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
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- Supplementary Text and Figures (594 KB)
Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Note 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.
- 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.