optimisation of exposure conditions for in vitro radiobiology experiments
TRANSCRIPT
SCIENTIFIC PAPER
Optimisation of exposure conditions for in vitroradiobiology experiments
Elizabeth Claridge Mackonis • Natalka Suchowerska •
Pourandokht Naseri • David R. McKenzie
Received: 23 October 2011 / Accepted: 28 February 2012 / Published online: 28 March 2012
� Australasian College of Physical Scientists and Engineers in Medicine 2012
Abstract Despite the long history of using cell cultures in
vitro for radiobiological studies, there is to date no study
specifically addressing the dosimetric implications of flask
selection and exposure environment in clonogenic assays.
The consequent variability in dosimetry between labora-
tories impedes the comparison of results. In this study we
compare the dose to cells adherent to the base of three
types of commonly used culture flasks or plates. The cells
are exposed to a 6MV clinical photon beam using either an
open or a half blocked field. The depth of medium in each
flask is varied with the medium surrounding the flask either
water or air. The results show that the dose to the cells is
more affected by the scattering conditions surrounding the
flasks than by the level of filling within the flask. It is
recommended that water or a water equivalent phantom
material is used to surround the flasks or plates to
approximate full scatter conditions at the cell layer. How-
ever for modulated fields, surrounding the 24 well plates
with water-equivalent material is inadequate because of the
large volume of air surrounding individual wells. Our
results stress the importance of measuring the dose for new
experimental configurations.
Keywords Radiobiology � Dosimetry � In vitro �Irradiation � MV
Introduction
In vitro studies are extensively used in research. They are
particularly valuable for hypothesis testing and evaluating
the response of a chosen cell line to radiation and/or che-
motherapy drugs. Consequently, an accurate knowledge of
dose to the cells for in vitro studies is essential. Repro-
ducibility of results across laboratories can only be
accomplished if the dose to cells is accurately measured
and reported. The use of different laboratory protocols for
in vitro cell irradiations can have dosimetric consequences,
making it invalid to compare results. For example, com-
parisons between laboratories of the bystander effect have
been difficult due to differences in experimental design [1].
Progress cannot be made until dosimetry has been elimi-
nated as a source of variability.
The increasing use of cell cultures to investigate the
effect of spatially modulated radiation fields, requires
clonogenic studies using adherent cell layers such that the
position of the cells in the flask can be correlated to the
dose received [2, 3]. However the requirements of good
cell biology practice are sometimes incompatible with
accurate and reproducible dosimetry. For example, good
biology would require cells to remain at a constant tem-
perature, which is often inconvenient when transporting
cells for irradiation. Reproducible dosimetry requires the
cells to be in conditions of electronic equilibrium. Sub-
merging the flasks containing the cells in a water bath does
not always achieve this, because air pockets are inherent in
their design. Furthermore, there is a high risk of contami-
nation if the flask cannot be made water tight.
E. Claridge Mackonis (&) � N. Suchowerska
Sydney Cancer Centre, Royal Prince Alfred Hospital,
Missenden Road, Camperdown, NSW 2050, Australia
N. Suchowerska � P. Naseri � D. R. McKenzie
School of Physics, University of Sydney, Sydney,
NSW 2006, Australia
123
Australas Phys Eng Sci Med (2012) 35:151–157
DOI 10.1007/s13246-012-0132-6
Most radiotherapy cancer treatments use megavoltage
photon beams, where the dose distribution depends
strongly on the composition and geometry of the materials
being irradiated. For radiation protection experiments, the
energy distribution which results from the experiment
geometry may also be important [4–6]. For in vitro studies,
there is a large choice of irradiation conditions (flasks, petri
dishes, multiwell plates, tubes) which result in different
radiation scattering conditions. Any air spaces above or
around the cells can compromise the conditions of elec-
tronic equilibrium, introducing uncertainty in the dose
delivered to the cells.
