LASER ABLATION OF SUB CELLULAR ORGANELLES & PROTEINS

(部分由NIH GMS 40198至C.Rieder提供支持)。在过去的时间里 几年来，我们一直在探索使用高能的用途 纳秒脉冲绿光(532 Nm)，从我们的脉冲 作为选择性破坏或“光烧蚀”的工具的NdYAG激光 活细胞内的特定结构。我们发现，在 适当的条件(具有高斯分布的高能光束--见 TRD项目以上)该系统可以用来摧毁任何结构 通过视频增强型DIC LM在活细胞内可见，无 杀死牢房！在过去的一年里，我们间接地进行了相同的细胞 免疫荧光和电子显微镜研究证实我们 可以随意切断或破坏微管、肌动蛋白和角蛋白 细丝、液泡、线粒体、中心体、动粒、 染色体等。在这样的切割条件下，激光束离开 一条约0.3米宽的光学致密的“嗅探”小径(Cole等人，1995)。 J.Microsc.SoC。1203-215)在出现的单元格中 EM级，作为一种凝聚和变性的不透明电子沉积 蛋白。目前，我们正在探索这背后的机制 光消融：更高能级的破坏是由 双光子效应，这是许多人感觉到的，还是由局部形成的 血浆。为了研究这个问题，我们正在使用激光切割 蛋白质在不同的环境条件下，例如缺乏 氧、自由基清除剂、在荧光孔存在的情况下等。 。今年我们还探索了使用绿色荧光的想法 蛋白质(GFP)作为标签以可视化原本看不见的细胞 结构，以便随后可以有选择地通过 激光。为了实现这一点，我们修改并重新设计了 基于DIC的激光显微镜上的EPI-荧光附着物 所有光学元件都直接从光路中移除 在滤镜立方体后面。相反，这些被放置在直角上。 通向那条路。然后我们添加了一个二色镜来结合激光 光束和激发波长(荧光成像所需) 它是由通常用来照明的汞灯产生的 DIC成像标本。目前，一个标准的荧光立方体 (没有激励器)用于对荧光标签成像并 放置样品，使标签位于激光上方。一种不同的 然后必须将筛选器立方体滑动到位，以便 兴趣可以被光消融。由于荧光GFP信号是 我们将一个或现有的SIT摄像机连接到显微镜上 通过二向色镜的摄像头端口。与过去一样，DIC图像是 由Pultek公司的一台CCD相机拍摄。两个摄像头都安装在 三轴定位器，以便于它们之间的配准，并 使其与激光束和透射光图像共面。 然后，我们进行了一个“概念验证”，在这个过程中，我们使用该系统 选择性地破坏活的PtK1细胞的中心体。(请参阅 亮点#5)Khodjakov，A.，R.W.Cole和C.L.Rieder(1997)A 技术的协同作用：将激光显微手术与绿色技术相结合 荧光蛋白(GFP)标记。细胞运动。赛托斯克尔，38：1-8。 书名/作者声明：[by]Khodjakov A. (1997)只有一个着丝粒的染色体片段可以 国会向主轴赤道进发。《细胞生物学》，136：229-240。

(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.