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

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(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.