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Deep-tissue focal fluorescence imaging with digitally time-reversed ultrasound-encoded light

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      Abstract

      Fluorescence imaging is one of the most important research tools in biomedical sciences. However, scattering of light severely impedes imaging of thick biological samples beyond the ballistic regime. Here we directly show focusing and high-resolution fluorescence imaging deep inside biological tissues by digitally time-reversing ultrasound-tagged light with high optical gain (~5×10 5). We confirm the presence of a time-reversed optical focus along with a diffuse background—a corollary of partial phase conjugation—and develop an approach for dynamic background cancellation. To illustrate the potential of our method, we image complex fluorescent objects and tumour microtissues at an unprecedented depth of 2.5 mm in biological tissues at a lateral resolution of 36 μm×52 μm and an axial resolution of 657 μm. Our results set the stage for a range of deep-tissue imaging applications in biomedical research and medical diagnostics.

      Abstract

      Fluorescence imaging is important for biomedical research and applications, but the turbidity of biological material prohibits deep tissue study. By combining ultrasound-encoding with digital time-reversal, Wang et al. perform focussed fluorescence imaging at a tissue depth of 2.5mm.

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      Most cited references 35

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      Optical coherence tomography.

      A technique called optical coherence tomography (OCT) has been developed for noninvasive cross-sectional imaging in biological systems. OCT uses low-coherence interferometry to produce a two-dimensional image of optical scattering from internal tissue microstructures in a way that is analogous to ultrasonic pulse-echo imaging. OCT has longitudinal and lateral spatial resolutions of a few micrometers and can detect reflected signals as small as approximately 10(-10) of the incident optical power. Tomographic imaging is demonstrated in vitro in the peripapillary area of the retina and in the coronary artery, two clinically relevant examples that are representative of transparent and turbid media, respectively.
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        Two-photon laser scanning fluorescence microscopy.

        Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.
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          Going deeper than microscopy: the optical imaging frontier in biology.

          Optical microscopy has been a fundamental tool of biological discovery for more than three centuries, but its in vivo tissue imaging ability has been restricted by light scattering to superficial investigations, even when confocal or multiphoton methods are used. Recent advances in optical and optoacoustic (photoacoustic) imaging now allow imaging at depths and resolutions unprecedented for optical methods. These abilities are increasingly important to understand the dynamic interactions of cellular processes at different systems levels, a major challenge of postgenome biology. This Review discusses promising photonic methods that have the ability to visualize cellular and subcellular components in tissues across different penetration scales. The methods are classified into microscopic, mesoscopic and macroscopic approaches, according to the tissue depth at which they operate. Key characteristics associated with different imaging implementations are described and the potential of these technologies in biological applications is discussed.
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            Author and article information

            Affiliations
            [1 ]Bioengineering and Electrical Engineering, California Institute of Technology , 1200 E California Boulevard, Pasadena, California 91125, USA.
            [2 ]Department of Clinical Research, London School of Hygiene and Tropical Medicine , Keppel Street, London WC1E 7HT, UK.
            [3 ]Electrical and Computer Engineering, Mechanical and Industrial Engineering, Northeastern University , 360 Huntington Avenue, Boston, Maryland 02115, USA.
            [4 ]These authors contributed equally to this work.
            Author notes
            Journal
            Nat Commun
            Nat Commun
            Nature Communications
            Nature Pub. Group
            2041-1723
            26 June 2012
            : 3
            : 928
            22735456 3621452 ncomms1925 10.1038/ncomms1925
            Copyright © 2012, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

            This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/

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