Pulse-shaping for Multiphoton FRET Microscopy in Vivo

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DESCRIPTION (provided by applicant): Pulse-shaping for Multiphoton FRET Microscopy In Vivo Multiphoton microscopy has proven to be an invaluable tool for imaging in scattering tissue and live animals. However, quantitative multiphoton studies of protein-protein interactions have been hindered by the expense and limited spectral range of the typical laser sources available for multiphoton microscopy. Broadband titanium-sapphire laser sources offer a stunning range of excitation wavelengths in a single laser source, allowing simultaneous excitation of multiple fluorophores. However, with broadband excitation comes a lack of excitation selectivity. Pulse-shaping offers an ideal solution for providing efficient and selective multiphoton excitation, with the added benefit of reduced photobleaching. This proposal aims to combine pulse-shaping with quantitative multiphoton FRET microscopy in vivo, introducing an exciting new tool to the research community. This new technology will permit rapid and quantitative imaging of many commonly-used two photon fluorophores and FRET pairs, including many fluorescent proteins. We will initially demonstrate multiplex fluorescence excitation and detection of commonly-used fluorescent proteins and FRET pairs in solution, employing spectral-unmixing and FRET stoichiometry algorithms to obtain quantitative measures of relative free and interacting protein concentrations. We will then employ the optimized pulse-shapes in live-cell imaging applications to provide quantitative measures of cell membrane receptor signaling. We will compare the pulse-shaping-based technology with one-photon FRET stoichiometry and quantitative multiphoton FRET imaging employing fluorescence lifetime methodology (FRET-FLIM). Compared to FRET-FLIM and current multiplex multiphoton imaging based on laser-tuning we anticipate orders of magnitude enhancement in image acquisition speed, enabling multiphoton FRET microscopy in vivo. In optimizing the pulse-shaping methodology for multiphoton FRET we will also gain insight into photobleaching and photodamage reduction mechanisms that will be widely applicable to other multiphoton imaging techniques.