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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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(A) Differential interference contrast (DIC), (B) pseudocolor single-photon fluorescence, (C) two-photon fluorescence (λ = 740 nm, 6.5 mW, filter 510−550 nm) images of HeLa cells stained with amine reactive probe 1. HeLa cells incubated for 5 h with amine-reactive probe 1. Filter Cube for SPM: Fluo-M-Blue (Exc 377/50, DM 409; Em 460/50), Scale bar 50 μm.
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fig3: (A) Differential interference contrast (DIC), (B) pseudocolor single-photon fluorescence, (C) two-photon fluorescence (λ = 740 nm, 6.5 mW, filter 510−550 nm) images of HeLa cells stained with amine reactive probe 1. HeLa cells incubated for 5 h with amine-reactive probe 1. Filter Cube for SPM: Fluo-M-Blue (Exc 377/50, DM 409; Em 460/50), Scale bar 50 μm.

Mentions: The utility of amine-reactive tag 1 as a 2PA biological marker was demonstrated by incubation with HeLa cells. Cells were incubated with a solution of 10−6 mol L−1 of chromophore, and after 1 and 5 h incubation times, images were taken with a modified Olympus Fluoview FV300 microscope system. Strong fluorescence was observed after 5 h of incubation, with a homogeneous coloration of the cytoplasm region. Differential interference contrast (DIC) and epifluorescence microscopic images of the stained cells are shown in Figure 3a,b. The low quantum yield of this probe (0.02) was an important characteristic, because after incubation, it appeared to have reacted spontaneously with a protein in HeLa cells, generating a bioconjugate with significantly enhanced fluorescence (Qy = 0.7), improving detection by single- and two-photon fluorescence microscopy imaging (Figure 3b). 2PFM images of the same amine-reactive fluorene 1 stained cells were collected on a modified Olympus Fluoview FV300 microscope system combined with a tunable Coherent Mira 900F Ti:sapphire laser. Two-photon induced fluorescence was observed predominantly from the cytoplasmic region, consistent with the images collected from epifluorescence imaging (Figure 3c).


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)

(A) Differential interference contrast (DIC), (B) pseudocolor single-photon fluorescence, (C) two-photon fluorescence (λ = 740 nm, 6.5 mW, filter 510−550 nm) images of HeLa cells stained with amine reactive probe 1. HeLa cells incubated for 5 h with amine-reactive probe 1. Filter Cube for SPM: Fluo-M-Blue (Exc 377/50, DM 409; Em 460/50), Scale bar 50 μm.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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

fig3: (A) Differential interference contrast (DIC), (B) pseudocolor single-photon fluorescence, (C) two-photon fluorescence (λ = 740 nm, 6.5 mW, filter 510−550 nm) images of HeLa cells stained with amine reactive probe 1. HeLa cells incubated for 5 h with amine-reactive probe 1. Filter Cube for SPM: Fluo-M-Blue (Exc 377/50, DM 409; Em 460/50), Scale bar 50 μm.
Mentions: The utility of amine-reactive tag 1 as a 2PA biological marker was demonstrated by incubation with HeLa cells. Cells were incubated with a solution of 10−6 mol L−1 of chromophore, and after 1 and 5 h incubation times, images were taken with a modified Olympus Fluoview FV300 microscope system. Strong fluorescence was observed after 5 h of incubation, with a homogeneous coloration of the cytoplasm region. Differential interference contrast (DIC) and epifluorescence microscopic images of the stained cells are shown in Figure 3a,b. The low quantum yield of this probe (0.02) was an important characteristic, because after incubation, it appeared to have reacted spontaneously with a protein in HeLa cells, generating a bioconjugate with significantly enhanced fluorescence (Qy = 0.7), improving detection by single- and two-photon fluorescence microscopy imaging (Figure 3b). 2PFM images of the same amine-reactive fluorene 1 stained cells were collected on a modified Olympus Fluoview FV300 microscope system combined with a tunable Coherent Mira 900F Ti:sapphire laser. Two-photon induced fluorescence was observed predominantly from the cytoplasmic region, consistent with the images collected from epifluorescence imaging (Figure 3c).

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