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      In vivo fluorescence lifetime optical projection tomography

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          Abstract

          We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos ( Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions.

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          The green fluorescent protein.

          R Tsien (1998)
          In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
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            FRET imaging.

            Elizabeth A Jares-Erijman, Thomas M. Jovin (2003)
            Förster (or Fluorescence) Resonance Energy Transfer (FRET) is unique in generating fluorescence signals sensitive to molecular conformation, association, and separation in the 1-10 nm range. We introduce a revised photophysical framework for the phenomenon and provide a systematic catalog of FRET techniques adapted to imaging systems, including new approaches proposed as suitable prospects for implementation. Applications extending from a single molecule to live cells will benefit from multidimensional microscopy techniques, particularly those adapted for optical sectioning and incorporating new algorithms for resolving the component contributions to images of complex molecular systems.
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              The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish

              Chris Hall, Maria Vega C Flores, Thilo Storm … (2007)
              Background How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable. Results We used regulatory regions of the zebrafish lysozyme C (lysC) gene to drive enhanced green fluorescent protein (EGFP) and DsRED2 expression in a manner that completely recapitulated the endogenous expression profile of lysC. Labeled cells were shown by co-expression studies and FACS analysis to represent a subset of macrophages and likely also granulocytes. Functional assays within transgenic larvae proved that these marked cells possess hallmark traits of myelomonocytic cells, including the ability to migrate to inflammatory sources and phagocytose bacteria. Conclusion These reporter lines will have utility in dissecting the genetic determinants of commitment to the myeloid lineage and in further defining how lysozyme-expressing cells participate during inflammation.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Biomed Opt Express
                Journal ID (publisher-id): BOE
                Title: Biomedical Optics Express
                Publisher: Optical Society of America
                ISSN (Electronic): 2156-7085
                Publication date (Electronic): 26 April 2011
                Publication date Collection: 01 May 2011
                Publication date PMC-release: 26 April 2011
                Volume: 2
                Issue: 5
                Pages: 1340-1350
                Affiliations
                [1 ]Photonics Group, Department of Physics, Imperial College London, SW7 2AZ, UK
                [2 ]Division of Cell and Molecular Biology, Department of Life Sciences, Imperial College London, SW7 2AZ, UK
                [3 ]Centre for Integrative Systems Biology, Department of Life Sciences, Imperial College London, SW7 2AZ, UK
                Author notes
                Article
                Publisher ID: 143678
                DOI: 10.1364/BOE.2.001340
                PMC ID: 3087590
                PubMed ID: 21559145
                SO-VID: 7bf6762c-ea66-4837-a730-8066a65a8215
                Copyright © ©2011 Optical Society of America
                License:

                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
                Date received : 4 March 2011
                Date revision received : 21 April 2011
                Date accepted : 21 April 2011
                Funding
                Funded by: UK Biotechnology and Biological Sciences Research Council
                Funded by: UK Engineering and Physical Sciences Research Council
                Funded by: Wellcome Trust
                Funded by: GlaxoSmithKline
                Categories
                Subject: Microscopy
                Custom metadata
                OpenAccess True
                OpenAccessEmbargo 0

                ScienceOpen disciplines: Vision sciences
                Keywords: (170.6960) tomography,(170.3650) lifetime-based sensing,(170.6920) time-resolved imaging,(170.6900) three-dimensional microscopy
                Data availability:
                ScienceOpen disciplines: Vision sciences
                Keywords: (170.6960) tomography, (170.3650) lifetime-based sensing, (170.6920) time-resolved imaging, (170.6900) three-dimensional microscopy

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