Currently there are no standard protocols for irradiating
cell cultures in vitro because of the variety of experimental
designs used. Some authors [2, 7–10] provide full details of
their experimental irradiation conditions, while others
simply state that ‘dosimetry’ had been performed, but
without detailed description [11]. Others calculate the dose
deviation from assumed full scatter conditions [12].Table 1
summarises the range of approaches to ‘dosimetry’ for in
vitro cell irradiations reported in the literature.
In this study, we report the measured dose in common
configurations for irradiating cell cultures exposed in
therapeutic mega-voltage photon beams. We measure the
dose to an adherent cell layer for a range of scatter con-
ditions. The effect of the following are addressed: the
medium surrounding the flask/plate (air, water, or water-
equivalent phantom), the culture conditions (for example,
the number and size of wells) and the level of filling of the
wells or the flask.
Method
The dose to the base of the flasks for the irradiation con-
ditions was measured using radiochromic film dosimetry
Table 1 Dosimetry for in vitro cell irradiations reported in the literature
Authors Flask/plate Irradiation setup Dosimetry
Seymour and Mothersill
[11]
T25 Co-60 beam at room temperature, no further
detail
None mentioned
Ling et al. [13] 34 mm cell
culture disks
Array of sources placed 12 mm above cell
layer
TLD and ionisation chamber measurements
Mu et al. [14] Lux 11 mm
tissue
culture disc
Custom phantom with growth medium and
controlled atmosphere, 35 cm from the
source
FeSO4- dosimetry
Suchoweska et al. [15] T75 Linac irradiation from beneath the flask in a
water bath. Full scatter.
Parallel plate ionisation chamber
measurements
Sterzing et al. [9] Cryotubes Linac irradiation in a cylindrical water-
equivalent phantom
Pin-point ionisation chamber measurements
Bromley et al. [16] 6-well Perspex surrounding plate, linac irradiation
from beneath. Air in plate.
Farmer-type chamber and radiographic film
measurements (film above) for full scatter
conditions
Moiseenko et al. [8] 2 ml plastic
vials then
plated
Linac irradiation in an acrylic cylindrical
phantom
IC-10 ionisation chamber measurement
Claridge-Mackonis et al.[3]
T75 Flask in a water bath, linac irradiation from
beneath. Full scatter.
GafChromic film measurements
Bewes et al. [17] T75 Linac irradiation from side with cells in water
bath. Full scatter.
Thimble chamber measurements
Keall et al. [12] 4-well Linac irradiation from above. Water
equivalent material above and below flask
with air in flask
Ionisation chamber measurements (full scatter)
and calculations
Altman et al. [7] 6-well Customised phantom. Assessment of
irradiation setup before experiment
TLD and radiographic film measurements
(film below)
Gow et al. [18] T25 Co-60 and linac 20 MeV irradiation from
above with polystyrene buildup
None mentioned
Xing et al. [19] Not specified Cs-137 Not specified
Hehlgans et al. [20] Not specified 200 kV irradiation A duplex dosimeter measurement
Butterworth et al. [2] T75 and T25 Flask in water bath on water equivalent
phantom, linac irradiation from below
Radiochromic film measurements
and 2D array
152 Australas Phys Eng Sci Med (2012) 35:151–157
123
(GAFCHROMICTM EBT and EBT2, International Spe-
cialty Products). To add further insight into the measure-
ments, Monte Carlo simulations of selected geometries
were carried out.
Irradiation and dosimetry
Three types of cell culture containers were considered: 24
well plates (Linbro Chemical Co, USA), 6 well plates and
T75 culture flasks (IwakiTM, Bibby-Sterlin Ltd., UK) were
used (Table 2). The T75 flasks and the multi-well plates
were constructed from polystyrene. The T75 flask had a
75 cm2 plating surface area, the 24 well plate had cylin-
drical wells 1.6 cm in diameter and the 6 well plate had
wells 3.5 cm in diameter. Each flask or plate was centred in
a 6MV photon beam produced by a Varian 21IXS linear
accelerator with two different beam arrangements: an open
field (30 9 30 cm) and a modulated field represented by a
half beam blocked to the central axis (30 9 15 cm) [16].
