In order to be able to """"""""photoablate"""""""" a chosen organelle or structure with nanosecond pulses of green (532 nm) laser light, the energy output of the laser must closely approximate a Gaussian distribution across the beam profile. It has been our experience that maintaining such a profile requires that the laser be constantly tuned and aligned (both within the laser head and also along the optical train) throughout the workday. To facilitate these operations we added a laser beam analyzer (LBA 300PC, Spiricon Inc) to our optical trapping/laser microsurgery workstation that graphically displays the beam profile in real time. Using this analyzer we confirmed that the laser profile changes on a day-to-day basis, and that the ability to photoablate cell structures is sensitive to these changes. In order to improve the long-term stability of the beam, and to generate reproducible experimental results, we inserted pinhole apertures of various sizes into different places within the laser cavity. This well known """"""""trick"""""""" allows us to now use only the most stable (central portion) of the laser beam during our ablation studies. The """"""""best"""""""" size and placement of the aperture was determined from viewing the beam on the laser analyzer to check for maxima, and also experimentally by using it to ablate different cellular structures. We found that a 1.4 mm aperture placed between the rod and the front mirror yielded the best performance. Before the pinhole was added the fit to Gaussian was approx. 0.7 were as once the aperture was in place it was 0.85. Because this modification significantly decreased the beam diameter, we added a beam expander/collimator to increase the diameter to that of the back aperture of the objective lens.
Showing the most recent 10 out of 252 publications
