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Amine-reactive fluorene probes: synthesis, optical characterization, bioconjugation, and two-photon fluorescence imaging.

Morales AR, Schafer-Hales KJ, Marcus AI, Belfield KD - Bioconjug. Chem. (2008)

Bottom Line: The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described.The conjugates displayed high chemical stability and photostability.The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The College of Optics and Photonics, University of Central Florida, P.O. Box 162366, Orlando, Florida 32816-2366, USA.

ABSTRACT
With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated.

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H1299 cells labeled with cyclic peptide bioconjugate 7: (A) DIC, (B) 1PM, (C) 2PM, (D) 2P-FLIM. Scale bar in (A) corresponds to 20 μm. (E) lifetime scale bar for (D). Pseudo color is used for (B), (C), and (D).
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fig6: H1299 cells labeled with cyclic peptide bioconjugate 7: (A) DIC, (B) 1PM, (C) 2PM, (D) 2P-FLIM. Scale bar in (A) corresponds to 20 μm. (E) lifetime scale bar for (D). Pseudo color is used for (B), (C), and (D).

Mentions: The efficacy of cyclic peptide-conjugate 7 was evaluated by incubation with H1299 lung tumor line cells (Figures 6 and 7). Additionally, fluorescence was observed predominantly from the cytoplasmic region of the cells, with the nucleus clearly outlined. Two-photon induced fluorescence was observed predominantly from the cytoplasmic region, consistent with the images collected from single fluorescence images (Figure 6c). Some differences were observed in the probe lifetime. This is a subject of further investigation.


Amine-reactive fluorene probes: synthesis, optical characterization, bioconjugation, and two-photon fluorescence imaging.

Morales AR, Schafer-Hales KJ, Marcus AI, Belfield KD - Bioconjug. Chem. (2008)

H1299 cells labeled with cyclic peptide bioconjugate 7: (A) DIC, (B) 1PM, (C) 2PM, (D) 2P-FLIM. Scale bar in (A) corresponds to 20 μm. (E) lifetime scale bar for (D). Pseudo color is used for (B), (C), and (D).
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651597&req=5

fig6: H1299 cells labeled with cyclic peptide bioconjugate 7: (A) DIC, (B) 1PM, (C) 2PM, (D) 2P-FLIM. Scale bar in (A) corresponds to 20 μm. (E) lifetime scale bar for (D). Pseudo color is used for (B), (C), and (D).
Mentions: The efficacy of cyclic peptide-conjugate 7 was evaluated by incubation with H1299 lung tumor line cells (Figures 6 and 7). Additionally, fluorescence was observed predominantly from the cytoplasmic region of the cells, with the nucleus clearly outlined. Two-photon induced fluorescence was observed predominantly from the cytoplasmic region, consistent with the images collected from single fluorescence images (Figure 6c). Some differences were observed in the probe lifetime. This is a subject of further investigation.

Bottom Line: The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described.The conjugates displayed high chemical stability and photostability.The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The College of Optics and Photonics, University of Central Florida, P.O. Box 162366, Orlando, Florida 32816-2366, USA.

ABSTRACT
With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated.

Show MeSH
Related in: MedlinePlus