Microbeam Irradiation of In Vivo Systems - raraf

Microbeam Irradiation of In Vivo Systems

Antonella Bertucci

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Medaka Fish Embryo

In collaboration with with Bill Dynan,

Medical College of Georgia

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Medaka Fish Embryo

Mid-brain

Stage 28

Eyes

• Well established system

• Optically clear

• Small size (a fertilized egg has a diameter of 1.2 mm)

• Also good model for embryonic development

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Orientation of embryo for

microbeam irradiation

In order to microbeam irradiated

specific organs, the embryo must

be appropriately oriented with

respect to the microbeam

Currently, each embryo is

oriented and aligned by hand

(finger) on the beamline, which

typically takes about 10 minutes

Proton

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An orientation / alignment system for fish embryos,

operated remotely by a trackball

Offset hinge

Trackball

This will increase microbeam

throughput approximately 10 fold

Proton

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Experimental Design

• Irradiate the mid-brain with 4.5

MeV protons (total penetration

of ~280 µm).

• Beam diameter 50 µm.

• 10,000 and 20,000 protons

deliver to the target area

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Results

250

200

150

100

n=23

50

20X water objective

0

0 5000 10000 15000 20000 25000

Number of Particles

• No significant difference with 10,000 protons versus unirradiated

control (p=0.816).

• Significant difference with 20,000 protons versus

unirradiated control (p=0.002) and 10,000 particles

(p=0.003).

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Conclusions

These data demonstrate that the Medaka fish

embryo may prove to be a suitable model for

microbeam in-vivo studies..

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C. Elegans

In collaboration Dr. Oliver Hobert

Howard Hughes Medical Institute,

Columbia University

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C. elegans

‣ body length ∼ 1 mm

‣ diameter ∼ 50 µm

‣ transparent body

‣ short life cycle (3.5 days from single-cell to adult)

‣ short life span (2-3 weeks)

‣ fully sequenced genome

‣ shares cellular and molecular structures and control

pathways with higher organisms

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Initial in vivo microbeam exposure of the

C. Elegans hsp-4::GFP stress response evaluation

Protons

Energy: 3 MeV

Beam diameter: 1 µm

Worms assayed 24 hrs

post irradiation

Bertucci A., et al, 2009

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Generation of a new C. elegans strain for

high precision microbeam targeting

hsp-4::GFP; dat-1::RFP

hsp-4::GFP

dat-1::RFP

hsp-4: heat shock protein expressed in

pharynx, spermathecae and tail

dat-1: a plasma membrane dopamine transporter

expressed in eight dopaminergic neurons

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Cell Targeting of Postdeirid neuron (PDE)

Only a discrete number

of cells are directly

traversed by protons.

Muscles

Intestine

Gonads

Neurons

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Experimental Design

• Four independent experiments

•Worms were imaged immediately before microbeam

irradiation

• 75 protons were delivered to one of the PDE neuron

•Control worms were mock-irradiated, by targeting the

microbeam just outside the worm (~ 200 μm)

•Worms (irradiated and control) were imaged 24 hours after

microbeam irradiation

GFP expression quantification:

QuantWorm

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Results

Control T 0

Control T 24 hs

75 H T 0

75 H T 24 hs

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Results

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Control : 63 worms

75 protons: 45 worms

Data pooled from 4 independent experiments

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Conclusions

• Microbeam irradiation with a 1µm diameter proton

microbeam induce distal stress response in unirradiated

areas.

• Microbeam irradiation of specific neurons is capable

of inducing both local and distal GFP over expression

in the C. elegans posterior intestine.

• These data demonstrate the suitability C. elegans as a

model for microbeam in-vivo studies.

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RARAF has Microbeam irradiation

facilities capable of targeting 3-D

systems which provide new tools to

investigate complex long-range

biological responses in

living organisms.