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      Cannula-based computational fluorescence microscopy

      1 , 2 , 2 , 3 , 4 , 1

      Applied Physics Letters

      AIP Publishing

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          Most cited references 14

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          Miniaturized integration of a fluorescence microscope

          The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals towards relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including semiconductor light source and sensor. This device enables high-speed cellular-level imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens.
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            Non-invasive imaging through opaque scattering layers.

            Non-invasive optical imaging techniques, such as optical coherence tomography, are essential diagnostic tools in many disciplines, from the life sciences to nanotechnology. However, present methods are not able to image through opaque layers that scatter all the incident light. Even a very thin layer of a scattering material can appear opaque and hide any objects behind it. Although great progress has been made recently with methods such as ghost imaging and wavefront shaping, present procedures are still invasive because they require either a detector or a nonlinear material to be placed behind the scattering layer. Here we report an optical method that allows non-invasive imaging of a fluorescent object that is completely hidden behind an opaque scattering layer. We illuminate the object with laser light that has passed through the scattering layer. We scan the angle of incidence of the laser beam and detect the total fluorescence of the object from the front. From the detected signal, we obtain the image of the hidden object using an iterative algorithm. As a proof of concept, we retrieve a detailed image of a fluorescent object, comparable in size (50 micrometres) to a typical human cell, hidden 6 millimetres behind an opaque optical diffuser, and an image of a complex biological sample enclosed between two opaque screens. This approach to non-invasive imaging through strongly scattering media can be generalized to other contrast mechanisms and geometries.
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              In vivo three-photon microscopy of subcortical structures within an intact mouse brain

              Two-photon fluorescence microscopy (2PM) 1 enables scientists in various fields including neuroscience 2,3 , embryology 4 , and oncology 5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of 2PM within the mouse brain to the cortical layer, and imaging subcortical structures currently requires the removal of overlying brain tissue 3 or the insertion of optical probes 6,7 . Here we demonstrate non-invasive, high resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue.
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                Author and article information

                Journal
                Applied Physics Letters
                Appl. Phys. Lett.
                AIP Publishing
                0003-6951
                1077-3118
                June 29 2015
                June 29 2015
                : 106
                : 26
                : 261111
                Affiliations
                [1 ]Department of Electrical and Computer Engineering, University of Utah, Salt Lake City, Utah 84112, USA
                [2 ]Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA
                [3 ]Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA
                [4 ]Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112, USA
                Article
                10.1063/1.4923402
                © 2015

                http://creativecommons.org/licenses/by/3.0/

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