Carbocyanine Dyes as Efficient Reversible Single-Molecule Optical Switch

Abstract

We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher beta-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of >90% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.

Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions

“…4A), indicating that this feature arises from emission fluctuations of the dye molecules themselves rather than conformational changes affecting the FRET coupling. This interpretation is supported by similar observations in singlemolecule studies of dye molecules (32,33). A three-exponential fit, keeping two exponentials fixed at 90 and 250 s to model the slow step, yielded a correlation time of 20 Ϯ 5 s for the fast process, shown as a solid line in Fig.…”

Photoswitching microscopy with standard fluorophores

“…Recently, we demonstrated a new photoswitching microscopy method that relies on the use of organic fluorophores and efficient oxygen removal as well as addition of millimolar concentrations (50-100 mM) ß-mercaptoehtylamine [7,11,15]. Because our approach uses conventional cyanine dyes (Cy5, Alexa 647) for cellular staining and is not relying on the proximity of two fluorophores, both being attached to an antibody in a specific ratio and distance as is required for STORM [8,16], it is marked as "direct," dSTORM.…”

Fluorescence nanoscopy. Methods and applications

“…Many green, red and NIR-emitting standard fluorophores (e.g. most carbocyanines, Alexa and Atto dyes [37,38,148,149]) can undergo switching. A recent report has evaluated 26 synthetic fluorophores on the basis of the emitted photons per switching event, on-off duty cycle, photostability and number of switching events, identifying as bestperforming dyes for STORM imaging Atto488 for the blue spectral window, Cy3B for the yellow window, Alexa 647, Cy5 and Dyomics 654 for the red and DyLight 750, Cy7 and Alexa 750 for the NIR window [150].…”