Research into disease mechanisms and cell signaling is increasing as tools that enable detection and visualization of these cellular and subcellular processes are developed. The utility of fluorescence reporter technologies has been recognized and widely adapted for both microscopy and small animal imaging applications. One such fluorescent reporter technology that has been used extensively for this type of research is Forster resonance energy transfer (FRET). FRET imaging applications enable detection of variations in the local fluorophore environment, specifically the proximity of another fluorophore and have been utilized in the exploration of macromolecular interactions. Quantitative FRET detection poses difficulties as FRET signal is affected by concentration changes and sample optical properties. However, using fluorescence lifetime imaging (FLIM) to detect FRET interactions can circumvent these challenges, as the lifetime of a fluorophore is independent of fluorophore concentration and light path length. Both FRET and FLIM microscopy are in widespread academic use although neither technique is used routinely for high content screening research since FRET is inherently not a quantitative measurement and FLIM measurements are typically slow at the cellular level. When three-dimensional, small animal imaging is desired these disadvantages are compounded.
In the study by McGinty, et al.,
FLIM on a conventional wide-field microscope was combined with optical projection tomography (OPT) to image live zebrafish embryos. The study demonstrates that live transgeneic zebrafish with myeloid cells that express green fluorescent protein (GFP) can be imaged using FLIM-OPT. The importance of FLIM-OPT is demonstrated by separating the strong tissue autofluorescence particularly visible in the yoke-sac from the GFP fluorescent myeloid cells—a distinction that would be difficult without the fluorescence lifetime. Data collection of FLIM-OPT images of the live zebrafish were completed in about 20 minutes, and thus any internal feature, such as the heart, that moves significantly during this period could not be accurately detected. FLIM-OPT data reconstruction by filter backprojection required an additional 20 minutes of processing time. Both the data acquisition and the processing times could be significantly optimized to enable fast imaging and data reconstruction in future iterations of the FLIM-OPT system. Such an optimized system could be used with appropriate FRET sensors to probe interactions in signaling pathways and allow correlation of three-dimensional motion with molecular signaling events, particularly in the area of immune response.
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