Microbeam Training Course RARAF 2011 MICROBEAM LABORATORY 3 Sample Image Capturing & Analysis RARAF...
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Transcript of Microbeam Training Course RARAF 2011 MICROBEAM LABORATORY 3 Sample Image Capturing & Analysis RARAF...
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
=