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Long-lived bright red emitting azaoxa-triangulenium fluorophores.

Maliwal BP, Fudala R, Raut S, Kokate R, Sørensen TJ, Laursen BW, Gryczynski Z, Gryczynski I - PLoS ONE (2013)

Bottom Line: Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns.Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization.ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, United States of America. bpmal001@gmail.com

ABSTRACT
The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful. Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36-0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images. ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins suggest that these probes will be highly useful to study slower intra-molecular motions and interactions among macromolecules. The fluorescence lifetime facilitated gating of cellular nanosecond autofluorescence should be of considerable help in in vitro and in vivo applications.

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Related in: MedlinePlus

Excitation and emission intensity and anisotropy spectra in 95% glycerol at -40°C.(A) ADOTA-ba (B) DAOTA-ba. The arrows indicate wavelengths of excitation and emission.
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pone-0063043-g005: Excitation and emission intensity and anisotropy spectra in 95% glycerol at -40°C.(A) ADOTA-ba (B) DAOTA-ba. The arrows indicate wavelengths of excitation and emission.

Mentions: The limiting excitation and emission anisotropy spectra were measured in 95% glycerol at -40°C, and are shown in Figure 5A (ADOTA-ba) and 5B (DAOTA-ba). The wavelengths to observe excitation and emission fluorescence and anisotropy spectra were 570 nm and 475 nm respectively in case of ADOTA. The corresponding wavelengths for DAOTA are 590 nm and 520 nm, respectively. Under these conditions, the fluorophores probes are completely immobilized when considering their fluorescence lifetimes. In case of ADOTA-ba, the limiting anisotropy has negative sign under the 430 nm peak and rises rapidly to a maximum value of 0.38 at main absorption peak. The shape of limiting anisotropy spectrum in case of DAOTA is similar to that seen with ADOTA and the maximum value of 0.36 is seen at the main absorption peak. In our instrumental set up for time-resolved anisotropy measurements, the limiting anisotropy values are 0.305 in case of ADOTA for 475 nm centered narrow diode laser and 0.326 in case of DAOTA for broader 520 nm centered LED. The observed high limiting anisotropies at absorption maxima are very similar to that seen in rhodamine and related red fluorophores [7]. Essentially similar results were obtained by Thyrhaug et al [18] for alkyl substituted ADOTA and DAOTA in frozen glycerol and PVA film. Thyrhaug et al also resolved underlying electronic transitions and their orientations in the molecular framework of these two fluorophores.


Long-lived bright red emitting azaoxa-triangulenium fluorophores.

Maliwal BP, Fudala R, Raut S, Kokate R, Sørensen TJ, Laursen BW, Gryczynski Z, Gryczynski I - PLoS ONE (2013)

Excitation and emission intensity and anisotropy spectra in 95% glycerol at -40°C.(A) ADOTA-ba (B) DAOTA-ba. The arrows indicate wavelengths of excitation and emission.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646960&req=5

pone-0063043-g005: Excitation and emission intensity and anisotropy spectra in 95% glycerol at -40°C.(A) ADOTA-ba (B) DAOTA-ba. The arrows indicate wavelengths of excitation and emission.
Mentions: The limiting excitation and emission anisotropy spectra were measured in 95% glycerol at -40°C, and are shown in Figure 5A (ADOTA-ba) and 5B (DAOTA-ba). The wavelengths to observe excitation and emission fluorescence and anisotropy spectra were 570 nm and 475 nm respectively in case of ADOTA. The corresponding wavelengths for DAOTA are 590 nm and 520 nm, respectively. Under these conditions, the fluorophores probes are completely immobilized when considering their fluorescence lifetimes. In case of ADOTA-ba, the limiting anisotropy has negative sign under the 430 nm peak and rises rapidly to a maximum value of 0.38 at main absorption peak. The shape of limiting anisotropy spectrum in case of DAOTA is similar to that seen with ADOTA and the maximum value of 0.36 is seen at the main absorption peak. In our instrumental set up for time-resolved anisotropy measurements, the limiting anisotropy values are 0.305 in case of ADOTA for 475 nm centered narrow diode laser and 0.326 in case of DAOTA for broader 520 nm centered LED. The observed high limiting anisotropies at absorption maxima are very similar to that seen in rhodamine and related red fluorophores [7]. Essentially similar results were obtained by Thyrhaug et al [18] for alkyl substituted ADOTA and DAOTA in frozen glycerol and PVA film. Thyrhaug et al also resolved underlying electronic transitions and their orientations in the molecular framework of these two fluorophores.

Bottom Line: Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns.Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization.ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, United States of America. bpmal001@gmail.com

ABSTRACT
The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful. Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36-0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images. ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins suggest that these probes will be highly useful to study slower intra-molecular motions and interactions among macromolecules. The fluorescence lifetime facilitated gating of cellular nanosecond autofluorescence should be of considerable help in in vitro and in vivo applications.

Show MeSH
Related in: MedlinePlus