advantages of a deterministic irradiation achieved by
Application of Microbeams to the Study of the Biological Effects of Low Dose Irradiation 577
targeting and analyzing cells individually have been recognized since the beginning of radiobio-
logical studies. Using basic setups, Zirkle and colleagues (Zirkle and Bloom, 1953) tried to correlate
radiation-induced cell damage to the type and energy of radiation, the number of ions per cell, and
even the subcellular compartment irradiated. Despite the limited control and precision offered by
their polonium-tipped syringe needle, important observations were made regarding the nuclear and
cytoplasmic sensitivity, strengthening the hypothesis that considerable benet could be achieved
with a deterministic irradiation approach. However, it was only in the last decade of the twenti-
eth century that improvements in radiation production, detection and delivery, image processing,
and micropositioning have the required precision and speed been achieved to successfully develop
radiobiological
microbeam facilities.
21.4 history, rationale, and key aspeCts
The rst microbeam experiment has to be attributed to Zirkle and Bloom (Zirkle and Bloom, 1953)
who in 1953 used a 2MV Van de Graaff accelerator and microcollimators to study the process
of cell division following proton exposure. Their collimator consisted of two metal plates with a
groove etched on one, clamped together to achieve a 2.5μm beam or adjustable cross slits to form
a nominal proton beam of any size from a few microns to a few millimeters. Later a 25μm micro-
beam was developed using the cyclotron facility at the Brookhaven National Laboratories using a
similar collimator approach and 11MeV/amu proton or 22MeV/amu deuterons (Baker etal., 1961),
to investigate radiation damage to mouse-brain cells (Zeman etal., 1959). These rst microbeams
were limited to relatively high doses and impossibility to control and/or determine the number of
particles reaching the samples. As a solution, the GSI Darmstadt microbeam (Kraske etal., 1990)
based on a UNILAC accelerator to collimate ions from carbon to uranium (1.4MeV/amu) used
plastic track detectors to monitor the particle traversals and, a posteriori, determine the traversals
experienced by each sample. Such an approach, however, was extremely time consuming and only
20
cells could be irradiated with a single ion during 10
h
of beam time.
The
incentive to develop modern microbeams came mainly from the necessity to evaluate the
biological effects of exactly one particle traversal in order to evaluate environmental and occupational
radiation risks, where only a few cells in the human body experience isolated traversals by charged
particles (Brenner etal., 1995) separated by intervals of months or years (see Figure 21.1). Due to the
random Poisson distribution of tracks, such scenarios cannot be simulated in vitro using conventional
broad eld techniques, and current excess cancer risks associated with exposure to very low doses of
ionizing radiation are estimated by extrapolating high dose data. These data obtained from in vitro
experiments or from epidemiological data from the atomic bomb survivors, however, suffer from
limited statistical power and are unable to resolve uncertainties from confounding factors, forcing the
adoption of the precautionary LNT model. Moreover, there is experimental evidence that non-linear
effects may play a considerable role at low doses. Genomic instability (Kadhim etal., 1995), hypersen-
sitivity (Marples and Joiner, 1993), and bystander effect (Prise etal., 2005; Morgan and Sowa, 2007),
for example, may increase the initial radiation risk, while the adaptive response (Boreham etal.,
2000) may act as a protective mechanism reducing the overall risks at low doses. Charged-particle
microbeams therefore represent a unique tool. They allow targeting of single cells and analysis of
the induced damage on a cell-by-cell basis, which is critical to assess the shape of the dose–response
curve in the low-dose region. A second driving question was related to the radiation-sensitivity of
the whole cell and the cell nucleus in particular. Traditional radiobiological theories were based on
the assumption that the DNA was the only critical target and that any biological effect observed
was entirely the result of DNA alteration. Following some observation of nonuniformity of cellular
response (Datta etal., 1976), the investigation of radiosensitivity across the cell nucleus was consid-
ered to be of great interest in improving radiobiology models. Modern microbeam facilities were
developed almost in parallel at the Pacic Northwest Laboratories (United States) (Braby and Reece,
1990), Columbia University (United States) (Michael etal., 1995), and the Gray Laboratory(UK)
578 Charged Particle and Photon Interactions with Matter
(Folkard etal., 1997a,b). They all adopted, at least initially, a collimating approach using either adjust-
able knife-edges, sets of microapertures, etched grooves, or microcapillaries to produce micron size
beams of proton and helium ions. The main difference between these microbeams and the previ-
ous facilities were the ability to a priori determine the number of particles to be delivered to each
sample, and the individual cell or part of it to be irradiated (see Figure 21.1). This has been achieved
using custom-designed radiation detectors (see below for details) coupled to fast electrostatic beam
defectors able to shut down the beam when the desired number of particles have been delivered. The
availability of accurate micropositioning stages and computer resources for automatically driving the
experiments were also essential elements.
