Microbeam Training Course RARAF 2011 MICROBEAM LABORATORY 3 Sample Image Capturing & Analysis RARAF Experiences – Micronuclei and DNA damage foci formation.

Microbeam Training Course RARAF 2011

MICROBEAM LABORATORY 3Sample Image Capturing & Analysis

• RARAF Experiences – Micronuclei and DNA damage foci formation

• Identifying micronuclei

Charles Geard & Brian Ponnaiya

RARAF Experiences Micronuclei and

DNA damage foci formation

0 1 2 3 4 5 6 7 80

0.2

0.4

0.6

0.8

Alpha particles per nucleus

Micr

onuc

lei p

er ce

ll pai

r

Track Segment

Human fibroblasts AG1522 G1 phase

Microbeam

Normal human keratinocytes: BrdU labeled & FITC immunofluorescence plus PI counterstain

for non-cycling cells, 24 hr after microbeam nuclear irradiation

Micronuclei per cell pair Micronucleus & bridge

Nucleoplasmic bridges & micronuclei in BrdU tagged normal human melanocyte cell pairs [highly motile]

Nucleoplasmic bridge extension in a labeled melanocyte pair 72 hr post microbeam particles to a

nuclear centroid

2

22

6 13

2.5Gy, 24hr

RGB display Pseudocolor

2.5Gy, 24hr

RGB display Pseudocolor

Identification of chromosomes involved in micronuclei by M-FISH

DNA ligase IV -/- cells

Susan Bailey, CSU

γ-H2AX TRF2 Merge

200

-pa

rtic

les

t ~ 1

0 m

in

400

-pa

rtic

les

t ~ 1

0 m

in

• TRF2 is not recruited to damage sites following exposure to high fluences of -particles

Results• Significant co-localization of DNA damage markers

can be seen following localized -particle exposure • Later time points (30m and 60m) show an

increase in the size of regions marked by γH2AX or MDC1

γH2AX MDC1 Merge

400

-pa

rtic

les

t = 3

0 m

in

Irradiated

Bystander

Irradiated Bystander

Keiji Suzuki, Nagasaki UniversityH33342 hit nuclei versus non-hit Cyto-orange tagged cells

Microbeam irradiation of cell nuclei in a circle format for gamma H2A-X expression in hit and bystander AL cells Tomoo Funayama Japan Atomic Energy Research Institute

3.2 Gy 1GeV Fe1h 30 min

3.2 Gy 1GeV Fe24 hrs

Criteria for identifying and scoring of micronuclei

• Morphologically identical but smaller than nuclei (82%)• Round or oval in shape (99%)• Diameter between 1/3rd and 1/16th of main nuclei (91%)• Not linked to main nucleus (73%)• May overlap or touch main nucleus (49%)• Same color as nucleus (85%)• Similar staining intensity as nucleus (83%)

Adapted from: Bonassi, et. al., Environmental and Molecular Mutagenesis 37:31-45 (2001)

A comparison of micronuclei scoring in 25 laboratories in 16 countries

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