Use of a microscope stage-mounted Nickel-63 microirradiator for real-time observation of the DNA double-strand break response.
ABSTRACT Eukaryotic cells begin to assemble discrete, nucleoplasmic repair foci within seconds after the onset of exposure to ionizing radiation. Real-time imaging of this assembly has the potential to further our understanding of the effects of medical and environmental radiation exposure. Here, we describe a microirradiation system for targeted delivery of ionizing radiation to individual cells without the need for specialized facilities. The system consists of a 25-micron diameter electroplated Nickel-63 electrode, enveloped in a glass capillary and mounted in a micromanipulator. Because of the low energy of the beta radiation and the minute total amount of isotope present on the tip, the device can be safely handled with minimum precautions. We demonstrate the use of this system for tracking assembly of individual repair foci in real time in live U2OS human osteosarcoma cells. Results indicate that there is a subset of foci that appear and disappear rapidly, before a plateau level is reached approximately 30 min post-exposure. This subset of foci would not have been evident without real-time observation. The development of a microirradiation system that is compatible with a standard biomedical laboratory expands the potential for real-time investigation of the biological effects of ionizing radiation.
- SourceAvailable from: George Iliakis[Show abstract] [Hide abstract]
ABSTRACT: DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with gamma-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of gamma-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.Nucleic Acids Research 10/2008; 36(17):5678-94. · 8.81 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The accurate repair of chromosomal double-strand breaks (DSBs) arising from exposure to exogenous agents, such as ionizing radiation (IR) and radiomimetic drugs is crucial in maintaining genomic integrity, cellular viability and the prevention of tumorigenesis. Eukaryotic cells have evolved efficient mechanisms that sense and respond to DSBs. The DNA DSB response is facilitated by hierarchical signaling networks that orchestrate chromatin structural changes, cell-cycle checkpoints and multiple enzymatic activities to repair the broken DNA ends. Sensors and transducers signal to numerous downstream cellular effectors which function primarily by substrate posttranslational modifications including phosphorylation, acetylation, methylation and ubiquitylation. In particular, the past several years have provided important insight into the role of chromatin remodeling and histones-specific modifications to control DNA damage detection, signaling and repair. This review summarizes recently identified factors that influence this complex process and the repair of DNA DSBs in eukaryotic cells.Nucleic Acids Research 02/2009; 37(5):1363-77. · 8.81 Impact Factor
- Experimental Cell Research 01/1959; 15(3):529-50. · 3.56 Impact Factor
Use of a microscope stage-mounted Nickel-63
microirradiator for real-time observation of the
DNA double-strand break response
Zhen Cao1,2, Wendy W. Kuhne1,3, Jennifer Steeb4, Mark A. Merkley1, Yunfeng Zhou2,
Jiri Janata4and William S. Dynan1,*
1Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA,
2Department of Chemotherapy and Radiation Oncology, Zhongnan Hospital, Wuhan University, Wuhan, Hubei
430071, PRC,3Savannah River National Laboratory, Savannah River Site, Aiken SC 29808 and4Department of
Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, GA 30332, USA
Received March 19, 2010; Revised April 29, 2010; Accepted May 1, 2010
nucleoplasmic repair foci within seconds after the
onset of exposure to ionizing radiation. Real-time
imaging of this assembly has the potential to
further our understanding of the effects of medical
and environmental radiation exposure. Here, we
describe a microirradiation system for targeted
delivery of ionizing radiation to individual cells
without the need for specialized facilities. The
electroplated Nickel-63 electrode, enveloped in a
glass capillary and mounted in a micromanipulator.
Because of the low energy of the beta radiation and
the minute total amount of isotope present on the
tip, the device can be safely handled with minimum
precautions. We demonstrate the use of this system
for tracking assembly of individual repair foci in real
time in live U2OS human osteosarcoma cells.
Results indicate that there is a subset of foci that
appear and disappear rapidly, before a plateau level
is reached ?30min post-exposure. This subset of
foci would not have been evident without real-time
observation. The development of a microirradiation
system that is compatible with a standard biomed-
ical laboratory expands the potential for real-time
investigation of the biological effects of ionizing
Ionizing radiation affects living tissue in a unique way, by
depositing energy along discrete, nanometer-scale tracks.
