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For in vivo imaging, the use of NIR fluorophores is essential. To minimize light-induced toxicity, it is preferable to use low-energy wavelengths in the near-infrared (NIR) spectral region ( λ=700–900 nm). Fluorophore dark toxicity must be low, so that cell viability is not compromised and normal cellular processes are unperturbed. Continuous live-cell imaging places very high demands on photostability of the fluorophore, as the same cell(s) are repeatedly imaged over time. Obtaining selective fluorescence responses for intracellular analytes is not trivial, as analyte selectivity observed in a controlled homogeneous environment of a cuvette does not necessarily translate to far more complex in vitro or in vivo settings. Stringent criteria are required, such as near-perfect response selectivity, exceptional photostability and low dark and light toxicities.
#Integrate stack nmr mestrenova series
Continuous recording of dynamic cellular events in real time may become feasible if the on/off fluorescence switching is reversible.ĭeveloping a responsive fluorophore suitable for real-time live-cell imaging poses a series of challenges. An innovative approach to enhance target-to-background signal ratio is to exploit a mechanism of selective fluorescence quenching in the background areas, while establishing the emitting potential of the fluorophore only in the ROI 12, 13. This can limit imaging to fixed cells or static snapshots, without the possibility of continuous data acquisition throughout the experiment. Poor ROI selectivity necessitates a time delay to allow background fluorophore clearance and/or a washing procedure between fluorophore administration and image acquisition. The major shortcomings of fluorescence imaging using molecular fluorophores are interference from nonspecific background fluorescence outside the region of interest (ROI), insufficient photostability and cytotoxicity. A century later, the use of fluorescence imaging as a technique to visualize specific regions of live cellular 1, 2, 3, 4 or whole organisms 5, 6 is often central to research programmes, with clinical applications such as fluorescence-guided surgery now emerging 7, 9, 10, 11. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools.Įhrlich’s use of synthetic dyes as a means of staining biological samples can be viewed as one of the foundation stones of modern scientific research. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D.
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Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to in vivo imaging.