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This work is supported by the UK Engineering and Physical Sciences Research Council for funding through grants EP/P030017/1 and EP/M000869/1, and has received funding from the European Union's Horizon 2020 Programme through the project Advanced BiomEdical OPTICAL Imaging and Data Analysis (BE-OPTICAL) under grant agreement no. 675512. Optical approaches have broadened their impact in recent years with innovations in both wide-field and super-resolution imaging, which now underpin biological and medical sciences. Whilst these advances have been remarkable, to date, the ongoing challenge in optical imaging is to penetrate deeper. TRAFIX is an innovative approach that combines temporal focusing illumination with single-pixel detection to obtain wide-field multiphoton images of fluorescent microscopic samples deep through scattering media without correction. It has been shown that it can image through biological samples such as rat brain or human colon tissue up to a depth of seven scattering mean-free-path lengths. Comparisons of TRAFIX with standard point-scanning two-photon microscopy show that the former can yield a five-fold higher signal-to-background ratio while signifcantly reducing photo bleaching of the specimen. Here, we show the first preliminary demonstration of TRAFIX with three-photon excitation imaging dielectric beads. We discuss the advantages of the TRAFIX approach combined with compressive sensing for biomedicine. Publisher PDF

This work has received funding from the European Union’s Horizon 2020 Programme through the project Advanced BiomEdical OPTICAL Imaging and Data Analysis (BEOPTICAL) under grant agreement no. 675512 and the UK Engineering and Physical Sciences Research Council (grants EP/P030017/1 and EP/R004854/1). We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 μm diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams. Postprint