When a track intersects DNA, damage can occur on both
DNA strands simultaneously, leading to an outright
chromosome break. Even one such break can cause
chronic genetic instability or cancer-associated gene re-
arrangements. It is thus not surprising that ionizing radi-
ation evokes complex biological defense and repair
mechanisms. A central aspect of the radiation response
is the self-assembly of nucleoplasmic repair foci, which is
characterized by specific histone modifications, accumula-
tion of DNA damage sensing and signal transduction
proteins, and assembly of the DNA repair machine
proper from pre-existing components. Repair foci begin
to appear shortly after irradiation and resolve over the
course of several hours (1,2)
Conventional methods for evoking this radiation
response require placing cells or tissues in the proximity
of a radiation source. These ‘off-line’ methods require
physical transfer of samples from the irradiator to a
microscope stage for observation, which precludes obser-
vation of early-stage assembly of repair foci in real time.
In addition, one of the most common methods for labora-
tory irradiation requires a high-activity137CsCl2source.
These sources face increasing restrictions because they are
perceived as threat to public health and safety in the event
of an accident or attack (3). An inexpensive replacement
technology using smaller amounts of isotope would
address this concern.
One approach to address the shortcoming of conven-
tional irradiation methods is to position a small radioiso-
tope source in proximity to the sample on a microscope
stage. One of the first examples of this approach was the
use of a polonium-coated tungsten microneedle to deliver
a particles to living cells (4). Recently, Steeb et al. (5)
described an updated version of the microirradiator
conceptbased onthe deposition
electroplating on a microelectrode enveloped by a glass
*To whom correspondence should be addressed. Tel: 706-721-1370; Fax: 706-721-8752; Email: email@example.com
Published online 19 May 2010Nucleic Acids Research, 2010, Vol. 38, No. 14e144
? The Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
capillary. Importantly, the diameter of the microelectrode
is on the same order as the size of a mammalian cell.
Concentrated deposition of isotope within this small
area allows for a high local radiation flux (106–109Bq/
cm2)) using subnanogram
addition, the electroplated surface can be recessed within
the capillary, which allows the glass walls to act as a col-
limator for the beam (5). The concept was implemented
using63Ni, a long-lived, low-energy b particle emitter. The
maximum range of the emissions is only ?60mm in water
or tissue, which allows the user to handle the device
without special radiological precautions.
Here, we describethe
microirradiator in a biological application. We mounted
the device in a micromanipulator on the stage of a decon-
volution microscope and used it to irradiate cultured
human cells. Cells were transfected with expression con-
structs for fluorescently-tagged 53BP1, a widely used
[reviewed in (6,7)]. We collected real-time image data,
which enabled quantitative characterization of the appear-
ance, disappearance and motion of these foci. Initial
studies suggest heterogeneous rates of formation and reso-
lution, which could not have been observed using a con-
ventional off-line radiation source.
amountsof isotope. In
break repair foci
MATERIALS AND METHODS
The microirradiator was fabricated as described (5) with
modifications as indicated below.63Ni is a low-energy b
particle emitter (maximum energy 67keV, average energy
17keV) with a 100-year half life. The maximum range of
the b particle emission in water is 60mm (average range,
30mm). A 25-mm Pt wire was threaded through a 0.5-cm
bore diameter borosilicate glass capillary, which was
flame-sealed and pulled in a glass electrode pulling appar-
atus at 800?C with a pull length of 3cm. The end of the
pulled Pt wire in glass was polished to produce a smooth
disk. After cleaning, the electrode was electrochemically
plated using a 50mCi63NiCl2 (1.6 ? 1013Bq/g) source
(NRD LLC, Grand Island, NY, USA). This source was
transformed into a pseudo Watts bath with the addition of
NiSO4and H3BO4for optimum Ni deposition. The Pt
surface was deposited and monitored by constant poten-
tial (?0.75V) until ample charge was passed (10?4C) to
past the capillary. After63Ni deposition, a micron layer
of Poly(3,4-ethylenedioxythiophene) (PEDOT) was de-
posited to ensure Ni did not interact with the cellular
medium. A 0.1-mM solution of PEDOT was dissolved
in 1mM tetraethylammonium perchlorate in acetonitrile
for 20s at 0.25V. Total activity of the microirradiator was
2000Bq, as measured by liquid scintillation counting. The
microirradiator was rinsed after each use and stored in a
63Ni had plated the surface, extending slightly
full-length 53BP1 open reading frame (ORF; locus
accession number BC112161) was purchased from Open
Biosystems (Huntsville, AL, USA). It was digested with
KpnI and XhoI to release the 53BP1 ORF together with
upstream and downstream untranslated regions (UTRs).
