microbeam training course raraf 2011
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Microbeam Training Course RARAF 2011. MICROBEAM LABORATORY 3 Sample Image Capturing & Analysis RARAF Experiences – Micronuclei and DNA damage foci formation Identifying micronuclei Charles Geard & Brian Ponnaiya. - PowerPoint PPT PresentationTRANSCRIPT
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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
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RARAF Experiences Micronuclei and
DNA damage foci formation
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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 pa
ir
Track Segment
Human fibroblasts AG1522 G1 phase
Microbeam
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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
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Nucleoplasmic bridges & micronuclei in BrdU tagged normal human melanocyte cell pairs [highly motile]
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Nucleoplasmic bridge extension in a labeled melanocyte pair 72 hr post microbeam particles to a
nuclear centroid
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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
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Susan Bailey, CSU
γ-H2AX TRF2 Merge20
0
-par
ticle
st ~
10
min
400
-p
artic
les
t ~ 1
0 m
in
• TRF2 is not recruited to damage sites following exposure to high fluences of -particles
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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
-p
artic
les
t = 3
0 m
in
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Irradiated
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Bystander
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Irradiated Bystander
Keiji Suzuki, Nagasaki UniversityH33342 hit nuclei versus non-hit Cyto-orange tagged cells
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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
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3.2 Gy 1GeV Fe1h 30 min
3.2 Gy 1GeV Fe24 hrs
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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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