Radiochromic film was placed directly below the flask or
plates, at 100 cm from the source, to measure the dose
representative of that received by the cells. The film was
supported by 2 cm of Virtual WaterTM (Standard Imaging,
USA) and a 1 cm of polymethylmethylacrylate (PMMA)
on the treatment couch. For the 6 well plates, additional
disks of film were placed inside at the base of each well.
The linac gantry was positioned at 180�, exposing the
flasks from beneath. This arrangement placed the cell layer
at a depth of 3.2 cm in the irradiation phantom and at
100 cm from the radiation source.
Five different irradiation setups were used to investigate
the effect of the scattering material surrounding the flasks
or plates (Fig. 1). In the first set of irradiations, the film was
covered with Virtual WaterTM slabs (Fig. 1a) to achieve
full scatter conditions. In the second and third irradiations,
the flasks were placed in a PMMA box. This box was either
filled with water or air (Fig. 1b, c). As the 24-well and
6-well plates are not water tight, they could not be used for
irradiations where the bath was filled with water. The
fourth and fifth irradiations used Virtual WaterTM slabs to
cover or surround the flasks (Fig. 1d, e).
To identify the effect of the scattering material within
the flasks or wells, they were one quarter, one half, three
quarters or completely filled with water. This set-up reflects
the experimental practice of complete filling of the flasks
for example with PBS (phosphate buffer solution) or partial
filling of flasks for example with growth medium.
The dosimetric measurements were initially performed
with EBT GafChromic film. This type of film was replaced
by EBT2 GafChromic film when EBT was no longer
available. Calibration films were irradiated using full
scatter conditions for each set of measurements. After 24 h
the films were scanned on an EpsonTM 10000XL scanner
(Seiko Epson Corporation, Japan) with a consistent film
orientation at 150 dpi. Using Mephysto MC2 software
(PTW, Germany), the 16-bit grey-scale film images were
calibrated and dose profiles measured at 0.4 mm resolu-
tion. Profiles were measured along the centre of the rows of
wells for the 6- and 24-well plates and at the centre and
edges of the T75 flasks. A minimum of three replicate films
were used for each experimental condition.
A 2-tailed Student’s t test was performed to test the
significance of the differences in measured dose. An
analysis of the errors in the measured doses was performed
following the method set out in the IAEA Technical Report
Series No. 398 [21]. Results were considered to be
Table 2 Shows the percentage difference in dose to the cells relative to the dose for full scatter conditions, for the irradiation setups of Fig. 1
Flasks/plate surrounded
by water (%)
Flasks/plate
in air (%)
Flasks/plate surrounded
by Virtual Water (%)
Flasks/plate covered
by Virtual Water (%)
T75
-2 -7 -3 -4
6 wells
N/A -9 ?1 -2
24 wells
N/A -13 -1 -3
All flasks in this table were filled with water. The combined uncertainty of each measurement is 2.4 %
Australas Phys Eng Sci Med (2012) 35:151–157 153
123
significant if the t-test showed a difference (P \ 0.05) and
the dose difference was greater than the combined uncer-
tainty (Table 3).
Monte Carlo simulation
The NRC version of Electron–Gamma-Shower Monte
Carlo method (EGSnrc) was used to calculate the dose to
the cell layer for a range of irradiation setups. The spec-
trum for a 6MV photon beam produced by a Varian 21EX
linear accelerator was kindly supplied by M. Williams,
Illawarra Cancer Care Centre. Electron and photon trans-
port parameters were selected to include pair production,
the photoelectric effect, Rayleigh scattering, Compton
scattering and Bremsstrahlung. The scattering properties of
each component material were provided by the EGSnrc
software. The scattering properties of Solid Water 457 [22]
were used for the Virtual Water in the experimental setup.
The global cutoff energies for electrons and photons were
0.521 and 0.001 MeV respectively. The incident field was
perpendicular to the phantom surface, as shown in Fig. 1e.
