Multipixel spectral imaging of green fluorescent protein (GFP) in COS-7 cells: folding kinetics and chromophore formation

Lior Greenbaum; Chana Rothmann; Judith Hanania; Zvi Malik

doi:10.1117/12.410631

18 December 2000 Multipixel spectral imaging of green fluorescent protein (GFP) in COS-7 cells: folding kinetics and chromophore formation

Lior Greenbaum, Chana Rothmann, Judith Hanania, Zvi Malik

Author Affiliations +

Proceedings Volume 4164, Laser Microscopy; (2000) https://doi.org/10.1117/12.410631
Event: EOS/SPIE European Biomedical Optics Week, 2000, Amsterdam, Netherlands

Abstract

Spectrally resolved imaging of Green fluorescent protein (GFP) expressed in living COS-7 kidney cells distinguished the subcellular localization and demarcated the processes of protein folding and chromophore formation. COS-7 kidney cells were transfected by a plasmid pEGFP-N1 plasmid followed by incubation for 15 hours for gen expression. At different intervals the cells were examined by fluorescence microscopy and analyzed by a spectral imaging system. After 7 hours, GFP was detected in the cytoplasm, concentrated in a localized form. Spectra of the initial GFP evinced several components that belong both tot he typical fluorescent signal as well as to unspecific fingerprints. At 10 and 15 hours, GFP was seen spread in the cytoplasm as well as in the nucleus, and the specific spectra of the GFP were dominant at the later time. The typical GFP spectral fingerprint is the result of protein folding and chromophore formation following internal oxidation reactions. This folding and chromophore formation process, up to final conformation, was detected by spectral imaging as localized in the nucleus rather than in the cytosol. Thus, the method of fluorescence microscopy combined with multiplex spectral imaging demonstrates distinct biochemical pathways leading to GFP conformation.

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Lior Greenbaum, Chana Rothmann, Judith Hanania, and Zvi Malik "Multipixel spectral imaging of green fluorescent protein (GFP) in COS-7 cells: folding kinetics and chromophore formation", Proc. SPIE 4164, Laser Microscopy, (18 December 2000); https://doi.org/10.1117/12.410631

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KEYWORDS

Green fluorescent protein

Imaging spectroscopy

Chromophores

Proteins

Luminescence

Microscopy

Kidney

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