This proposal seeks to acquire the first and currently only commercial microscope that has been specifically designed to have the improved sensitivity and reduced electronic noise necessary for monitoring the behavior of proteins in living cells (e.g. ligand binding receptors or proteins forming inclusions in degenerating cells). In combination with analysis methods developed at the LFD (Laboratory for Fluorescence Dynamics, UCI), this instrument reaches new levels of detection and allows measurements that were previously not possible. These capabilities are having a strong impact on the research endeavors at UCI and in particular are enabling several medically relevant projects to proceed. For example, Dr. Lee, breast cancer;Drs. Glabe and Marsh, neurodegeneration, Dr. Nelson, cancer profiling, Digman, cell migration, Gratton, protein trafficking in cells just to name a few. The acquisition of a Zeiss LSM 710 by the Optical Biological Core (OBC) facility at the Developmental Biology Center (DBC) represents an essential goal that would enable users to apply state-of-the-art live cell imaging methods to numerous emerging and fundamental biological problems. The permanent addition of this instrument at the OBC would allow multiple users access to vastly improved detectors that have significant higher signal to noise ratio, availability of multiple laser lines for one and two- photon image experiments or simultaneous imaging, moveable emission wavelength selection for broad or narrow collection and non-descanned detection. In addition, the twin gate beam splitter and high- transmission dichroic wheels allow users to use an array of laser wavelengths for fluorescence excitation. Since four lines can be used simultaneously, the complete system allows for maximum photon collection. The instrument will also have a FLIM detection unit. A microscope with all these features is not available on campus. In addition, an advanced feature of the LSM 710 not found in other commercial instruments includes the application of the Raster-scan Image Correlation Spectroscopy (RICS) technique to cells, a technique developed by Zeiss in collaboration with Dr. Enrico Gratton and Dr. Michelle Digman, the PI of this proposal and the scientific director of the OBC. We developed the RICS, Number and Molecular Brightness (N&B) and phasor FLIM techniques and have made them accessible for the standard commercial microscope. We thus have the expertise and enthusiasm to apply these techniques to an array of different problems in the areas of Medical Sciences, Biochemistry, Pharmacology, Nanotechnology, and Biology as illustrated in the """"""""projects"""""""" section of this proposal. This application seeks funds to purchase an LSM 710 to replace the instrument that has been on temporary loan and that has proven to be essential for many projects and in high demand.
The acquisition of the first available commercial instrument capable of high sensitivity, dynamic and quantitative imaging of proteins and fluorescent particles in cells will greatly enable progress on a large number of NIH funded projects ranging from cancer profiling to the study of growth factor ligands interacting with receptors in tissues. The microscope will be housed in a facility that is available to all and that currently has users from 60 PI laboratories in 20 departments across 4 schools. The sensitive filter-less detector and the use of two-photon excitation with FLIM capabilities will allow studies of molecular interactions in living cells and in tissues.
