Our current single-particle
microbeam system features an extensively upgraded spatial resolution
of 0.6 μm (diameter) for the beam. Current technology developments
focus on adding and improving imaging techniques, upgrading the
capabilities of our existing system, and on developing new
functionalities for the future.
Among these new developments are a
novel neutron microbeam, the ability
to irradiate non-adhering cells via
flow and shoot technology
and optoelectronic tweezers,
and the additional imaging capabilities of our
EMCCD
camera and
simultaneous immersion Mirau interferometery.
Neutron Microbeam
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Schematic
of the neutron microbeam. The proton microbeam is incident on a
lithium target, and neutrons are produced with a highly
forward-peaked angular distribution. |
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A significant number of
individuals are occupationally exposed to low doses of neutrons,
mostly low-energy neutrons. These low-energy neutrons produce
biological damage in a fundamentally different way from most photon
or high-energy charged particle irradiations. Both the x rays and
the high-energy charged particles that our users study damage DNA
primarily through atomic ionization, i.e. production of electron
vacancies in DNA, directly or via free radicals. By contrast, the
primary DNA damage mechanism for low-energy neutrons is via direct
knockout of protons in the DNA. In order to better understand
possible damage response mechanisms such as the bystander effect
from neutrons on biological systems, we are developing a neutron
microbeam.
Our neutron microbeam design is
based on the existing charged particle microbeam technology at RARAF.
The principle of the neutron microbeam is to use the proton beam
with a micrometer-sized diameter impinging on a very thin lithium
fluoride target system. From the kinematics of the 7Li(p,n)7Be
reaction near the threshold of 1.881 MeV, the neutron beam is
confined within a narrow, forward solid angle. Calculations show
that the neutron spot using a target with a 17 µm thick gold backing
foil will be less than 20 µm in diameter for cells attached to a 3.8
µm thick propylene-bottomed cell dish in contact with the target
backing. The neutron flux will roughly be 2000 per second based on
the current beam setup at RARAF Singleton accelerator. The dose rate
will be about 200 mGy/min. By reducing the target thickness to the
minimum necessary, the production of resonance gamma rays in the
thin target will be limited. The principle of this neutron microbeam
system has been preliminarily tested at RARAF using a collimated
proton beam.
Predicted neutron yields,
microbeam diameters, neutron energies, and dose rates for
different proton energies.
Selected proton energy and characteristics of the predicted
neutron microbeam are highlighted. |
Proton Energy
(MeV) |
Neutron Yield
(per nC) |
Maximum Neutron
Angle (Deg.) |
Neutron Beam
Diameter (μm) |
Mean Neutron
Energy (keV) |
Dose Rate
(cGy/min) |
|
1.882 |
270 |
9 |
8.1 |
30.0 |
8 |
|
1.883 |
800 |
13 |
10.5 |
30.2 |
14 |
|
1.884 |
1440 |
16 |
12.5 |
30.5 |
18 |
|
1.885 |
2230 |
19 |
14.7 |
30.7 |
20 |
|
1.886 |
3170 |
21 |
16.2 |
31.0 |
23 |
FAST (Flow and Shoot
Technology)
At
present RARAF microbeams irradiate cells adhered to a thin membrane.
The cells are individually targeted either by being mechanically
moved to the location of the microbeam using a precision mechanical
stage, or by deflecting the beam slightly using the Point and Shoot
system. These methods only permit irradiation of cells that can be
made to adhere to the membrane and preclude the irradiation of
hematopoietic cells, which by nature do not adhere to any surface.
To expand our throughput
capabilities we are building a novel microbeam using Flow-And-Shoot
Technology (FAST). In this system, cells will undergo controlled
flow through a microfluidic channel intersecting the microbeam path.
They will be imaged and tracked in real time, using a high-speed
camera and targeted for irradiation, using the existing
Point-and-Shoot system.
We have manufactured several
polydimethylsiloxane (PDMS) microfluidic chips (shown right), using
soft lithography techniques. The cross-section of the channel has a
width of 200 μm and height of 20 μm, so that the cells, when
targeted by the microbeam, flow in the immediate vicinity of the
microbeam exit window. The flow rate is controlled by a syringe
pump. The open-bottomed chips are sealed to the microbeam exit
window by vacuum applied in a second channel surrounding the cell
flow channel.
SRIM simulations of this geometry
indicate a beam broadening of <1 μm at the top of the channel, due
to scattering, for a 5 μm diameter, 5 MeV He++ beam and negligible
broadening for a 4 MeV proton beam.
Development of online cell
tracking and targeting software is underway.