The phantom consisted of a 3 cm thick slab of Solid Water
upon which the simulated square culture flask of dimen-
sions 2.5 9 2.5 9 2.3 cm3 was positioned. A flask wall
thickness and a cell layer thickness of 1 mm each were
used. Simulated flask contents were: filled with water, half
filled with water (10 mm depth) or quarter filled with water
(5 mm depth). When partial filling of the flask was used,
the remaining contents of the flask were assumed to be air.
The number of histories per data point was varied from
2 9 108 to 5 9 109 to achieve an acceptable level of
uncertainty. Quoted uncertainties are standard deviations for
each data point, except where the mean of the entire data set
has been calculated. In such cases the uncertainties are the
standard deviations of the data points from the mean.
Results
The dose profiles measured using film from below the cell
layer for the 24 well plate irradiated with an open field
(Fig. 2) show the insignificant effect of different depths of
medium inside the wells (the mean dose difference is less
Surrounding material either water or air
Water
Cells
Air
Solid
h0.1cm
3cm
2.3cm
2.5cm
20cm
17cm
Cell layer
6 MV
(b)(a)
(e)(d)
(c)
Flask Virtual Water
PMMA Water Couch‘tennis racquet’
(f)
Fig. 1 The five different irradiation designs used in this study with a
T75 flask and the geometry used in the Monte Carlo simulation. For
the experimental setups, the scattering materials are supported by the
carbon fibre tennis racquet couch top and a PMMA sheet and the
flasks are irradiated from below on central axis. The arrows show the
position of the film under the flask. a full scatter conditions in Virtual
Water, used as a reference, b the flask placed in a water bath, c the
flask surrounded by air, d the flask ‘‘covered’’ with Virtual Water
slabs, e the flask surrounded by Virtual Water slabs f the configuration
for the Monte Carlo simulation
Table 3 The error analysis showing an example of the uncertainty analysis performed
Component Type Std. Unc. (%) Comment
Noise in film readout A 1.7 From noise observed in uniform region of the film
Stability of scanner B 0.8 Based on ±2 % with triangular distribution
Height of flask/plate and film at linac B 0.1 Based on 1 mm error with rectangular distribution
Positioning of film on scanner B 0.4 Based on maximum 1 cm difference resulting in maximum
1 % readout difference (triangular distribution)
Film sheet differences B 1.4 Based on maximum ±7 cGy difference between calibration films,
a triangular distribution and 2 Gy dose
Linac dose stability B 0.4 Based on a maximum difference of 1 % and a triangular
distribution
Combined uncertainty 2.4
154 Australas Phys Eng Sci Med (2012) 35:151–157
123
than the combined uncertainty of 2.4 %). The dose profile for
other flask or plate designs also did not vary significantly
with medium depth. Note that the dose profile for the 24 well
plate does not appear to have features associated with the
individual wells. The dose to the cells relative to full scatter
conditions, for open field irradiation are summarised in
Table 2.
Figure 3 summarises the measured dose for the 3 flask and
plate designs with different levels of filling. No significant
difference was observed (greater than ± 2.4 %) between
any of the measurements for a given irradiation setup.
For the 6-well plate, the dose in the medium at the bottom
of the wells was compared to the dose beneath the wells for a
half-blocked beam irradiation (Fig. 4a). The dose profiles
measured inside the wells were consistent in shape with the
dose below the wells. In both the open and shielded regions
of the field, the dose distribution changes significantly
(P \ 0.01 and difference[2.4 %) when the air surrounding
the flasks or plates is replaced by water. This is consistent
with Fig. 3 and is seen for all levels of filling.
Figure 4 shows the dose distributions for the 6-well plate
in air compared to full scatter conditions. The dose distri-
butions for the 6-well plate surrounded or covered with
Virtual Water are shown in Fig. 5a and compared to the full
scatter results obtained in Virtual Water in the absence of a
flask or plate. No significant difference between full scatter
conditions and the conditions created using Virtual Water
around the plate were found (differences \2.4 %). This
result was also observed for the T75 flask. However, in the
blocked region of the field (Fig. 5b), the dose profiles below
the 24-well plates are higher than for the full scatter condi-
tions (P \ 0.01). There was a measured dose difference from
full scatter conditions of at least 10 cGy averaged from 20 to
70 mm from the edge of the blocked region.
