Laser microbeam induced DNA damages and repair

 
Our research is basicaly focused on following questions

1. How DNA is damaged and repaired after irradiation with laser microbeam of different qualities?
2. How DNA double strand break repair machinery is organized in space and time in living cell nuclei?
3. Does DNA double strand breaks are repaired differently in gene rich (centre of nuclei) and gene poor (periphery of nuclei) regions?

Different laser microtools are widely used in medicine and biology for different purposes: diagnosis, therapy and research. But it is also known that intensive laser light may induce photodamage to living cells. Our aim is to study how does a variation of different laser light parameters such as dose, pulse energy and wavelength influence DNA damage induction and repair. For that purpose we build a new setup which comprises of a tunable femtosecond Ti:Sa laser system (Tsunami, Spectra Physics) coupled into an Axiovert 135M microscope (Carl Zeiss) via the epifluorescence illumination path. This setup enables micro-irradiation in a broad part of the spectrum using the fundamental (750-1000 nm), frequency-doubled (370-500 nm) and frequency-tripled (250-330nm) irradiation within the same system. Dose and pulse energies are tuned by changing pulse repetition rate and neutral filters respectively.

Highly focused laser beams allow very high resolution in space (x,y,z) and time and additionally they can be directed to the preselcted sub nuclear locus at user defined points of time, thus allows detection of fast events (~ 1 s) when combining it with live cell imaging and ~3 min when combining with immuno-histochemistry.
Laser microbeam accuracy in X,Y, and Z directions. Red - laser microbeam at 420 nm induced PCD's possibly due to two photon absorption. Green - phosphorylated H2AX as a marker for double strand breaks	Double strand breaks induced by laser microbeam at 420 nm after irradiation with low dose but high pulse repetition rate (2600 pulses per µm). Green - gamma H2AX; Red - Rad51; Blue-cell nucleus

Additionaly UV-A laser microbeam coupled into confocal microscope is used for observation of repair processes in living cells.
	NBS1-GFP accumulation to the laser microbeam induced DNA damage in living cell.