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      Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast

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          Abstract

          Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes.

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          Most cited references28

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          Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins.

          Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.
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            OPTICAL PHASE CONJUGATION FOR TURBIDITY SUPPRESSION IN BIOLOGICAL SAMPLES.

            Elastic optical scattering, the dominant light interaction process in biological tissues, prevents tissues from being transparent. While scattering may appear stochastic, it is in fact deterministic in nature. We show that, despite experimental imperfections, optical phase conjugation (lambda = 532 nm) can force a transmitted light field to retrace its trajectory through a biological target and recover the original light field. For a 0.69 mm thick chicken breast tissue section, we can enhance point source light return by approximately 5x10(3) times and achieve a light transmission enhancement factor of 3.8 within a collection angle of 29 degrees . Additionally, we find that the reconstruction's quality, measured by the width of the reconstructed point source, is independent of tissue thickness (up to 0.69 mm thick). This phenomenon may be used to enhance light transmission through tissue, enable measurement of small tissue movements, and form the basis of new tissue imaging techniques.
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              Two-photon absorption properties of fluorescent proteins.

              Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.
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                Author and article information

                Journal
                Biomed Opt Express
                Biomed Opt Express
                BOE
                Biomedical Optics Express
                Optical Society of America
                2156-7085
                27 July 2012
                01 August 2012
                27 July 2012
                : 3
                : 8
                : 1955-1963
                Affiliations
                [1]Department of Chemistry, Columbia University, New York, NY 10027, USA
                Author notes
                Article
                169563
                10.1364/BOE.3.001955
                3409713
                22876358
                9a681118-e1ec-449c-b225-cf13c5ec72c4
                ©2012 Optical Society of America

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 Unported License, which permits download and redistribution, provided that the original work is properly cited. This license restricts the article from being modified or used commercially.

                History
                : 30 May 2012
                : 17 June 2012
                : 19 June 2012
                Funding
                Funded by: Columbia University
                Funded by: Kavli Institute for Brain Science
                Categories
                Microscopy
                Custom metadata
                True
                0

                Vision sciences
                (190.4180) multiphoton processes,(180.2520) fluorescence microscopy,(180.4315) nonlinear microscopy

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