IMPROVEMENTS & UPGRADES TO LASER MICROSURGERY & OPTICAL TRAPPING WORKSTATION

为了能够“光消融”选定的细胞器或 结构由纳秒脉冲的绿色(532 Nm)激光组成， 激光的能量输出必须非常接近于高斯 横跨光束轮廓的分布。根据我们的经验， 保持这样的轮廓需要不断地调谐激光器 并且对准(在激光头内和沿光学 火车)在整个工作日。为了促进这些操作，我们 增加了一个激光光束分析器(LBA 300PC，思创公司)到我们的光学 捕捉/激光显微手术工作站，图形显示 实时的波束轮廓。使用这个分析仪，我们确认了 激光配置文件每天都在更改，并且能够 光烧蚀细胞结构对这些变化非常敏感。按顺序 以提高光束的长期稳定性，并产生 可重复性的实验结果，我们插入了针孔孔 不同大小的激光腔内的不同位置。这 众所周知的“技巧”允许我们现在只使用最稳定的(中央 部分)在我们的消融研究中。“最好的” 孔的大小和位置是通过查看 光束放在激光分析仪上以检查最大值，还 实验上用它来消融不同的细胞结构。 我们发现，在杆子和前部之间有一个1.4毫米的口径 Mirror的表现最好。在添加针孔之前， 近似符合高斯分布。0.7与光圈进入时一样 排名是0.85。因为这一修改大大减少了 为了增大光束直径，我们增加了扩束器/准直器 直径与物镜后光圈的直径一致。

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.