We request funds to purchase a shared two-photon fluorescence correlation spectroscopy (PCS) system. This system will have four initial major users, all of whom have qualifying funded NIH projects that specifically include the use of PCS. These projects all require quantitation of protein interactions and cover a range of systems including kinases, transporters, and chaperones. Preliminary data for these funded projects was acquired using the installed Zeiss LSM 510 Confocor II, but this existing instrument has two major limitations. First, it is in the laboratory of Dr. Anne Kenworthy, and is used extensively for all of her confocal and PCS projects. The Confocor II is available to other labs only through collaborative agreements and not as fee-for-service. As the Kenworthy lab grows, this system will be difficult to access for our needed PCS experiments. Second, the Confocor II is limited to confocal detection, and cannot be adapted for two-photon excitation (because it already has the 405 nm laser option installed). As described in this proposal, two-photon excitation facilitates data analysis and interpretation, because of the well-defined focal volume generated by the non-linear excitation. The proposed instrument will be part of the Cell Imaging Shared Resource. All major users will have access to the instrument and training through the Resource. In addition, new users will also be able to use this instrument. The Resource has an extensive track record of education, training, and productivity with over 220 lab groups at Vanderbilt University. Over the last 11 years, we have introduced shared access to confocal microscopy, live cell imaging, two photon excitation, total internal reflection (TIRF) microscopy, and deconvolution microscopy. These techniques began with use by the more biophysical laboratories, but rapidly became widely used by the general biomedical research community. We foresee a similar broadening in PCS use over the next few years. We have chosen the ISS Alba instrument because of its flexibility, which allows different lasers for excitation and multiple analysis modes. In particular, we require the Photon Counting Histogram (PCH) analysis to extract the maximal amount of information from molecular interaction experiments. While conventional autocorrelation analysis separates species by diffusion coefficient (making it difficult to distinguish between monomers and dimers), the PCH analysis separates species based on molecular brightness. This analysis will greatly facilitate all of our on-going projects. Finally, the open Alba data structure allows other analysis methods to be applied as they are developed.
| Yirdaw, Robel B; McHaourab, Hassane S (2012) Direct observation of T4 lysozyme hinge-bending motion by fluorescence correlation spectroscopy. Biophys J 103:1525-36 |
| Kremers, Gert-Jan; Hazelwood, Kristin L; Murphy, Christopher S et al. (2009) Photoconversion in orange and red fluorescent proteins. Nat Methods 6:355-8 |
| Easley, Christopher J; Rocheleau, Jonathan V; Head, W Steven et al. (2009) Quantitative measurement of zinc secretion from pancreatic islets with high temporal resolution using droplet-based microfluidics. Anal Chem 81:9086-95 |