Optical Manipulation of Cells
On the other hand, Optoelectronic
Tweezers (OET), initially developed by
our collaborators at Berkeley, have been proven to be a powerful
tool for parallel manipulation of multiple cell-sized objects. An
OET system, integrated into the microbeam endstation, will allow
real-time manipulation of single cells before, during and after
irradiation, of specific interest to many of our users. This design
will allow for a new class of bystander experiments, where cells are
irradiated and then brought into contact with other (irradiated or
non-irradiated) cells for controlled time periods and then sorted
out into subgroups for separate analysis based on location in the
irradiation dish or based on morphology.
Our goal is to be able to load
cells into a microfluidics channel, place them in prescribed
locations within the Point-and-Shoot field of fire, irradiate
specific cells and then dispense single cells or groups of cells,
into separate vials, keeping track of which cell is which.
To date, we have created a set of
OET electrodes with the guidance of our Berkeley collaborators in
the cleanroom at Columbia University. As a preliminary test, we
manufactured the devices on 1mm thick glass slides. Future work will
focus on reducing the thickness of the bottom substrate to allow
charged particles to easily pass through.
The OET consists of two parallel
plate Indium Tin Oxide (ITO) electrodes. One electrode is covered
with a 1 μm thick layer of hydrogenated amorphous silicon (a-Si:H),
a thin film semiconductor that acts as a photoconductive layer. When
light is focused on the surface of the a-Si:H, its conductivity
increases by several orders of magnitude. By patterning a dynamic
image with a computer and a projector, a reconfigurable virtual
electrode is created. In order to prevent evaporation of the fluid
and to easily control the spacing between the parallel plates, a
rubber gasket was placed between the electrodes, and a plastic clamp
was used to hold the two plates together, as shown above in figure
1. Initial tests to manipulate a 19 μm diameter bead by projecting a
red square pattern outline in the OET device found a maximum
velocity approximately 50 μm/s.
Trials on live cells are commencing.
Electron-Multiplied Charge-Coupled Device (EMCCD)
We have upgraded
our microbeam end station with the addition of an electron-multiplied
charge-coupled device (EMCCD) camera. The EMCCD camera is operating as
a direct replacement of our old ICCD (intensified CCD) camera for
normal microbeam operations and we are exploring a reduction of the UV
illumination of the fluorescent stain (Hoechst 33342) that binds to
the DNA in the cell nuclei and is used for target identification. We
are working with our biological microbeam users in the further
reduction of the amount of nuclear stain used – which can have
deleterious effects on its own. Requiring fewer fluorescence photons
to be generated in the sample for target detection allows the
reduction of the amount of stain and the intensity of the UV
illumination required to perform targeted irradiations.
The EMCCD camera has given us an
opportunity to reduce potentially confounding factors of UV exposure
and stain toxicity in our sensitive experiments, such as bystander
effects or genomic instabilities, where very slight effects can have
huge ramifications on the experimental results.
The EMCCD technology allows the
multiplicative gain of the image pixels directly at the readout of the
CCD sensor that is mounted in a vacuum housing and cooled to -70 C by
a Peltier cooler, reducing the thermal electrical noise in each pixel
to less than 1 electron per read. This allows a single photon to
generate a large enough signal to be read as part of an image, making
extreme low-light imaging possible on a single sensor without a
coupled intensifier. The EMCCD camera technology also allows us to
acquire enough signal from the sample in our present setup to target
our samples in 1 image frame, where previously several frames had to
be aggregated, thus increasing the speed and reliability of the sample
targeting.
Simultaneous Immersion Mirau Interferometric Imaging
Because the endpoints our
collaborators wish to measure after microbeam exposure are
increasingly complex, there is much demand to avoid confounders such
as the potential stress induced by fluorescent staining or the
phototoxicity of UV light. This is a non-trivial task because all
our imaging approaches necessitate epi-illumination, as transmitted
light imaging is not consistent with microbeam geometries.
In order to provide no-stain
imaging we have developed Immersion Mirau Interferometry, which uses
temporal phase shifting. However, the interference patterns needed
to reproduce the image are acquired at different times. If even very
low frequency vibrations exist, they prevent maintaining the
desirable phase shifts between consecutive frames. Therefore we have
devised a method to acquire all interference images at the same
time, a process we call Simultaneous Immersion Mirau Interferometry
(SIMI).
Simultaneous interferometric
imaging is achieved through incorporating polarization optics into
the interferometric attachment. The interferograms are combined with
the background image to reconstruct the intensity map of the
specimen. The figure below demonstrates a result of imaging of 3T3
cells in medium with an experimental SIMI arrangement. Our results
show that this system produces images of a quality that is
sufficient to perform targeted cellular irradiation experiments.