Carlsbad, CA, USA) was first modified by insertion of
fragment was inserted between these two sites. To delete
unwanted 50-UTR sequences, a 520ntN-terminal segment
of the 53BP1 ORF was amplified using primers d(GGCG
CTCGAGATGGACCCTACTGGAAGT) and d(GGCG
CATATGGCACAGTATTTTCC), digested with XhoI
and NdeI to create cohesive ends, and substituted for
the corresponding XhoI–NdeI fragment in the natural
cDNA, resulting in the promoterless pENTR/53BP1
vector. To insert an N-terminal fluorescent tag, the
EYFP coding sequence was amplified, together with
flanking Kozak consequence sequence, using primers
GCGAGG) and d(GCGCTCGAGCTTGTACAGCTCG
TCCATGC). The product was cleaved with NotI and
XhoI and inserted between the NotI and XhoI sites of
pENTR/53BP1, upstream of and in frame with the
53BP1 ORF. The resulting construct was transferred
using phage lambda recombinase into pcDNA-DEST40
sequence of the expression construct is provided in
Cells and electroporation conditions
U2OS 2-6-3 cells (8) were maintained in high glucose
DMEM supplemented with GlutaMAX-1 (Invitrogen),
10% fetal bovine serum, 100U/ml penicillin, 100mg/ml
Electroporation was performed using 3mg of plasmid
DNA and 40mg of sheared salmon sperm DNA carrier
(Ambion, Austin, TX, USA) using a Gene Pulser II
(BioRad, Hercules, CA, USA). Electroporation was per-
formed in a 0.4-cm cuvette containing 120ml of culture
medium, with settings of 170V and 975mF, resulting in
a 60–90ms time constant. Following electroporation, cells
were seeded into glass bottom culture dishes (MatTek,
Ashland, MA, USA) and incubated at 37?C to allow
protein expression. Irradiation and imaging were per-
formed 24–48h post-electroporation.
Conventional c irradiation and cell imaging
EYFP-53BP1 and H2B-diHcRed, a nuclear marker (9).
irradiator (Gammacell40 Exactor, MDS Nordion,
Ottawa, ON, Canada) at a dose rate of 0.84Gy/min,
then transferred to the WeatherStation environmental
chamber of an Applied Precision Deltavision microscope,
where incubation was continued at 37?C in a humidified,
5% CO2atmosphere. Live cell images were collected using
a 60X Plan Achro oil objective beginning 25–30min
post-irradiation. Filter sets were as follows: EYFP,
500nm excitation/535nm emission; diHcRed, 572nm
e144 Nucleic Acids Research, 2010,Vol.38, No. 14PAGE 2 OF 8
excitation/632nm emission. Z stacks of 15–24 images were
collected using a 0.4-mm step size and deconvoluted using
softWoRx software. Projection images were prepared and
nuclear foci were scored manually. Analysis was restricted
to cells that were positive for both EYFP and the diHcRed
nuclear marker (>80% of the population). To determine
the dose response, regression analysis was performed
using SigmaPlot v11.0 software (Systat Software, San
Jose, CA, USA).
63Ni microirradiator b-irradiation
WeatherStation environmental chamber of the Applied
Precision Deltavision microscope to facilitate precise pos-
itioning of the microirradiator tip. The microirradiator
probe was secured in an acrylic electrode holder. Cells
were electroporated, incubated 24–48h, transferred into
the environmental chamber, and the microirradiator was
positioned directly over a target cell, as determined by
observation through the Deltavision optics. Time-lapse
exposures were performed, with 15–24 Z-stack sections
per timepoint, 0.4-mm section thickness, 0.5s exposure
time and auto-focusing in the EYFP channel. Images
were deconvoluted, projection images were prepared and
foci were scored manually. Foci present prior to irradi-
ation were excluded.
