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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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Fluorescence lifetime imaging (FLIM) of the 4T1 endothelial cell line.(A) Autofluorescence image (B) Autofluorescence image after time gating.
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pone-0063043-g017: Fluorescence lifetime imaging (FLIM) of the 4T1 endothelial cell line.(A) Autofluorescence image (B) Autofluorescence image after time gating.

Mentions: The results with ADOTA-C16 are shown in Figures 15 A–B and Figure 16 A–B, while that of DAOTA-Arg are in Figure 17A–B and 18 A–B. A breast cancer endothelial cell line 4T1 was used to collect these images. All four panes in the Figures 15, 16, 17, and 18 are made up of three parts. The left most panel shows photon counts and associated fluorescence lifetimes for cell autofluorescence. The middle part is an intensity image and the right most is a lifetime image of same cells. The auto-fluorescence signal from the cells can be satisfactorily described by two lifetimes of 1.3 ns and 3.2 ns. We can see that after 10 ns almost all of the fluorescence has disappeared (Figure 15A). We therefore used a gate of 10 ns and discarded all the photons collected before this point to construct time gated FLIM images.The results of this 10 ns time gating are shown in panel B of the figure 15. The left most image has few residual photons as result of the near complete elimination of autofluorescence signal with the time gating. The intensity (middle) and the lifetime (right most) images are now essentially black. Figure 16 panels A and B depict the FLIM images collected from cells that were stained with 5 nM ADOTA-C16 solution. We now observe presence of an additional relatively long-lived component of 15.6 ns, which also is the dominant source of photons (Pane A). As expected the middle image is bright with fluorescence signal covering significant parts of cell surface. As this long-lived component affects the average lifetimes calculated for each pixel it results in making the false color in the lifetime image appear more yellow/red (right most figure). When time gating is applied as before, the two short-lived components attributed to auto-fluorescence are eliminated leaving only the long-lived ADOTA signal (Pane B left most panel). The intensity is significantly reduced in middle panel and fluorescence is now confined to cell membrane area. The color of the FLIM image in right most part is now noticeably redder, as the blue was removed with the time gating. It should be emphasized that 10 ns time gating not only eliminates auto-fluorescence but also the initial part of ADOTA fluorescence decay. The result is dimmer fluorescence images. However, we also obtain a significantly improved signal contrast when compared to non-gated images (right most panels A vs B)


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)

Fluorescence lifetime imaging (FLIM) of the 4T1 endothelial cell line.(A) Autofluorescence image (B) Autofluorescence image after time gating.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063043-g017: Fluorescence lifetime imaging (FLIM) of the 4T1 endothelial cell line.(A) Autofluorescence image (B) Autofluorescence image after time gating.
Mentions: The results with ADOTA-C16 are shown in Figures 15 A–B and Figure 16 A–B, while that of DAOTA-Arg are in Figure 17A–B and 18 A–B. A breast cancer endothelial cell line 4T1 was used to collect these images. All four panes in the Figures 15, 16, 17, and 18 are made up of three parts. The left most panel shows photon counts and associated fluorescence lifetimes for cell autofluorescence. The middle part is an intensity image and the right most is a lifetime image of same cells. The auto-fluorescence signal from the cells can be satisfactorily described by two lifetimes of 1.3 ns and 3.2 ns. We can see that after 10 ns almost all of the fluorescence has disappeared (Figure 15A). We therefore used a gate of 10 ns and discarded all the photons collected before this point to construct time gated FLIM images.The results of this 10 ns time gating are shown in panel B of the figure 15. The left most image has few residual photons as result of the near complete elimination of autofluorescence signal with the time gating. The intensity (middle) and the lifetime (right most) images are now essentially black. Figure 16 panels A and B depict the FLIM images collected from cells that were stained with 5 nM ADOTA-C16 solution. We now observe presence of an additional relatively long-lived component of 15.6 ns, which also is the dominant source of photons (Pane A). As expected the middle image is bright with fluorescence signal covering significant parts of cell surface. As this long-lived component affects the average lifetimes calculated for each pixel it results in making the false color in the lifetime image appear more yellow/red (right most figure). When time gating is applied as before, the two short-lived components attributed to auto-fluorescence are eliminated leaving only the long-lived ADOTA signal (Pane B left most panel). The intensity is significantly reduced in middle panel and fluorescence is now confined to cell membrane area. The color of the FLIM image in right most part is now noticeably redder, as the blue was removed with the time gating. It should be emphasized that 10 ns time gating not only eliminates auto-fluorescence but also the initial part of ADOTA fluorescence decay. The result is dimmer fluorescence images. However, we also obtain a significantly improved signal contrast when compared to non-gated images (right most panels A vs B)

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