UV Microspot Irradiator

We have integrated a UV microspot irradiator into our microbeam system. Where traditional UV laser microbeam design involves an elongated laser path of exposure, our system is an improvement over the conventional design because it utilizes multiphoton excitation to produce a micro-volume of effective UV radiation - the defining characteristic behind our term UV microspot. What makes our UV microspot unique is that it is integrated within the Microbeam II charged-particle cell-irradiation platform to provide a cocktail of photon and particle irradiations within one system.

To demonstrate the capabilities of RARAF’s UV microspot irradiator, the Columbia University crown logo was irradiated onto a live single cell nucleus. The cells for this demonstration were HT1080 human Fibro Sarcoma cells with nuclei containing GFP-tagged XRCC1 (repair protein for single-strand DNA damage). Provided by our user David Chen, UT Southwestern, Dallas, Texas, these cells were plated on Petri dishes and kept under physiological conditions during the irradiation and imaging phases. Of the two cell nuclei visible in the 3D multiphoton microscopy image above, the lower of the two cell nuclei was irradiated using a Titanium Sapphire laser tuned to 976 nm, which “acts as” 488 nm and 325 nm in the two- and three-photon modes, respectively. The crown pattern was drawn by precision stage motions to 59 irradiation locations. Each location received 16 mW of laser power for 1 second in an elliptical volume, 0.65 µm radial by 2.8 µm axial full width half maximum (FWHM), corresponding to the point spread function of the laser through our 60X NA 1.0 water-dipping objective. Typical spacing between points was 1 µm. As DNA damage occurred in the cell nucleus, XRCC1 repair protein formed foci at the damage sites. Following irradiation, multiphoton microscope z-stack imaging of the GFP concentration in the nucleus revealed the crown pattern in the irradiated cell nucleus. This 3D image was processed with the deblurring program AutoQuant and the image size is 58.1 µm wide by 55.8 µm high.