To track and quantify individual foci, a region of
interest was defined within
exported in TIF format, and loaded into DeCyder 6.5
Automated spot detection was performed, foci (‘spots’)
were matched across the time-lapse images, and spot
volumes were determined and plotted as a function of
Microscope stage-mounted irradiation system
The microirradiator was made by electrochemical depos-
ition of an ?1-mm thick layer of63Ni on a 25mm-diameter
microelectrode wire enveloped in a glass capillary (5). The
configuration of the device used here was similar to the
‘recessed disk’ design reported earlier (5), except that the
device was prepared without etching the tip of the
microelectrode, allowing63Ni to be plated directly onto
a flush, polished Pt surface. This design change eliminates
concern over attenuation of activity due to trapping of
medium or debris in the cavity of the ‘recessed disk’ con-
figuration. The isotopic enrichment of the63Ni was higher,
resulting in a more highly active probe surface. In
addition, a polymer PEDOT coating was applied to
prevent direct contact between the Ni surface and the
WeatherStation environmental chamber of an Applied
Precision Deltavision microscope (Figure 1A), using a pre-
cision micromanipulator with an acrylic electrode holder
(Figure 1B). The micromanipulator allows precise manual
adjustment of the position along three orthogonal axes.
A bend was introduced in the capillary such that the ca-
pillary tip is coaxial with the optical path (Figure 1C). A
dissecting microscope view showing the flush tip is shown
in Figure 1D. The microirradiator was positioned so that
the tip of the capillary was slightly above the target cell
and was visible through the microscope optics (Figure 1E).
The plane of the active surface is parallel to the plane of
the culture dish.
Calibration of biological reporter system
The EYFP-53BP1 expression construct was as described
in ‘Materials and Methods’ section (Figure 2A) and
consists of an intrinsically fluorescent enhanced yellow
fluorescent protein domain joined to full-length 53BP1.
It is essentially identical to constructs used in previous
live-cell imaging studies [e.g. (10–12)]. The U2OS 2-6-3
recipient cells (8) are derived from the U2OS osteosar-
coma cell line, which has been widely used in prior
studies of 53BP1 foci formation (11,13).
To calibrate the number of foci per unit dose of radi-
ation in this system, cells were exposed to 0–3Gy of137Cs
radiation and imaged 25–30min post-exposure, at which
time the response has reached a maximum. H2B-diHcRed
(9) was co-expressed as a registration marker (14). Live
cell images were collected (Figure 2B) and nuclear foci
were scored manually using 8–24 nuclei for each dose
point (Figure 2C). Regression analysis indicated forma-
tion of 27.4 foci per nucleus per Gy. The slope of the
53BP1 dose response curves is 1.4-fold greater than
reported in a prior live-cell imaging study (12). This is
consistent with the fact that the hypertriploid U2OS
cells contain about 1.5-fold more DNA per nucleus than
the quasi-diploid HT1080 cells in the prior study and thus
present a larger target for radiation damage.
Real-time observation of microirradiator-induced foci
Results of several microirradiator experiments are shown
in Figure 3. Cells were electroporated with EYFP-53BP1
and H2B-diHcRed expression constructs. After 24–48h,
they were transferred to the stage of the Deltavision
microscope, where they were maintained in growth
medium in a humidified 5% CO2atmosphere at 37?C.
The microirradiator was positioned above a target cell
(Figure 3A). Z-stack images were collected at 5min inter-
vals. The irradiator was withdrawn after the 16min
timepoint and cells were allowed to recover for a further
46min. Images were deconvoluted, projections were
prepared and foci were counted manually. Results for
three individual cells are presented in Figure 3. In all
three instances, foci appear with no discernible lag and
increase to a maximum ?35min after the probe was
withdrawn and 50min after the start of the experiment.
The absolute number of foci varied between experiments,
perhaps reflecting slight differences in the position of the
probe. In order to pool the data from the three experi-
ments, we normalized the data to the percent of maximum
response in each experiment. We calculated the mean and
SD to obtain the data in the rightmost panel of Figure 3B.
PAGE 3 OF 8Nucleic Acids Research, 2010,Vol.38, No. 14 e144
Figure 1. Microscope stage-mounted microirradiation system. (A) Gross view of microirradiation system. Device is mounted on a micromanipulator
housed within a heated, humidified, environmental chamber of the Applied Precision Deltavision microscope. (B) Close-up view showing attachment
of device to the micromanipulator. (C) Detail showing the bend in enclosing capillary, which allows positioning of the active surface directly above
the target cell. (D) View of the microirradiator tip in dissecting microscope. (E) Close-up view through Deltavision microscope optics. Cell has been
transfected with EYFP-53BP1 and H2B-diHcRed expression plasmids as described in ‘Materials and Methods’ section. Scale bar, 10 microns.
