This proposal is to develop a simultaneous two-photon excitation and confocal laser scanning microscope optimized for multi-color fluorescence imaging of living cells and tissues. Over the last decade, it has become apparent that the cell cannot be regarded as a 'bag of chemicals' that carries out biological reactions. The exact three-dimensional arrangement of the cellular components is tremendously important, as are the time-dependent changes in this arrangement during the cell cycle and upon interaction with a variety of cell activators (neurotransmitters, hormones, growth factors, etc.). Recent advances, such as two-photon excitation microscopy and the Green Fluorescent Protein (GFP), now allow high resolution imaging of living cells with minimal effects on cell viability. Considerable eff ort has been devoted over the last five years to develop quantitative laser scanning microscopies (imaging of NAD(P)H, Indo- 1, and GFP by two-photon excitation, and imaging of GFP mutants by confocal microscopy), and now have determined optimal parameters for each of these imaging modes. However, to understand the relationships between cellular arrangement, dynamics, and function, it is necessary to examine individual cells from multiple perspectives. Multi- parameter imaging of living. cells has been used effectively with widefield microscopy. For imaging of thick tissues, though, an optical sectioning microscopy, such as confocal or two- photon excitation, is needed. The proposed instrument will be built around a Zeiss LSM510 confocal microscope, added with lasers for two-photon excitation and optimized excitation of GFP mutants. A non-descanned detection channel will be installed for optimized detection of two-photon excited fluorescence. Installation of this detector will permit testing true photon counting electronics and different detectors, such as a cooled PMT or avalanche photodiode. This new instrument will allow researchers to combine multiple imaging modes that they have optimized separately. As an example, they can use two-photon excitation imaging to monitor cellular metabolic activity (by NAD(P)H autofluorescence) and confocal imaging to GEP fusion proteins to study specific protein translocations that may affect metabolism. Currently, these two experiments must be done on two separate instruments which prohibits direct correlations at the single cell level. With the proposed instrument, such experiments can he performed on the same cell at the same time by confocal imaging of GFP and two- photon excitation imaging of NAD(P)H. An alternative imaging method is the use of two-photon excitation to image both GFP and NAD(P)H by utilizing dual emission filters to measure each flluorophore simultaneously. The new instrument will work in either configuration, and can thus be used to rapidly determine the preferable method for each application. Once this instrument is developed and tested, it will become part of the Vanderbilt University Cell Imaging Resource. The resource has three staff members, and is currently used by more than 70 labs from the medical center and 4 from other campus units. It contains two microscopes, a confocal laser scanning microscope and a conventional fluorescence microscope with an ultralow light level CCD camera, plus several computers for data analysis and high quality image output. Inclusion of this new microscope in the imaging resource will allow its efficient administration, facilitate the transfer of new developments to a large number of researchers, and create a unique, full-service resource dedicated to live cell imaging.