The initial success of these rst microbeam facilities was related to the ability to measure radia-
tion effects at very low doses with great accuracy. The Columbia charged-particle microbeam was
used to measure the oncogenic transforming efciency of the nuclear traversal of exactly one α
particle (Miller etal., 1999). It was found to be signicantly lower than that predicted by a Poisson-
distribution delivery by an average of one α particle. Such ndings suggested that multiple tra-
versals dominate the biological response, and extrapolation from multiple particle traversals may
overestimate the single traversal risk. However, similar experiments (Hei etal., 1997) highlighted
how even a single α-particle traversal has considerable toxic (∼20%) and mutagenic probability
(average 110 mutants per 10
5
survivors). Similarly, using 3.2MeV protons, Prise and coworkers at
the Gray Laboratory (Prise etal., 2000) used the micronucleus assay as a measure of predomi-
nantly lethal chromosome damage showing a linear dose response in the range 1–30 protons per
nucleus with a single proton responsible for micronuclei formation in less than 2% of exposed
cells. A single-particle traversal was also shown to induce a signicant increase in the proportion
of aberrant human T-lymphocyte cells, 12–13 population doublings after exposure (Kadhim etal.,
2001). The unstable phenotype indicated by the high level of chromatid-type aberrations suggested
that a single α particle through the cell nucleus can also induce genomic instability. Moreover, by
targeting the cytoplasm, microbeams have shown that intracellular signaling between the cyto-
plasm and the nucleus can also cause DNA damage, undermining therefore the fundamental para-
digm of radiobiology that considers direct DNA exposure a prerequisite for the manifestation of
Conventional Microbeam
Nuclear
(A)
(B)
(C)
Non-nuclear
Figure 21.1 Schematic showing differences between conventional radiation exposure experiments and
those using a microbeam. (A) depicts the ability to deliver uniform numbers of charged-particle tracks to indi-
vidual cells, including that of a single track. (B) represents the ability to target radiation to different subcellular
locations.
(C) represents the ability to deliver localized radiation to dened depths within tissue.
Application of Microbeams to the Study of the Biological Effects of Low Dose Irradiation 579
the radiation effects. This is well summarized in the work of Wu and colleagues (Wu etal., 1999),
where cytoplasmic irradiation by α particles was found to induce mutations in human hamster
hybrid (A
L
) cells, indicating that the cytoplasm is also a critical target. By comparing the mutant frac-
tion induced by nuclear and cytoplasmic α-particle traversals for an equitoxic dose, cytoplasmic
irradiation was found to be as much as seven times more mutagenic (due to the low cell killing)
than nuclear exposure and therefore potentially more harmful. Moreover, it was noticed that the
spectrum of aberrations induced by the cytoplasm irradiation was quite different from that pro-
duced by nuclear irradiation, suggesting that different mutagenic mechanisms may be involved.
The importance of cytoplasmic irradiation has also been highlighted by other microbeam experi-
ments directed at investigating the bystander phenomenon. Using both α particles (Shao etal.,
2004) and soft x-rays, increased micronuclei formation and decreased clonogenic survival have
been measured following the irradiation of one or a few cells through their cytoplasm. This nding
showed that direct DNA damage is not required for switching on of important intra cell-signaling
mechanisms. Another advantage offered by the microbeam approach is the possibility of assessing
radiation damage on a cell-by-cell basis, thus avoiding the statistical uncertainty that affects some
conventional assays. For conventional assays, accurate measurements of the radiation effect may
be limited by both the uncertainty in the number of samples exposed and the dose delivered. This
is a particular problem in the low-dose region, where only small effects are expected. However,
using the Gray Laboratory charged-particle microbeam, it has been possible to precisely measure
the survival of V79 cells exposed to 3.2MeV protons at doses below 1Gy (Schettino etal., 2001).
As a result of the precise particle delivery system and knowing the number of cells exposed, it was
possible to detect small variations in the initial slope of the survival curve indicating the presence
of a hypersensitivity region that had been shown previously using x-rays (Marples and Joiner, 1993)
and proving that microbeams are ideally suited to investigate cell survival at very low doses.
Since then, microbeams have established themselves as a powerful, often unique, radiobiological
tool facilitating our understanding of a variety of biological responses. For example, the micro-
beam approach is considered particularly appropriate to investigate the so-called bystander effect
(Morgan and Sowa, 2007), where radiation damage is being detected in unexposed samples follow-
ing the irradiation of neighboring cells. Even though this type of study could be undertaken using
basic shielding arrangements, comprehensive investigations could only be done using the precise
pre-selected dose delivery and the individual cell system assay offered by the microbeam. Using
the Gray Laboratory microbeam (Prise etal., 1998), it was possible to demonstrate that the level
of bystander damage is independent of the number of cells targeted (up to 25%) and of the number
of particle traversals leading to a binary all-or-nothing interpretation of the bystander response
(Schettino etal., 2005). Bystander studies have now been extended to tissues and 3D cell models in
order to understand the role of cell-to-cell communication and the implication of tissue architecture
on radiation response. It is anticipated that the new generation of microbeams, employing longer-
range radiations and deep-tissue imaging techniques, may offer a unique contribution. Moreover,
increasing scientic interest toward subcellular targets and signaling pathways that regulate the
various radiobiological responses greatly benets from the use of microbeam facilities (see Figure
21.1). Only by using facilities that are able to localize the radiation dose to specic areas of interest,
is it possible to address where the triggering effects for specic biological responses (Lyng etal.,
2006) (such as apoptosis or the bystander effect) are initiated and whether the nonuniformity of the
damage
induction may be responsible for the differential damage expression.
Following
the success of the rst microbeams at the end of the twentieth century, considerable inter-
est has been generated in the scientic communities regarding microbeam technologies, resulting in a
sharp increase in the number of facilities worldwide (Gerardi etal., 2005). Many different experimental
setups have been exploited in order to assess the damage induced following irradiation by a micrometer
(or submicrometer in some cases) beam that targets individual cells or part of cells. Some of the most
popular approaches are reviewed and discussed below. In general, however, there are a few basic require-
ments that a microbeam facility has to fulll in order to perform accurate radiobiological experiments.