Figure 2. Calibration EYFP-53BP1 reporter system. (A) EYFP-53BP1 primary structures. EYFP coding sequence is joined to full-length 53BP1,
with Tudor and BRCT domains indicated. EYFP1-53BP coding sequence was inserted into pcDNA-DEST40 (Invitrogen) for expression under
control of the CMV promoter. (B) Dose response to calibrated doses of137Cs reference radiation. Cells were co-transfected with EYFP-53BP1 WT
and H2B-diHcRed. Live-cell images were collected 30min post-irradiation. (C) Quantification of dose response. Foci were scored using 8–24
individual nuclei at each dose point, and linear regression was performed to obtain the slope of the dose-response curve.
e144 Nucleic Acids Research, 2010,Vol.38, No. 14PAGE 4 OF 8
The result is a smooth curve showing an increase with
approximately linear kinetics until a plateau is reached.
No foci were seen in control cells imaged under the
(Supplementary Figure S1)
To estimate the radiation dose based on foci numbers to
radiation dose, we compared the maximum number of foci
attained in each experiment in Figure 3 to the calibration
curves in Figure 2. We did not make an adjustment for the
different radiation types (high energy g rays versus b par-
ticles) as they may be considered as radiobiologically
equivalent. We also did not adjust for dose rate. Because
of this, the number of foci in Figure 3 may be slightly
underestimated (i.e. a lower dose rate affords more time
for the earliest foci to be resolved before the maximum is
attained). Subject to these caveats, comparison with the
standard curve indicates that the apparent dose delivered
by the microirradiator in a 16min exposure was 2.8, 2.0
and 3.6Gy for cells 1, 2 and 3, respectively. The variability
between cells could be due to differences in the intrinsic
sensitivity of different cells (e.g. position in the cell cycle
or, perhaps, to small differences in the positioning of the
probes. These doses equate to dose rates range of 0.12–
0.22Gy/min. This is about an order of magnitude less
than predicted by modeling a source with this activity
and geometry (‘Discussion’ section), and the reason for
the difference is still to be determined.
Although an effort was made to minimze the light
exposure during imaging, we were aware of the possibility
that phototoxicity might itself result in repair foci forma-
tion. We therefore performed a mock irradiation under
the same conditions, but without the microirradiator.
No time-dependent accumulation of foci was seen above
the background present at the beginning of the experiment
(Supplementary Figure S1).
Dynamic behavior of individual foci
The time course in Figure 3B shows that the time between
initial traversal of a radiation track and appearance of foci
is variable, as some foci appeared immediately after the
onset of irradiation, whereas others did not appear until
30min after microirradiator was removed. It also follows
from inspection of the shape of the curve that the time
required for resolution of foci must also be variable. If the
foci appeared at variable times post-exposure, but all were
long-lived (i.e. persistent throughout the experiment), then
a plot of foci versus time should be strongly sigmoidal.
This was not the case. Instead, the system rapidly ap-
proached a steady state, showing a quasilinear response
over the first 50min. To account for this steady state,
some of the foci that appear rapidly must also disappear
To test this prediction, we tracked the behavior of indi-
vidual foci over time. Figure 4A shows the same nuclei as
in Figure 3A, but with expansion of a region of interest.
Eighteen foci (‘spots’) were matched manually across the
time series and intensities were quantified as described in
Supplementary Figure S2). Figure 4B shows plots of
spot volumes as a function of time. Several patterns are
seen in these data. Foci 7, 9 and 11 appear and disappear
rapidly, losing at least half their peak intensity within
10min. Foci 1, 2, 5, 10, 12 and 15 decay with intermediate
kinetics, losing half their peak intensity within 15–30min.
Figure 3. Real-time imaging of microirradiator-induced 53BP1 foci. (A) Representative images of same cell collected at indicated time points.
Microirradiator was introduced 1min prior to the first time point and withdrawn after the 16min. Z-stacks containing 15–24 individual images
were collected at each time point and deconvoluted. Projections are shown. PO, probe on; PF, probe off. (B) Quantification of
microirradiator-induced foci from three independent experiments. Cell 1 corresponds to images in Panel A. Rightmost panel shows pooled data
from the three experiments. Data were normalized to maximum response= 100%, averaged, and plotted. Error bars denote SD.
