(Supported in part by NIH GMS 40198 to C. Rieder). Over the past several years we have explored the utility of using high energy nanosecond pulses of green (532 nm) light, obtained from our pulsed NdYAG laser, as a tool for selectively destroying or """"""""photoablating"""""""" specific structures within the living cell. We found that under the appropriate conditions (high beam energies with a Gaussian profile-see above TRD project) the system can be used to destroy any structure visible within the living cell by video enhanced DIC LM, without killing the cell!. Over the past year we conducted same cell indirect immunofluorescence and electron microscopic studies to verify that we could sever or destroy, at will, microtubules, actin and keratin filaments, vacuoles, mitochondria, centrosomes, kinetochores, chromosomes, etc. Under such cutting conditions the laser beam leaves an ~0.3 (m wide optically-dense """"""""sniglet"""""""" trail (Cole et al., 1995. J. Microsc. Soc. Amer., 1203-215) in the cell which appears, at the EM level, as an electron-opaque deposit of coagulated and denature protein. Currently we are exploring the mechanism behind this photoablation: is the destruction at higher energy levels caused by a two photon effect, which many feel, or by the formation of a local plasma. To investigate this issue we are using the laser to cut protein under different environmental conditions, e.g., lack of oxygen, free radical scavengers, in the presence of fluoropores, etc. . This year we also explored the idea of using green fluorescent protein (GFP) as a tag to visualize otherwise invisible cellular structures so that they could be subsequently selectively destroyed by the laser. To accomplish this we modified and re-designed the epi-fluorescent attachment on the DIC based laser microscope so that all of the optical elements were removed from the light path directly behind the filter cube. These were placed, instead, at right angles to that path. We then added a dichroic mirror to combine the laser beam and the excitation wavelength (needed for fluorescent imaging) which was generated by the Hg lamp that is normally used to illuminate the specimen for DIC imaging. Currently, a standard fluorescent cube (without the exciter) is used to image the fluorescent tag and to position the specimen so that the tag is over the laser. A different filter cube must then be slid into position so that the structure of interest can be photoablated. Since the fluorescence GFP signal is weak we coupled one or our existing SIT cameras to the microscopes camera port via a dichroic mirror. As in the past the DIC images are captured on a on a Pultek CCD camera. Both cameras are mounted on three axis positioners to facilitate registration between them, and to make both coplanar with the laser beam and transmitted light image. We then conducted a """"""""proof of concept"""""""" in which we used the system to selectively destroy the centrosome in living PtK1 cells. (Please see Highlight #5) Khodjakov, A., R.W. Cole and C.L. Rieder. (1997) A synergy of technologies: combining laser microsurgery with green fluorescent protein (GFP)-tagging. Cell Motil. Cytoskel., 38:1-8. Khodjakov, A., R.W. Cole, B.F. McEwen, K. F. Buttle, and C.L. Rieder. (1997) Chromosome fragments possessing only one kinetochore can congress to the spindle equator. J. Cell Biol., 136:229-240.
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