Single-molecule characterization of a bright and photostable deep-red fluorescent squaraine-figure-eight (SF8) dye

Squaraine Figure Eight (SF8) dyes are a unique class of deep-red fluorescent dyes with self-threaded molecular architecture that provides structural rigidity while simultaneously encapsulating and protecting the emissive fluorochrome. Previous cell microscopy and bulk phase studies of SF8 dyes indicated order of magnitude en-hancements in photostability over conventional pentamethine cyanine dyes such as Cy5. Studies conducted at the single molecule level now reveal that these ensemble level enhancements carry over to the single molecule level in terms of enhanced emission quantum yields, longer times to photobleaching, and enhanced total photon yields. When compared to Cy5, the SF8-based dye SF8(D4)2 possesses a three-fold larger single molecule emission quantum yield, exhibits order of magnitude longer average times before photobleaching, and exhibits twenty times larger photon yields. Additional features such as water solubility, fluorochrome encapsulation to protect it against nucleophilic attack, and selective biomarker targeting capability make SF8-based dyes prom-ising candidates for biological labeling and microscopy applications and single molecule tracking.

Automated summary

Squaraine Figure Eight (SF8) dyes are deep-red fluorescent dyes with unique self-threaded molecular architecture, providing structural rigidity and protection for the fluorochrome. Studies have shown that SF8 dyes have significantly higher photostability and improved emission quantum yields, longer times to photobleaching, and enhanced total photon yields at the single molecule level compared to conventional dyes. SF8-based dye SF8(D4)2 exhibits three-fold larger emission quantum yield, order of magnitude longer average times before photobleaching, and twenty times larger photon yields compared to Cy5. These features, along with water solubility, fluorochrome encapsulation, and biomarker targeting capability, make SF8-based dyes promising for biological labeling, microscopy, and single molecule tracking applications.