PAGE 5 OF 8Nucleic Acids Research, 2010,Vol.38, No. 14 e144
Foci 3, 6, 14, 16, 17 and 18 were stable for the duration of
the experiment. Foci 4, 8 and 13 showed somewhat irregu-
Clearly, it will be of interest to collect longer and
finer-grained time series, to analyze larger numbers of
foci, and to perform a more computationally intensive
analysis using the original 3D imaging data, rather than
the 2D projections that were analyzed here. Even this
limited analysis, however, suggests that foci fall into
several classes, with some appearing and disappearing
rapidly and others that are stable over the period of
We describe here the first biological application of a novel
microirradiator that is designed specifically for compati-
bility with a standard cell biology laboratory environment.
The device is characterized by small physical dimensions,
radiation flux sufficient for many types of biological ex-
periments and minimal radiological hazards. We demon-
strate the use of the device in experiments to track and
characterize the induction of repair foci in irradiated cells.
The microirradiator is a potential replacement for a
self-contained137CsCl2irradiator in certain cell and mo-
lecular biology research applications, particularly where
biological response is measured at the single cell level.
The device is made from inexpensive, commercially avail-
able materials, and the low total activity mitigates the
public health and security concerns that have been
raised for high-activity37CsCl2radiation sources (3).
Because the microirradiator can be mounted directly to
a microscope stage, it provides a capability for real-time
observation of the early stages of the radiation response.
This early response has not been well studied, particularly
for sparsely radiation such as g rays and X-rays.
Preliminary analysis has
behavior of repair foci. In particular, there is a class of
rapidly resolved foci that would not be apparent in experi-
ments using conventional irradiation methods. The exist-
ence of a rapidly resolved subset of foci is significant
because it may account for a small, but unexplained, dis-
crepancy between the best estimate of the number of
double strand breaks determined by physical methods
(?30 DSBs per Gy per diploid genome) (15,16) and the
number of breaks estimated by careful counting of
ionizing radiation-induced foci (?10–25 foci per Gy per
diploid genome) (6,12) reviewed in (1). Several explan-
ations for the heterogeneous behavior of repair foci may
be contemplated, including heterogeneity in the structure
of the radiation-induced DNA breaks and the existence of
competing repair or signaling pathways. Potentially, these
knockdown of individual DNA processing and repair
enzymes in the target cells.
The present dose rate, estimated to be in the range of
0.12–0.22Gy/min, is sufficient for many experiments to
investigatethe biology of
response. A higher dose rate would be useful for experi-
ments that require synchronous induction of large
numbers of DSBs. The thickness of the nickel layer is
sufficient that self-absorption is a limiting factor in
controlling surface flux density, so that deposition of
more radioisotope would not result in a corresponding
increase in dose rate. The specific activity of the
nickel-63 used here is the highest that is commercially
available. However, the activity as stated by the vendor
equates to <10% isotopic enrichment, so improvement is
We have attempted to compare the dose rate estimated
from the biological response to the predicted dose rate
based on probe activity (as determined by liquid scintilla-
tion counting) and modeling as described in (5) (refer to
Supplementary Data for details). A discrepancy between
this predicted dose rate (2.65Gy/min) and the estimated
dose rate based on biological response suggests either that
there is a flaw in the modeling or that the b emissions are
attenuated in some unanticipated way. Further insight
may be provided by experiments currently underway to
characterize b particle flux and emission spectrum using
solid-state scintillation counting (J.S. and J.J., unpub-
It is useful to compare the microirradiator to two other
approaches that are currently in use to introduce targeted
DNA damage into samples on a microscope stage. One of
these is the particle microbeam. More than 50 years ago,
Zirkle and Bloom (17) described the use of a Van de Graaf
generator and microaperture to deliver an intense proton
beam to a 2.5-mm diameter spot within living cells. It is
now estimated that there are 30 operational microbeam
facilities worldwide (18). Microbeam facilities have been
developed that are capable of delivering precisely targeted
heavy particles, electrons or ultrasoft X-ray photons, with
varying degrees of real-time imaging capability [e.g. (19–
23)]. As currently configured, the microirradiator lacks the
capabilities for precision targeting and particle counting
that are found at the most advanced microbeam facilities.
