Cyanine fluorophore derivatives with enhanced photostability

Abstract

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.

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 (τ_on) 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.

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) τ_on 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). τ_on 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.

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. (c–e) 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.