IDBR - Development of an Ultrafast Phase-shaping Contrast Microscope

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Project AbstractThe project will develop an Ultrafast Phase-Shaping Contrast Microscope (UPSCM) to provide enhanced imaging capabilities and new modes of imaging contrast for biological research. The UPSCM will exploit the amplitude and phase-shaping of a broadband laser in a versatile instrument that will have three modes of operation to meet a wide range of imaging needs. The first imaging modality will permit rapid and quantitative multiphoton fluorescence resonance energy transfer (MFRET) imaging. The proposed MFRET imaging modality is orders of magnitude faster than current technology, opening up new opportunities for studying protein-protein interactions in animal and tissue models and will be readily extended to video-rate imaging speeds. The second imaging modality of the UPSCM will be two-color pump-probe contrast microscopy. This modality will exploit pump-probe contrast mechanisms including stimulated emission, excited state absorption and ground-state bleach to enable sensitive imaging of nonfluorescent and weakly fluorescent endogenous species. Finally, the third imaging modality will employ a single shaped laser pulse designed to provide molecular contrast as well as insight into the energy landscape of the molecule.Multiphoton microscopy has revolutionized our ability to visualize the biological world. Multiphoton excitation markedly enhances the depth of imaging into tissues, and provides novel modes of contrast. Multiphoton fluorescence microscopy has been widely used and has been shown to increase signal-to-background for fluorescence while reducing photodamage and photobleaching, permitting more sensitive detection of fluorescence signals over extended periods of time. However, in a typical multiphoton fluorescence microscope, a tunable femtosecond laser is used to image a single fluorescent probe, making the simultaneous observation of multiple chemical species difficult. The proposed UPSCM will employ an extremely broadband femtosecond laser to allow selective and simultaneous multiphoton fluorescence imaging of commonly used fluorescent probes. This methodology will facilitate studies of protein-protein interactions in transgenic animals, enabling new studies of cellular signaling in healthy and diseased animals. In addition to enhanced multiphoton fluorescence imaging, the UPSCM will provide novel, sensitive and selective contrast for nonfluorescent endogenous species that will be broadly applicable to imaging studies aimed at improving current understanding of basic cellular processes and methods for disease diagnosis.