The portability of the microirradiator and its potentially
low cost of manufacture provide offsetting advantages,
however, for many applications. Localized ultraviolet ir-
radiation (or, equivalently, 2-photon absorption in the
near infrared range) provides another approach that is
usefulin analysisof the
[reviewed in (24), see also (25)]. The value of the
approach is its ability to induce dense, near-instantaneous
damage in a precisely localized region. This is also,
however, its main limitation, as the dense DNA damage
has no clear physiological equivalent, whereas the
low-energy b particles from the nickel-63 microirradiator
are expected to produce damage that is similar in type and
distribution to that from common medical and environ-
mental exposure sources. Thus, the characteristics of the
Ni-63 microirradiator make it a useful complement to
other technologies that are currently in use.
In principle, it should be possible to adapt the same
microirradiators using other radioisotopes. For example,
252Cf could be used as a source of fission neutrons or
241Am as a source of a particles. Both of these isotopes
are used industrially and are potentially available for con-
struction of research microirradiators. The cellular and
e144Nucleic Acids Research, 2010,Vol.38, No. 14PAGE 6 OF 8
Figure 4. Tracking of individual 53BP1 foci. (A) Images are from nuclei from Cell 1 in Figure 3. Inset panels show enlargement of region of interest
marked by white rectangle. Eighteen individual foci were identified manually and are marked. (B) Quantification of image intensity for individual
foci. The 2D image projections were loaded in DeCyder v6.5 (GE Healthcare, Buckinghamshire, UK) and automated spot detection was performed
(Supplementary Figure S2). Volumes corresponding to integrated signals for each spot were quantified. Plots show image intensity for individual foci
as a function of time. The same arbitrary units are used in all panels. Time is indicated in min.
PAGE 7 OF 8 Nucleic Acids Research, 2010,Vol.38, No. 14e144
molecular response to high LET radiation, such as
neutrons and a particles, has not been as extensively
studied as low LET radiation, in part because convenient
sources are not available outside the radiation community.
Concentrated deposition of isotope on a very small surface
in the microirradiator allows experiments to be performed
using very small total amount of isotope, mitigating health
and safety concerns that pose barriers to access to nuclear
Supplementary Data are available at NAR Online.
We thank Dr David Spector and Dr Ileng Kumaran (Cold
Spring Harbor Laboratory, Cold Spring Harbor NY) for
the gift of U2OS-2-6-3 cells, the members of the
Nanomedicine Center for Nucleoprotein Machines for
helpful discussions, Dr Rhea-Beth Markowitz for scientif-
ic editorial support, and the Medical College of Georgia
Imaging Core Facility for their services.
National Institutes of Health Roadmap for Biomedical
Research (US Public Health Service Award EY018244);
US Department of Energy Low Dose Radiation Research
Program (DE-SC0002343); National Institutes of Health
National Research Service Award (ES015663 to W.K.);
Georgia Research Alliance (GRA) Eminent Scholar
Challenge Grant (to W.S.D. and J.J.). Funding for open
access charge: US Public Health Service.
Conflict of interest statement. None declared.
1. Kinner,A., Wu,W., Staudt,C. and Iliakis,G. (2008) Gamma-H2AX
in recognition and signaling of DNA double-strand breaks in the
context of chromatin. Nucleic Acids Res., 36, 5678–5694.
2. Pandita,T.K. and Richardson,C. (2009) Chromatin remodeling
finds its place in the DNA double-strand break response.
Nucleic Acids Res., 37, 1363–1377.
3. Radiation Source Use and Replacement: Abbreviated Version
(2008). National Academies Press, Washington, D.C.
4. Munro,T.R. (1958) Alpha irradiation of parts of single cells in
tissue cultures. Exp. Cell. Res., 15, 529–550.
5. Steeb,J., Josowicz,M. and Janata,J. (2009) Nickel-63
Microirradiators. Anal. Chem., 81, 1976–1981.
6. Costes,S.V., Chiolo,I., Pluth,J.M., Barcellos-Hoff,M.H. and
Jakob,B. (2010) Spatiotemporal characterization of ionizing
radiation induced DNA damage foci and their relation to
chromatin organization. Mutat. Res., [8 January 2010, Epub
ahead of print].
7. FitzGerald,J.E., Grenon,M. and Lowndes,N.F. (2009) 53BP1:
function and mechanisms of focal recruitment. Biochem. Soc.
Trans., 37, 897–904.
