Modern research in molecular, cellular, and developmental biology requires the precise measurement of cellular or subcellular activity in two or three dimensions. The development of probes for the noninvasive assay of activity has contributed greatly to our understanding of the complex hierarchies of pathways that regulate cellular functions. Equally significant has been the development of microscope imaging technologies that can directly follow the changes in molecular content and distribution which underlie a wide range of biological processes in living cells. Many available fluorescence microscopic techniques are yielding limited detail about the organization and dynamics of complex cellular structures, primarily because of the slow speed with which conventional image detectors capture and process high-resolution images. Impaired by UV-excited fluorophores, one-photon confocal laser scanning microscopy (OCLSM) techniques are often incompatible with high- resolution (aberration-free) 3-D imaging and greatly reduce cell or tissue viability with repeated scanning. In contrast, the new two- or three-photon excitation laser scanning microscope (TELSM), which uses a red-wavelength femtosecond laser, surmounts some of the problems of OCLSM and provides both sensitivity and high resolution without a confocal aperture. The proposed Multi-photon Excitation Fluorescence Imaging Microscopy (MEFIM) system will afford a unique, state-of-the- art opportunity to integrate advances in optical microscopy, low-light video detection, and two- or three-dimensional image analysis in measuring the fluorescent signals from a living cell. The system's modular nature will also provide the flexibility to meet varying optical and detection requirements of multiple users. This instrumentation will play a vital role in current research programs at the Advanced Cellular Imaging Facility (ACIF) of the University of Virginia and will provide novel opportunities to meet future research n eeds.
