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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

ADOTA-C16 in DOPC.(A) Fluorescence intensity decay (B) anisotropy decay.
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pone-0063043-g012: ADOTA-C16 in DOPC.(A) Fluorescence intensity decay (B) anisotropy decay.

Mentions: The results from fluorescence lifetime measurements in DOPC for both ADOTA and DAOTA hexadecyl derivatives are given in Table 5. The intensity and anisotropy decays for ADOA-C16 are shown in Figures 12A and 12B and of DAOTA-C16 in Figures 13A and 13B. The lifetime of main component was 18.5 ns in case of ADOTA-C16 and 14.6 ns for DAOTA C16 derivative. There is also a small population with a short lifetime, suggesting some heterogeneity of the environment. The heterogeneity in the intensity decays when in lipid environment in otherwise single lifetime probe has been observed with several fluorophores. Among possible explanations are presence of micro-heterogeneity in vesicles and less than random distribution of the fluorophore [33], [35]. Table 6 has results from anisotropy decays in DOPC. We can satisfactorily describe the anisotropy decays with two correlation times and a residual anisotropy often known as r∞. As expected in case of a disordered lipid DOPC the values of r∞, which reflects “order”, is small. It is less than 10% of total anisotropy in case of ADOTA-C16 and even less around 5% in case of DAOTA-C16. The recovered anisotropy of 0.278 and 0.246 for ADOTA and DAOTA respectively are smaller than their limiting anisotropy values due to tail fitting of anisotropy decays in the presence of nanosecond probe motions. We are time gating initial 150 ps and 1 ns in case of ADOTA-C16 and DAOTA-C16, respectively. If we were to keep this under-estimation of faster component in mind, the probes would seem to experience a faster and almost order of magnitude slower motions of equal amplitudes. It should be noted that our model of sum of correlation times and a residual anisotropy analysis [23] is often applied due to its simplicity. Models that are more physical are available which also tend to be more complex [24, 32, 34, and 35]. These results also compare favorably to those seen with long lifetime fluorescent fatty acids [33], [34]. The fluorescence lifetimes of these fatty acids are similar to the azaoxa-triangulenium fluorophores. However, though anchored in head-group/interface region by their carboxylic group the fluorescent part of the fatty acid is located in hydrocarbon core.


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)

ADOTA-C16 in DOPC.(A) Fluorescence intensity decay (B) anisotropy decay.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063043-g012: ADOTA-C16 in DOPC.(A) Fluorescence intensity decay (B) anisotropy decay.
Mentions: The results from fluorescence lifetime measurements in DOPC for both ADOTA and DAOTA hexadecyl derivatives are given in Table 5. The intensity and anisotropy decays for ADOA-C16 are shown in Figures 12A and 12B and of DAOTA-C16 in Figures 13A and 13B. The lifetime of main component was 18.5 ns in case of ADOTA-C16 and 14.6 ns for DAOTA C16 derivative. There is also a small population with a short lifetime, suggesting some heterogeneity of the environment. The heterogeneity in the intensity decays when in lipid environment in otherwise single lifetime probe has been observed with several fluorophores. Among possible explanations are presence of micro-heterogeneity in vesicles and less than random distribution of the fluorophore [33], [35]. Table 6 has results from anisotropy decays in DOPC. We can satisfactorily describe the anisotropy decays with two correlation times and a residual anisotropy often known as r∞. As expected in case of a disordered lipid DOPC the values of r∞, which reflects “order”, is small. It is less than 10% of total anisotropy in case of ADOTA-C16 and even less around 5% in case of DAOTA-C16. The recovered anisotropy of 0.278 and 0.246 for ADOTA and DAOTA respectively are smaller than their limiting anisotropy values due to tail fitting of anisotropy decays in the presence of nanosecond probe motions. We are time gating initial 150 ps and 1 ns in case of ADOTA-C16 and DAOTA-C16, respectively. If we were to keep this under-estimation of faster component in mind, the probes would seem to experience a faster and almost order of magnitude slower motions of equal amplitudes. It should be noted that our model of sum of correlation times and a residual anisotropy analysis [23] is often applied due to its simplicity. Models that are more physical are available which also tend to be more complex [24, 32, 34, and 35]. These results also compare favorably to those seen with long lifetime fluorescent fatty acids [33], [34]. The fluorescence lifetimes of these fatty acids are similar to the azaoxa-triangulenium fluorophores. However, though anchored in head-group/interface region by their carboxylic group the fluorescent part of the fatty acid is located in hydrocarbon core.

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