1. 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

2. RARAF Experiences Micronuclei and DNA damage foci formation

3. Human fibroblasts AG1522 G1 phase Microbeam

4. 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

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

7. Nucleoplasmic bridge extension in a labeled melanocyte pair 72 hr post microbeam particles to a nuclear centroid

9. 2 2 2 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

12. Susan Bailey, CSU γ-H2AXTRF2Merge 200  -particles t ~ 10 min 400  -particles t ~ 10 min TRF2 is not recruited to damage sites following exposure to high fluences of  -particles

13. 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 γH2AXMDC1Merge 400  -particles t = 30 min

14. Irradiated

15. Bystander

16. IrradiatedBystander Keiji Suzuki, Nagasaki University H33342 hit nuclei versus non-hit Cyto-orange tagged cells

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

18. 3.2 Gy 1GeV Fe 1h 30 min 3.2 Gy 1GeV Fe 24 hrs

20. 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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