Monte Carlo simulation results
The results of the Monte Carlo simulations are shown in
Table 4. The calculated dose below the cell layer, at the
position of the film, is compared to the experimental results
measured with film placed below the wells. The calcula-
tions show that the difference in dose due to flask filling is
very small (\1 %) and agrees with the experimental results
which show no significant difference. However the dose is
significantly decreased, by approximately 3 %, when air
replaced water as the surrounding medium. The calculated
reduction in dose is less than is observed experimentally.
The results of Table 4 confirm that the use of film below
the well to measure the dose to the cell layer results in a
negligible error (\1 %).
Fig. 2 The measured dose profiles below the 24-well plate as a
function of distance from the beam centre. The plate is surrounded by
air. The level of well filling has a negligible effect on the dose
deposited. The thick horizontal lines demonstrate the positions of the
wells. Fully filled corresponds to a depth of 1 cm of medium
Fig. 3 The percentage deviation in dose from full scatter conditions
for all the flask and plate types. No significant differences were
observed within measurement uncertainty for the same irradiation
setup as the level of medium was changed. However the measured
dose was strongly dependent on the choice of surrounding scattering
material (air or water). The 1 cm depth is approximately equivalent to
a full well for the 24-well or 6-well plates
Fig. 4 Measured dose profiles across the wells of the 6-well plate for
a half beam blocked field from film placed either below or at the
bottom, inside the wells. The profiles are measured with the flask
surrounded by air. The inset shows the central region in more detail
Australas Phys Eng Sci Med (2012) 35:151–157 155
123
Discussion
Our results show that the single most important factor that
determines the dose received by a layer of cells is the
scattering material surrounding the culture flask or plate.
This result applies for both an open field and for the open
portion of a half-beam blocked field. Depending on the
flask or plate design, experimental measurements show that
the dose to the cell layer is reduced by between 7 and
13 % ± 2.4 % when the surrounding medium is changed
from water to air. Based on the cell survival curves for
primary fibroblast (AGO-1522) and human prostate cancer
(DU-145) cells [2], this could result in a survival fraction
error of approximately 7 and 10 % respectively. Monte
Carlo simulation confirms that when the flask is surrounded
by air, a dose deficit occurs both in the film layer and in the
adherent cell layer. The reduction in dose is caused by the
removal of back scattered radiation. The results also show
that blocks of water-equivalent phantom material can
provide equivalent scattering conditions to liquid water
despite unavoidable gaps around the perimeter of the flask
or plate. These results agree with the calculations of Keall
et al. [12] reporting a perturbation in dose of \3 % when
flasks are covered with water-equivalent material leaving
air inside and to the sides of the culture flasks.
In the shielded portion of the half blocked field, when air
surrounded the flask a dramatic reduction in dose was
observed compared to full scatter conditions (67 and 69 %
for the shielded wells in Fig. 4). For the T75 and 6-well
flasks, blocks of water-equivalent phantom material can
provide equivalent scattering conditions to liquid water in
the shielded portion of the field. However this does not
apply for the 24-well plates where the dose in the shielded
half of the field was still significantly lower due to the
larger proportion of air surrounding the wells gaps leading
to decrease in scatter. For experiments investigating cells
survival in the shielded parts of a modulated field, these
findings emphasise the importance of dosimetric validation
of experimental designs including the flask with the exact
configuration of the surrounding scattering medium.
There is a small discrepancy between the doses pre-
dicted by the Monte Carlo simulation below the flask
compared to the measured values. The lower dose observed
experimentally may be explained by the differences in
geometry between simulation and experiment. In the
idealised geometry of the simulation, only a single well or
Fig. 5 a The dose profiles below the wells of the 6-well plate when it
is surrounded by Virtual Water or covered by Virtual Water. b The
dose profiles below the wells of the 24-well plate, when it is
surrounded by Virtual Water or covered by Virtual Water. In each
plot, the thick horizontal lines demonstrate the positions of the wells.
