Cyanine fluorophore derivatives with enhanced photostability

Journal name:
Nature Methods
Volume:
9,
Pages:
68–71
Year published:
DOI:
doi:10.1038/nmeth.1774
Received
Accepted
Published online

Fluorescence applications requiring high photostability often depend on the use of solution additives to enhance fluorophore performance. Here we demonstrate that the direct or proximal conjugation of cyclooctatetraene (COT), 4-nitrobenzyl alcohol (NBA) or Trolox to the cyanine fluorophore Cy5 dramatically enhanced fluorophore photostability without otherwise affecting its native spectral characteristics. Such conjugation is a powerful means of improving the robustness of fluorescence-based applications demanding long-lived, nonblinking fluorescence emission.

At a glance

Figures

  1. Enhancement of Cy5 photophysical properties through direct coupling to TSQs.
    Figure 1: Enhancement of Cy5 photophysical properties through direct coupling to TSQs.

    (a) General schematic of TSQ-conjugated fluorophore derivatives. (b) Average dwell times in the on state (τon) with individual TSQs in solution (TSQ, solution) or directly conjugated to Cy5 (Cy5-TSQ). Error bars, s.d. (n ≥ 6 movies from at least two independent experiments). (c) Representative traces of Cy5 fluorescence under direct excitation in the absence of TSQ (Cy5) and with COT, NBA or Trolox directly coupled to the dye.

  2. Role of proximity in the enhancement of Cy5 fluorescence with indirectly coupled TSQs.
    Figure 2: Role of proximity in the enhancement of Cy5 fluorescence with indirectly coupled TSQs.

    (a) Schematic of the TSQ proximity experiment. DNA duplexes were created with one strand labeled with Cy5 (red) at the 5′ end and TSQs (blue) 2, 5 and 8 nucleotides from the 3′ end on the complementary strand. Views from the side (top) and above (bottom) are shown. (b) τon for constructs in which an individual TSQ was linked proximally to the Cy5 fluorophore 2 base pairs distal to the terminus of the DNA duplex (TSQ-2) (top). τon examined with Trolox attached at positions 2, 5 and 8 nucleotides away from the terminus of the DNA duplex (bottom). Error bars, s.d. (n = 3 movies). (c) Spatial distributions sampled by Cy5 and proximal TSQs determined using molecular dynamics simulations of DNA duplexes with Cy5 (red) labeled at the 5′ end of one strand and one molecule of Trolox at one of three locations on the DNA helix: 2, 5 and 8 nucleotides (nt) from the 3′ end of the complementary strand. Isosurfaces of the spatial distribution sampled by Cy5 and Trolox are shown as translucent clouds about the DNA helix.

  3. Photostability of Cy5 and Cy5-TSQ conjugates in the presence of oxygen and in living cells.
    Figure 3: Photostability of Cy5 and Cy5-TSQ conjugates in the presence of oxygen and in living cells.

    (a,b) Photostability of Cy5- and Cy5-TSQ–conjugated DNA duplexes surface-immobilized at a saturating density and in the presence (a) and absence (b) of an oxygen-scavenging system. Decay curves were fit to a single exponential process, and the time constants are reported in Supplementary Table 1. (ce) Single-molecule total internal reflection fluorescence image sequences of living CHO cells containing dopamine D2 receptors labeled with Cy5 (c), labeled with Cy5-COT (d) and labeled with Cy5-COT and imaged in deoxygenated solution containing 1 mM PCA and 50 nM PCD (e). Scale bar, 5 μm.

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Author information

  1. These authors contributed equally to this work.

    • Daniel S Terry &
    • Zhou Zhou

Affiliations

  1. Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York, USA.

    • Roger B Altman,
    • Zhou Zhou &
    • Scott C Blanchard
  2. Tri-Institutional Training Program in Computational Biology and Medicine, Weill Medical College of Cornell University, New York, New York, USA.

    • Daniel S Terry &
    • Scott C Blanchard
  3. Tri-Institutional Training Program in Chemical Biology, Weill Medical College of Cornell University, New York, New York, USA.

    • Qinsi Zheng &
    • Scott C Blanchard
  4. Department of Psychiatry, Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York, USA.

    • Peter Geggier,
    • Rachel A Kolster,
    • Yongfang Zhao &
    • Jonathan A Javitch
  5. Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York, USA.

    • Jonathan A Javitch
  6. Department of Biochemistry, Weill Medical College of Cornell University, New York, New York, USA.

    • J David Warren &
    • Scott C Blanchard

Contributions

S.C.B., R.B.A. and D.S.T. designed in vitro experiments. Z.Z. and J.D.W. synthesized TSQ-conjugated fluorophores. R.B.A. made and purified complexes. R.B.A. performed single-molecule and bulk imaging experiments. Q.Z. performed bulk fluorescence and singlet-oxygen measurements. D.S.T. performed simulations. D.S.T., R.B.A. and S.C.B. analyzed in vitro data. S.C.B., J.A.J., and P.G. designed live-cell imaging experiments. Y.Z. and R.A.K. designed and constructed receptor constructs. P.G. performed the in vivo imaging and analyzed data. R.B.A., D.S.T. and P.G. designed figures. R.B.A. and S.C.B. wrote the manuscript, which all authors edited.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (594 KB)

    Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Note 1

Movies

  1. Supplementary Video 1 (27 MB)

    Photostability of Cy5-TSQ conjugates when linked to DNA duplexes. Shown are single-molecule TIRF movies of Cy5 (top left), Cy5-COT (top right), Cy5-NBA (bottom left), Cy5-Trolox (bottom right) taken at 10 frames s−1 with oxygen scavenging (as in Fig. 1). The movie is played at five times actual imaging speed. The elapsed time (in min:s) is displayed in the top left corner.

  2. Supplementary Video 2 (6 MB)

    Photostability of Cy5 and Cy5-COT–labeled dopamine D2 receptors (D2Rs) in living CHO cells (top left). Single-molecule TIRF video sequence of Cy5 labeled D2Rs. In a deoxygenated environment Cy5-labeled D2 receptors show enhanced photostability at the cost of higher blinking rates (top right). A direct linkage of COT to Cy5 improves the photostability (bottom left). The greatest photostability of D2Rs was observed by labeling with Cy5-COT and imaging in a deoxygenated environment (bottom right). All image sequences were recorded at a rate of 25 frames s−1.

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