Abstract
Covalent heterodimers of the Cy3 and Cy5 fluorophores have been prepared from commercially available starting materials and characterized at the single-molecule level. This system behaves as a discrete molecular photoswitch, in which photoexcitation of the Cy5 results in fluorescence emission or, with a much lower probability, causes the Cy5 to enter into a long-lived, but metastable, dark state. Photoinduced recovery of the emissive Cy5 is achieved by very low intensity excitation (5 W cm(-2)) of the Cy3 fluorophore at a shorter wavelength. A similar system consisting of proximal, but not covalently linked, Cy3 and Cy5 has found application in stochastic optical reconstruction microscopy (STORM), a single-molecule localization-based technique for super-resolution imaging that requires photoswitching. The covalent Cy3-Cy5 heterodimers described herein eliminate the need for probabilistic methods of situating the Cy3 and Cy5 in close proximity to enable photoswitching. As proof of principle, these heterodimers have been applied to super-resolution imaging of the tubular stalk structures of live Caulobacter crescentus bacterial cells.
🔬 Techniques
✨ Fluorophores
🏛️ Research Organizations (ROR)
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📊 Figures
Figure 1
Absorption (solid) and fluorescence emission (dashed, u03bb ex =516 nm) spectra of Cy3-Cy5 covalent heterodimers 4 and 5 (in water; 3.7 u00b5M for absorption; 37 nM for fluorescence) before photodarke...
Figure 2
Representative SM fluorescence time traces of 5 covalently bound to an aminosilane-terminated glass coverslip. The molecules were imaged continuously beginning at t = 0 s with a red laser (633 nm, 150...
Figure 3
(A) Representative SM fluorescence time trace of 5 -labeled BSA showing reactivation numbers 12u201316, denoted by the red dashed lines. (B) Fluorescence images at times 1u20133 corresponding to the p...
Figure 4
Super-resolution fluorescence image of C. crescentus stalks (yellow) with 30.3 nm resolution superimposed on a white-light image of the cells. As discussed in the Supporting Information , the Caulobac...
CHART 1
Structures of Reactive Cyanine Dyes and Covalent Heterodimers
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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