Our
multiphoton microscope allows us to use the RARAF microbeam for
in
vivo microbeam studies. Researchers at RARAF are now irradiating
small, living organisms, such as the C. elegans
nematode. With its genome fully characterized in the literature, this
organism offers an ideal model for particle-irradiation effects. The worms
undergo anesthesia prior to microbeam irradiation. Immobilization of the
worms is important for multiphoton microscopy, where image exposure can
range from 0.5 seconds to 2 seconds. The movie shows the pharynx section
of a wild-type C. elegans specimen. Second harmonic
generation (red) and autofluorescence (blue) were used to produce this
movie. Following the anesthesia period and the multiphoton imaging
process, the C. elegans specimen regained normal motion and health.
This is a testament to the non-damaging quality of multiphoton microscopy.
By successfully imaging optical sections within a live, small organism,
the microbeam-integrated multiphoton imaging system at RARAF offers users
an array of imaging modes for 3D samples.
In radiation experiments using wild type C. elegans,
imaging modes that do not require added fluorescent markers are required.
Autofluorescence (AF) and second harmonic generation (SHG) offer two
non-stain imaging modes available with the multiphoton microscope at
RARAF. The movie demonstrates this capacity through a composite image
of the pharynx section of a wild type C. elegans specimen. The
red components were imaged using SHG and the blue portions originate
from AF signal. Light with an incident wavelength of 780 nm is used,
while both SHG and AF signals are gathered simultaneously.
The first RARAF microbeam irradiation studies with
C. elegans were recently published. For microbeam studies, C. elegans is an
ideal organism to study since the diameter of its body is ~ 50 µm and its
full length is ~ 1 mm -- small enough to be compatible with microbeam
studies. Additionally, the adult organism has only 959 somatic cells, and
its anatomy is invariant from one animal to the next.
In particular, we used C. elegans strain SJ4005
hsp-4::gfp(zcls4)V, which has a GFP reporter for the hsp-4 heat-shock
gene. Heat shock proteins (HSPs) are a ubiquitous family of gene products
present in cells under unstressed conditions that are expressed in much higher
concentration in the presence of stress. HSP responses can be
induced by diverse stressing agents including heat, UV irradiation, γ-rays
irradiation and chemicals.
In this study, young adult C. elegans hermaphrodites
were used. Before exposure, worms were individually imaged using an
epifluorescent microscope. For microbeam irradiations,
worms were anesthetized and placed in a customized microbeam dish with
a micro cover-slip for individual exposure. Animals were irradiated
at the tail, in the center of the GFP expression region, with 0, 25,
50, and 75 3-MeV protons (LET 12.5 keV/µm, max. penetration 140 µm).
The microbeam diameter
was 1 µm. (This is indicated by a red dot on the figure below). Control
worms were mock-irradiated, by targeting the microbeam just outside
the worm (~ 200 μm) and using the same set-up time for anesthetic
exposure and concentrations. After exposure, the micro cover-slips were
removed using a micro aspirating pump, and worms were washed with buffer
and re-cultured in standard agar/covered Petri dishes. Based on well
established protein kinetic studies and our preliminary expression time
studies, we selected a twenty four hour time point for GFP expression
evaluation.
Microbeam exposed worms showed a different GFP stress
response in terms of intensity and localization, compared with the control
group. Increase in expression was noted only after delivering 50 and
75 protons. Mock irradiated worms did not exhibit any GFP stress response
after 24 hours (Fig. a). No apparent stress response was detected when
25 protons were delivered (Fig. b). However, when the worms were exposed
to 50 or 75 protons, a strong stress response in the posterior intestine
was observed, between the spermatheca and the microbeam targeted area
(Fig. c-d). A one micrometer diameter proton beam was able to induce
tail region in situ GFP over-expression as well as distal stress
response as far as >150 µm away from the irradiated spot. No stress
response was seen in other regions of the body of exposed worms, and
only basal levels of GFP expression were detected at the pharynx and
spermathecae regions.
Sugimoto previously used wild type and a mutant strain C. elegans as an
in vivo model for microbeam irradiation. However, in those
experiments C.
elegans were irradiated with a collimated ~ 20-50 µm diameter microbeam
of 220 MeV carbon particles (LET of 120 keV/µm). Worms were
exposed to 1500 carbon ions delivered to an extended worm body area. While
these irradiation techniques are useful to study organ or region exposures,
they do not allow the exposure of a restricted number of cells or small
tissue sections in a living organism. Our technique is capable of
delivering, with high accuracy, small numbers of particles at the
sub-cellular and cellular level. Many phenomena under study using
microbeams,
such as the bystander phenomenon, must involve cell-to-cell communication,
extracellular environment, and functional integrity. The capability to
target individual tissue components is quite compelling for such studies.
We show that low doses of protons delivered at
the tail region, where different organs and cell populations are located,
are capable of inducing both local and distal GFP over-expression. This
response is remarkable since the microbeam used is estimated to traverse a
series of spatially co-localized cell structures. How this spatially
restricted stressor is capable of inducing a distal stress response is not
understood. However, it is worth noting that among the organs potentially
irradiated are the intestine and the stomato-intestinal muscle. Cell-cell
communications as well as tissue damage signaling are potential mechanisms
involved in these responses. Belyakov
et al suggested that autocrine/paracrine
mechanisms or juxtacrine signaling are potential bystander pathways to
explain long range bystander phenomena in complex tissues. Future
studies will be focused on the identification of cell targets and signal
mechanisms using different biological endpoints.
Use of this targeted irradiation method provides a
new tool to investigate complex long-range biological responses such as
the bystander effect in living organisms. Future developments include a microfluidics-based worm clamp for microbeam irradiation
without anesthesia as well as microbeam modulation techniques to adjust
the position of the Bragg peak to different locations within the target
organism.
More detail can be found in
A. Bertucci, R.D.J. Pocock, G. Randers-Pehrson, and D.J. Brenner.
Microbeam irradiation of the C. elegans nematode. Int. J. Radiat.
Res. 2009 (in press).