The dose profile for full scatter conditions is shown for comparison
Table 4 The dose to film placed below a flask and to cell layers adherent to the bottom of a flask calculated using Monte Carlo simulation for the
geometry shown in Fig. 1e
Surrounding material Flask filling Monte Carlo
film layer (%)
Experimental film
layer (%)
Monte Carlo cell
layer (%)
Water Full 0.0 ± 0.1 -2 ± 2 0.5 ± 0.1
Water 1.0 cm -0.7 ± 0.1 2 ± 2 0.2 ± 0.1
Water 0.5 cm -0.4 ± 0.1 2 ± 2 -0.2 ± 0.1
Air Full -3.4 ± 0.1 -7 ± 2 -3.5 ± 0.1
Air 1.0 cm -3.2 ± 0.1 -6 ± 2 -3.2 ± 0.1
Air 0.5 cm -3.3 ± 0.1 -6 ± 2 -3.2 ± 0.1
The Monte Carlo results are compared with the experimental measurements for a T75 flask. The results are shown as a percentage deviation from
full scatter conditions
156 Australas Phys Eng Sci Med (2012) 35:151–157
123
flask is modelled with no neighbouring wells. Therefore,
the lack of lateral scatter to the film due to the air gaps
between the wells was not modelled.
For the conditions used in this study, the filling of flasks or
plates plays an insignificant role in determining the dose to
the cell layer. No significant difference was found for dif-
ferent filling of any of the flasks or plates in either the open or
half-beam blocked fields for any configuration of surround-
ing material. Monte Carlo simulation confirms that when the
flask is surrounded by water, only a very small change (a
maximum of 0.7 %) in dose occurs in both the film layer and
the adherent cell layer when the flask filling level is changed.
Taken together, these results allow the experimenter to
choose the level of flask filling to suit experimental needs.
Conclusion
We present the results of dosimetric measurements and
Monte Carlo simulations for typical experimental designs
used in in vitro exposures of cells. The results show that the
material surrounding a flask or plate is an important factor
determining the dose to the cells, while the level of filling
of the flask is relatively unimportant.
We show that Virtual Water can be used instead of
liquid water as a surrounding material, without affecting
the dose to the cell layer, except in shielded regions of
modulated fields. As an example we have shown that even
by surrounding the 24-well plate with water-equivalent
material it is not possible to assume that dose profiles for
modulated fields will replicate those for full scatter con-
ditions. It is therefore essential to perform dosimetric
measurements as a precursor to radiobiological experi-
ments. These measurements should be performed at the
location of the cells, particularly where air gaps are
inherent in the plate design.
Acknowledgments The authors acknowledge funding from the
NSW Cancer Council in support of this research.