8. Kumaran,R.I. and Spector,D.L. (2008) A genetic locus targeted
to the nuclear periphery in living cells maintains its
transcriptional competence. J. Cell. Biol., 180, 51–65.
9. Gerlich,D., Beaudouin,J., Kalbfuss,B., Daigle,N., Eils,R. and
Ellenberg,J. (2003) Global chromosome positions are transmitted
through mitosis in mammalian cells. Cell, 112, 751–764.
10. Pryde,F., Khalili,S., Robertson,K., Selfridge,J., Ritchie,A.M.,
Melton,D.W., Jullien,D. and Adachi,Y. (2005) 53BP1 exchanges
slowly at the sites of DNA damage and appears to require RNA
for its association with chromatin. J. Cell. Sci., 118, 2043–2055.
11. Jakob,B., Splinter,J., Durante,M. and Taucher-Scholz,G. (2009)
Live cell microscopy analysis of radiation-induced DNA
double-strand break motion. Proc. Natl. Acad. Sci. USA, 106,
12. Asaithamby,A. and Chen,D.J. (2009) Cellular responses to DNA
double-strand breaks after low-dose gamma-irradiation.
Nucleic Acids Res., 37, 3912–3923.
13. Schultz,L.B., Chehab,N.H., Malikzay,A. and Halazonetis,T.D.
(2000) p53 binding protein 1 (53BP1) is an early participant in
the cellular response to DNA double-strand breaks. J. Cell. Biol.,
14. Rabut,G. and Ellenberg,J. (2004) Automatic real-time
three-dimensional cell tracking by fluorescence microscopy.
J. Microsc., 216, 131–137.
15. Ruiz de Almodovar,J.M., Steel,G.G., Whitaker,S.J. and
McMillan,T.J. (1994) A comparison of methods for calculating
DNA double-strand break induction frequency in mammalian
cells by pulsed-field gel electrophoresis. Int. J. Radiat. Biol., 65,
16. Iliakis,G.E., Cicilioni,O. and Metzger,L. (1991) Measurement of
DNA double-strand breaks in CHO cells at various stages of the
cell cycle using pulsed field gel electrophoresis: calibration by
means of 125I decay. Int. J. Radiat. Biol., 59, 343–357.
17. Zirkle,R.E. and Bloom,W. (1953) Irradiation of parts of
individual cells. Science, 117, 487–493.
18. Gerardi,S. (2009) Ionizing radiation microbeam facilities for
radiobiological studies in Europe. J. Radiat. Res., 50(Suppl. A),
19. Randers-Pehrson,G., Geard,C.R., Johnson,G., Elliston,C.D. and
Brenner,D.J. (2001) The Columbia University single-ion
microbeam. Radiat. Res., 156, 210–214.
20. Gerardi,S. (2006) A comparative review of charged particle
microbeam facilities. Radiat. Prot. Dosimetry., 122, 285–291.
21. Folkard,M., Schettino,G., Vojnovic,B., Gilchrist,S.,
Michette,A.G., Pfauntsch,S.J., Prise,K.M. and Michael,B.D.
(2001) A focused ultrasoft x-ray microbeam for targeting cells
individually with submicrometer accuracy. Radiat. Res., 156,
22. Kim,E.H., Sun,G.M. and Jang,M. (2006) An electron microbeam
cell-irradiation system at KIRAMS: performance and preliminary
experiments. Radiat. Prot. Dosimetry, 122, 297–300.
23. Sowa,M.B., Murphy,M.K., Miller,J.H., McDonald,J.C.,
Strom,D.J. and Kimmel,G.A. (2005) A variable-energy electron
microbeam: a unique modality for targeted low-LET radiation.
Radiat. Res., 164, 695–700.
24. Kong,X., Mohanty,S.K., Stephens,J., Heale,J.T., Gomez-
Godinez,V., Shi,L.Z., Kim,J.S., Yokomori,K. and Berns,M.W.
(2009) Comparative analysis of different laser systems to study
cellular responses to DNA damage in mammalian cells.
Nucleic Acids Res., 37, e68.
25. Suzuki,K., Yamauchi,M., Oka,Y., Suzuki,M. and Yamashita,S.
(2010) A novel and simple micro-irradiation technique for
creating localized DNA double-strand breaks. Nucleic Acids Res.,
[12 April 2010, Epub ahead of print].
e144Nucleic Acids Research, 2010,Vol.38, No. 14PAGE 8 OF 8