Use of a microscope stage-mounted Nickel-63 microirradiator for real-time observation of the DNA double-strand break response.
Eukaryotic cells begin to assemble discrete, nucleoplasmic repair foci within seconds after the onset of exposure to ionizing radiation. Real-time imaging of this assembly has the potential to further our understanding of the effects of medical and environmental radiation exposure. Here, we describe a microirradiation system for targeted delivery of ionizing radiation to individual cells without the need for specialized facilities. The system consists of a 25-micron diameter electroplated Nickel-63 electrode, enveloped in a glass capillary and mounted in a micromanipulator. Because of the low energy of the beta radiation and the minute total amount of isotope present on the tip, the device can be safely handled with minimum precautions. We demonstrate the use of this system for tracking assembly of individual repair foci in real time in live U2OS human osteosarcoma cells. Results indicate that there is a subset of foci that appear and disappear rapidly, before a plateau level is reached approximately 30 min post-exposure. This subset of foci would not have been evident without real-time observation. The development of a microirradiation system that is compatible with a standard biomedical laboratory expands the potential for real-time investigation of the biological effects of ionizing radiation.
Cao
Zhen
Z
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA.
Kuhne
Wendy W
WW
Steeb
Jennifer
J
Merkley
Mark A
MA
Zhou
Yunfeng
Y
Janata
Jiri
J
Dynan
William S
WS
D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004566:Electrodes; D005456:Fluorescent Dyes; D006801:Humans; D008164:Luminescent Proteins; D008846:Micromanipulation; D008853:Microscopy / Q000295:instrumentation*; D009532:Nickel; D011839:Radiation, Ionizing; D011868:Radioisotopes
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011868:Radioisotopes; D009532:Nickel