Epicocconone is a heterocyclic natural product from the fungus Epicoccum nigrum that fluoresces weakly in the green (520 nm). However, cells exposed to epicocconone rapidly absorb the dye and become bright orange fluorescent because the natural product reacts reversibly with proteins. The orange fluorescence is enhanced in lipophilic environments, allowing the visualization of membranous organelles and lipid rafts but does not stain oligonucleotides. As the unconjugated dye has no orange fluorescence, there is no need to wash out the excess fluorophore. Epicocconone is a neutral, non-toxic, small molecule that appears to diffuse readily into live of fixed cells without the need for permeabilization. These features enable the real-time imaging of live cells and the study of organelle movements. Cells stained with epicocconone are excitable by common lasers (UV, 405, 488, and 532 nm) and its long Stokes' shift allows multiplexing applications with more common short Stokes' fluorophores using a single light source.
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REFERENCES
R. P. Haugland (2005). Handbook of a Guide to Fluorescent Probes and Labeling Technologies, 10th ed., Molecular Probes, Eugene, OR, USA.
D. B. Zorov, E. Kobrinsky, M. Juhaszova, and S. J. Sollott (2004). Examining intracellular organelle function using fluorescent probes from animalcules to quantum dots. Circ. Res. 95, 239–252.
M. A. Haidekker, T. Ling, M. Anglo, H. Y. Stevens, J. A. Frangos, and E. A. Theodorakis (2001). New fluorescent probes for the measurement of cell membrane viscosity. Chem. Biol. 8, 123–131.
M. H. Teiten, L. Bezdetnaya, P. Morlière, R. Santus, and F. Guillemin (2003). Endoplasmic reticulum and Golgi apparatus are the preferential sites of Foscan® localisation in cultured tumour cells. Brit. J. Cancer 88, 146–152.
C. R. Parish (1999). Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol. Cell Biol. 77, 499–508.
A. Grützkau, S. Krüger-Krasagakes, H. Kögel, A. Möller, U. Lippert, and B. M. Henz (1997). Detection of intracellular interleukin-8 in human mast cells: Flow cytometry as a guide for immunoelectron microscopy. J. Histochem. Cytochem. 45, 935–946.
A. J. Janecki, M. Janecki, S. Akhter, and M. Donowitz (2000). Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts. J. Histochem. Cytochem. 48, 1479–1492.
G. Jedd and N-H. Chua (2002). Visualization of peroxisomes in living plant cells reveals acto-myosin-dependent cytoplasmic streaming and peroxisome budding. Plant Cell Physiol. 43, 384–392.
A. M. Derfus, W. C. W. Chan, and S. N. Bhatia (2004). Probing the cytotoxicity of semiconductor quantum dots. Nano Lett. 4, 11–18.
D. S. Y. Yeo, R. Srinivasan, M. Uttamchandani, G. Y. J. Chen, Q. Zhu, and S. Q. Yao (2003). Cell-permeable small molecule probes for site-specific labeling of proteins. Chem. Commun. 23, 2870–2871.
G. Gaietta, T. J. Deerinck, S. R. Adams, J. Bouwer, O. Tour, D. W. Laird, G. E. Sosinsky, R. Y. Tsien, and M. H. Ellisman (2002). Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507.
C. Bolte, C. Talbot, Y. Boutte, O. Catrice, N. D. Read, and B. Satiat-Jeunemaitre (2004). FM-dyes as experimental probes for dissecting vesicle trafficking in living plants. J. Microsc. 214, 159–173.
R. Srinivasan, S. Q. Yao, and D. S. Y. Yeo (2004). Chemical approaches for live cell bioimaging. Comb. Chem. High Throughput Screening 7, 597–604.
J. Fukuda, H. Ishimine, and Y. Masaki (2003). Long-term staining of live Merkel cells with FM dyes. Cell Tiss. Res. 311, 325–332.
B. Zanella, N. Calonghi, E. Pagnotta, L. Masotti, and C. Guarnieri (2002). Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy. Biochem. Biophys. Res. Commun. 290, 1010–1014.
L. Kuerschner, C. S. Ejsing, K. Ekroos, A. Shevchenko, K. I. Anderson, and C. Thiele (2005). Polyene-lipids: A new tool to image lipids. Nat. Methods 2, 39–45.
D. A. Veal, P. Bell, H. Brown, H-Y. Choi, and P. Karuso (2003). Fluorophores from fungi. Microbiol. Aust. 24, 12–14.
P. J. L. Bell, D. Deere, J. Shen, B. Chapman, P. H. Bissinger, P. V. Attfield, and D. A. Veal (1998). A flow cytometric method for rapid selection of novel industrial yeast hybrids. Appl. Environ. Microbiol. 64, 1669–1672.
B. C. Ferrari, P. V. Attfield, D. A. Veal, and P. J. Bell (2003). Application of the novel fluorescent dye Beljian red to the differentiation of Giardia cysts. J. Microbiol. Methods 52, 133–135.
P. J. L. Bell and P. H. Karuso (2003). Epicocconone, a novel fluorescent compound from the fungus Epicoccum nigrum. J. Am. Chem. Soc. 125, 9304–9305.
D. R. Coghlan, J. A. Mackintosh, and P. Karuso (2005). Mechanism of reversible fluorescent staining of protein with epicocconone. Org. Lett. 7, 2401–2404.
R. Freshney (1987). Culture of Animal Cells: A Manual of Basic Techniques. Alan R. Liss, New York.
National Cancer Institute Therapeutics Development Program, NCI ID No. D734922/1; Compound ID. Epicocconone (35597); Test date Jan. 31 2005.
Acknowledgments
We thank to Ning Xu at Australian Proteomic Analysis Facility (Sydney, Australia) for providing HCT-116 (wild type), Daniel Bain, Jim Mackintosh and Dan Coghlan for assistance and helpful discussions.
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Choi, HY., Veal, D.A. & Karuso, P. Epicocconone, A New Cell-Permeable Long Stokes' Shift Fluorescent Stain for Live Cell Imaging and Multiplexing. J Fluoresc 16, 475–482 (2006). https://doi.org/10.1007/s10895-005-0010-7
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DOI: https://doi.org/10.1007/s10895-005-0010-7