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. 2012;7(12):e51980.
doi: 10.1371/journal.pone.0051980. Epub 2012 Dec 14.

Ultra-bright and -Stable Red and Near-Infrared Squaraine Fluorophores for in Vivo Two-Photon Imaging

Free PMC article

Ultra-bright and -Stable Red and Near-Infrared Squaraine Fluorophores for in Vivo Two-Photon Imaging

Kaspar Podgorski et al. PLoS One. .
Free PMC article


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.

Conflict of interest statement

Competing Interests: The authors have read the journal’s policy and have the following conflict: Dr. Ewald Terpetschnig works for SETA BioMedicals, which produces and commercializes the dyes characterized in this manuscript. All characterization reported here was carried out by Podgorski and Haas independently of this company. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.


Figure 1
Figure 1. Squaraine derivatives with large two-photon action cross-sections in the NIR window.
a) Structure of squaraine-rotaxanes. Squaraines contain a characteristic squarylium core flanked by nucleophilic motifs, forming an electron Donor-Acceptor-Donor structure. The macrocyclic ‘cage’ sterically shields the more reactive squarylium core, increasing its stability. b) Two-photon action cross-sections of squaraine derivatives. Dye names with a prefix of K8 denote squaraines, K9 prefixes denote squaraine-rotaxanes. Cross-sections were obtained by ratiometric imaging and calculated using emission spectra measured from BSA-conjugate (K8-1342), IgG-conjugate (K8-1384), or free dye (all others).
Figure 2
Figure 2. Brightness and photostability of the squaraine-rotaxane SeTau-647 measured in vitro and in vivo.
a) Fluorescence emission spectrum of SeTau-647 (shaded curve) and two-photon action cross-section of SeTau-647, compared to published cross-sections of a bright organic dye (Rhodamine B, red) and fluorescent protein (eGFP, green). For SeTau-647, data in closed circles were obtained by ratiometric fluorescence measurement, and data in open circles were obtained by Z-scan, which is less sensitive to single-photon phenomena. b) Simultaneous two-photon photobleaching of SeTau-647 and Alexa 488 at 920 nm in water. Dashed lines are fitted mono-exponential decay curves. The abscissa is the product of illumination time with the two-photon action cross-section of each dye, proportional to number of photons emitted per dye molecule. c-e) Photobleaching of SeTau-647 and Alexa 594 dextran conjugates in neuronal dendrites in vivo. c) Neurons were electroporated with 1 mM Alexa 594- (top) or SeTau-647- ( bottom ) dextran, and distal dendritic segments were imaged at the minimum laser intensity which allowed clear visualization. The 1st (left) and 60th (right) consecutive images are shown. d) Timecourse of photobleaching in example dendritic segments loaded with Alexa 594 (red) or SeTau-647 (blue) indicating. Lines are fitted monoexponential decay curves. Intensities have been normalized to the maximum and minimum of the fit curve. e) Distribution of fitted photobleaching rate (τ) for neurons loaded with Alexa 594 or SeTau-647. Each data point is the median fit bleaching rate over all dendritic segments imaged in one neuron. Horizontal bars denote the mean of each distribution.
Figure 3
Figure 3. Cellular and subcellular labelling with SeTau-647 in vivo.
a) Z-projection images acquired 3 hours (left) and 5 days (right) after single-cell electroporation with SeTau-647-dextran. Images were acquired at an excitation power of 10 mW at the back aperture of the objective (<8 mW at the focal plane). b) Targeted electroporation of a squaraine-tagged PDZ binding peptide labels postsynaptic density protein PSD-95. ( top ) Z-projection of a neuron filled with OGB-1 (green) and the squaraine-tagged peptide (red). ( bottom ) Expanded views of dendritic regions numbered above show punctate labelling of SeTau-647 (arrows). c) Anti-PSD-95 puncta colocalize with SeTau puncta in labelled tectal neuron dendrites. ( left ) Anti-PSD-95 immunostaining (green) of brain section containing dendrites of neurons labelled with SeTau PDZ-binding peptide (red) and Cascade Blue dextran (blue) as a space filler. ( right ) Expanded views of dendritic regions numbered at left. Scale bars: 10 µm.

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