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Ultra-bright and -stable red and near-infrared squaraine fluorophores for in vivo two-photon imaging.

ABSTRACT: Fluorescent dyes that are bright, stable, small, and biocompatible are needed for high-sensitivity two-photon imaging, but the combination of these traits has been elusive. We identified a class of squaraine derivatives with large two-photon action cross-sections (up to 10,000 GM) at near-infrared wavelengths critical for in vivo imaging. We demonstrate the biocompatibility and stability of a red-emitting squaraine-rotaxane (SeTau-647) by imaging dye-filled neurons in vivo over 5 days, and utility for sensitive subcellular imaging by synthesizing a specific peptide-conjugate label for the synaptic protein PSD-95.

SUBMITTER: Podgorski K

PROVIDER: S-EPMC3522634 | biostudies-other | 2012

REPOSITORIES: biostudies-other

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Ultra-bright and -stable red and near-infrared squaraine fluorophores for in vivo two-photon imaging.

Podgorski Kaspar K Terpetschnig Ewald E Klochko Oleksii P OP Obukhova Olena M OM Haas Kurt K

PloS one 20121214 12

Fluorescent dyes that are bright, stable, small, and biocompatible are needed for high-sensitivity two-photon imaging, but the combination of these traits has been elusive. We identified a class of squaraine derivatives with large two-photon action cross-sections (up to 10,000 GM) at near-infrared wavelengths critical for in vivo imaging. We demonstrate the biocompatibility and stability of a red-emitting squaraine-rotaxane (SeTau-647) by imaging dye-filled neurons in vivo over 5 days, and utili ...[more]

PMID: 23251670

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Project description: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.

Dataset Information

Ultra-bright and -stable red and near-infrared squaraine fluorophores for in vivo two-photon imaging.

Publications

Ultra-bright and -stable red and near-infrared squaraine fluorophores for in vivo two-photon imaging.

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