FLUORESCENCE DECONVOLUTION LIGHT MICROSCOPY W/ SAME CELL CORRELATIVE LM & 3D EM

（由NIH/NCRR/P41 RR 01219赠款和NIH GMS R 01支持） 40198至C.L.R.）。我们正在探索如何确定 LM反卷积程序的准确性。 最近我们购买了一个API 三角洲视觉系统，使我们能够三维重建分布 在一个非常高分辨率的样本内的荧光。 在这个 现在，我们正在探索这个系统在定量分析方面的准确性。 分析。 为了确定这一点，我们正在研究 使用Delta Vision系统检测试样中的微管，然后 将相同的样本带到HVEM直接计数 微管 我们发现初级纤毛是一个很好的测试 试样 这种纤毛几乎存在于成体的每一个细胞中 生物体，包括人类，并来自最古老的中心粒 在细胞内。 它由9个双线微管组成， 排列成一个圆圈。 初级纤毛的功能仍然存在 模棱两可 纤毛长2-20 μ m， 已知双联体微管的数量逐渐增加， 从细胞表面（并因此沿着纤毛）进一步减少。 然而，没有明确的长度与双重数的关系。 由于袋鼠鼠肾上皮细胞（PtK）的初级纤毛 突出到肾腔中， 解释流体流动引起的弯曲的模型。 PtK细胞 不仅可以选择性地染色初级纤毛， 荧光显微镜，但也准备在这样的盖玻片， 初级纤毛的方向垂直于其平面 面 这使得人们可以使用宽视场荧光显微镜 去卷积以光学方式沿纤毛的长度沿着对其进行切片， 来估计微管数量是如何随着长度而变化的。 这些发现可以通过连续厚切片来证实。 用于HVEM或IVEM断层扫描的相同细胞。

(Supported by the NIH/NCRR/P41 RR 01219 grant and by NIH GMS R01 40198 to C.L.R.). We are exploring ways to determine the fidelity and accuracy of LM deconvolution programs. Recently we purchased an API Delta Vision system that allows us to 3-D reconstruct the distribution of fluorescence within a specimen at a very high resolution. At this time we are exploring just how accurate this system for quantitative analyses. To determine this we are examining the distribution of microtubules in test specimens using the Delta Vision system, and then taking the same specimen to the HVEM to directly count the number of microtubules. We find that the primary cilium is an excellent test specimen. This cilium is found in almost every cell in the adult organism, including humans, and is derived from the oldest centriole within the cell. It is comprised of 9 doublet microtubules that are arranged in a circle. The function of primary cilium remains ambiguous. The length o f the c ilium can range from 2-20 (m, and it is known that the number of doublet microtubules progressively decreases further from the cell surface (and thus along the cilium). However, there is no clear-cut length vs. doublet number relationship. Since the primary cilia of kangaroo rat kidney epithelial cells (PtK) protrude into the lumen of the kidney there are proposed mathematical models that explain bending in response to fluid flow. In PtK cells it is possible to not only selectively stain primary cilia for fluorescence microscopy, but also to prepare the coverslips in such a way the primary cilia are orientated perpendicular to its flat surface. This allows one to use wide field fluorescence microscopy and deconvolution to optically section the cilium along its length-and to estimate how microtubule numbers change as a function of length. These findings can then be confirmed by serially thick sectioning the same cell for HVEM or an IVEM tomography.