References
1. Sjostedt S, Bezak E (2010) Non-targeted effects of ionising
radiation and radiotherapy. Australas Phys Eng Sci Med 33:
219–231
2. Butterworth KT, McGarry CK, Trainor C, Sullivan JM, Hounsell
AR, Prise KM (2011) Out-of-field cell survival following expo-
sure to intensity-modulated radiation fields. Int J Radiat Oncol
Biol Phys 79:1516–1522
3. Claridge Mackonis E, Suchowerska N, Zhang M, Ebert M,
McKenzie DR, Jackson M (2007) Cellular response to modulated
radiation field. Phys Med Biol 52:5469–5482
4. Kellerer AM, Roos H (2005) Are all photon radiation similar in
larger absorbers?-a comparison of electron spectra. Radiat Prot
Dosim 113:245–250
5. Pattison JE, Hugtenburg RP, Charles MW, Beddoe AH (2001)
Experimental simulation of A-bomb gamma ray spectra for
radiobiology studies. Radiat Prot Dosim 92:125–136
6. Harder D, Petoussi-Henß N, Regulla D, Zankl M, Dietze G
(2004) Radiat Prot Dosim 109:291–295
7. Altman MB, Vesper BJ, Smith BD, Stinauer MA, Pelizzari CA,
Aydogan B, Reft CS, Radosevich JA, Chmura SJ, Roeske JC
(2009) Characterization of a novel phantom for three-dimensional
in vitro cell experiments. Phys Med Biol 54:N75–N82
8. Moiseenko V, Duzenli C (2007) In vitro study of cell survival
following dynamic MLC intensity-modulated radiation therapy
dose delivery. Med Phys 34:1514–1520
9. Sterzing F, Munter MW, Schafer M, Haering P, Rhein B, Thil-
mann C, Debus J (2005) Radiobiological investigation of dose-
rate effects in intensity-modulated radiation therapy. Strahlenther
Onkol 181:42–48
10. McGarry CK, Butterworth KT, Trainor C, O’Sullivan JM, Prise
KM, Hounsell AP (2011) Temporal characterization and in vitro
comparison of cell survival following the delivery of 3D-con-
formal, intensity-modulated radiation therapy (IMRT) and volu-
metric modulated arc therapy (VMAT). Phys Med Biol
56:2445–2457
11. Seymour CB, Mothersill C (1989) Lethal mutations, the survival
curve shoulder and split-dose recovery. Int J Radiat Biol
56:999–1010
12. Keall P, Chang M, Benedict S, Thames H, Vedam SS, Lin P-S
(2008) Investigating the temporal effects of respiratory-gated and
intensity-modulated radiotherapy treatments delivery on in vitro
survival: an experimental and theoretical study. Int J Radiat
Oncol Biol Phys 71:1547–1552
13. Ling CC, Li WX, Anderson LL (1995) The relative biological
effectiveness of I-125 and Pd-103. Int J Radiat Oncol Biol Phys
32: 373–378
14. Mu X, Lofroth P-O, Karlsson M, Zackrisson B (2003) The effect
of fraction time in intensity modulated radiotherapy: theoretical
and experimental evaluation of an optimisation problem. Radio-
ther Oncol 68:181–187
15. Suchowerska N, Ebert MA, Zhang M, Jackson M (2005) In vitro
response of tumour cells to non-uniform irradiation. Phys Med
Biol 50:3041–3051
16. Bromley R, Davey R, Oliver L, Harvie R, Baldock C (2006) A
preliminary investigation of cell growth after irradiation using a
modulated X-ray intensity pattern. Phys Med Biol 51:3639–3651
17. Bewes JM, Suchowerska N, Jackson M, Zhang M, McKenzie DR
(2008) The radiobiological effect of intra-fraction dose-rate
modulation in intensity modulated radiation therapy (IMRT).
Phys Med Biol 56:3567–3578
18. Gow MD, Seymour CB, Byun S-H, Mothersill CE (2008) Effect
of dose rate on the radiation-induced bystander response. Phys
Med Biol 53:119–132
19. Xing L, Sun X, Deng X, Kotedia K, Urano M, Koutcher JA, Ling
CC, Li GC (2009) Expression of the bifunctional suicide gene
CDUPRT increases radiosensitization and bystander effect of
5-FC in prostate cancer cells. Radiother Oncol 92:345–352
20. Hehlgans S, Lange I, Eke I, Cordes N (2009) 3D cell cultures of
human head and neck squamous cell carcinoma cells are radio-
sensitized by the focal adhesion kinase inhibitor TAE226.
Radiother Oncol 92:371–378
21. International Atomic Energy Agency (2000) absorbed dose
determination in external beam radiotherapy. Technical report
series no 398, IAEA, Vienna. pp 210–214
22. Lui L, Prasad SC, Bassano DA (2003) Evaluation of two water-
equivalent phantom materials for output calibration of photon and
electron beams. Med Dosim 28(4):267–269
Australas Phys Eng Sci Med (2012) 35:151–157 157
123