Stimulated emission reduced fluorescence (SERF) for breaking and extending the fundamental imaging-depth of two photon microscopy

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 DESCRIPTION (provided by applicant): Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting fluorescence photons originated from a spatially confined excitation volume. However, such optical sectioning capability eventually breaks down when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast (S/B~1) defines a fundamental imaging-depth limit (around 1 mm for mouse brain tissues), which cannot be overcome by simply increasing excitation power. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from their excited states. The resulting SERF image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall third-order nonlinear dependence on the incident laser intensity. We plan to (1) build the first SERF microscope in the lab; (2) construct in vitro 3D issue phantoms, and use them to test and validate the imaging performance of the new SERF microscope; and (3) apply SERF microscopy on animal brain, and demonstrate high-contrast ultra-deep tissue imaging in vivo. SERF microscope is technically straightforward and easy to build, as only a red CW laser beam (together with the associated modulation electronics) is needed to be added onto a standard two-photon fluorescence microscope. As supported by numerical simulations, the proposed technique is expected to extend the imaging depth limit of two-photon microscopy by a factor of 1.8.