Wide-field super-resolved DEEP 3-D microscopy

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1134561WagnerThe authors propose to develop several new capabilities of the novel Doppler Encoded Excitation Pattern (DEEP) microscopy technique that they have recently demonstrated for wide-field Biophotonics imaging applications. DEEP microscopy employs traveling-wave structured illumination and a sensitive high-speed single-element detector (instead of an imaging detector array) to sequentially probe the complex spatial frequencies (Fourier coefficients) of the sample using either fluoresced or scattered light. Tomographic algorithms are utilized to synthesize either an extended depth of field (DOF) 2D or, alternatively, with additional Fourier components a full 3D image. The authors have previously demonstrated that by sequentially illuminating a fluorescent sample with traveling frequency-swept sinusoidal intensity fringe patterns of diverse orientations, the DEEP technique can be used to synthesize a 2D image of a ¡­ mm3 volume that resolves diffraction limited wavelength-scale features, all in focus simultaneously, thereby providing a 1000-fold increase in DOF over a conventional microscope. Three new directions enhancing DEEP microscopy for wide-field biophotonic imaging applications will be pursued: high-speed 3D tomographic imaging multiplexed patterns created with crossed acoustooptic Bragg cells; using illumination coherence gating to extend the depth of high-resolution fluorescence imaging in strongly scattering biological tissue by utilizing broadband light; and super-resolution DEEP imaging microscopy exploiting fluorescence saturation. Each of these research directions will demonstrate a novel wide-field imaging modality for studying biological processes faster, deeper, and smaller than possible with existing techniques.