Targetable Conformationally Restricted Cyanines Enable Photon‐Count‐Limited Applications**

Abstract

Cyanine dyes are exceptionally useful probes for a range of fluorescence‐based applications, but their photon output can be limited by trans‐to‐cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA‐nanoantenna, in single‐molecule Förster‐resonance energy transfer (FRET) applications, far‐red fluorescence‐lifetime imaging microscopy (FLIM), and single‐molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.

Photochemical Mechanisms of Fluorophores Employed in Single‐Molecule Localization Microscopy

“…It was, however, photoactivatable after incubation in NaBH 4 ‐containing buffer and was used for TRABI‐BP SMLM. Cy5B has since been used in PALM and shown to give significantly improved resolution (13.5 nm) compared to Cy5 (17.7 nm) [22a] . These reductively caged and caging‐group‐free (PaX) strategies avoid the release potentially biologically active or toxic molecules into the sample.…”

Photochemical Mechanisms of Fluorophores Employed in Single‐Molecule Localization Microscopy

“…[21] In addition to the crucial ability of fluorophores to switch between bright (emissive) and dark (non-emissive) states, other fluorophore properties such as photon count, fluorophore stability, and duty cycle influence the localization precision of individual molecules as well as the resolution achieved. [22] A wide range of switchable fluorophores have been utilized for SMLM, including synthetic organic fluorophores and fluorescent proteins. Small-molecule fluorophores are generally preferred over fluorescent proteins because they tend to have higher quantum yields and greater photostability.…”

Heptamethine Cyanine Dyes with Ultra‐Efficient Excited‐State Nonradiative Decay for Synergistic Photothermal Immunotherapy

“…[ 12 ] Several molecular design strategies have been used to improve the photostability of cyanine dyes during PTT. [ 13 ] Installing a sterically shielded group at the meso position of cyanine dyes [ 14 ] or electron‐withdrawing groups (EWGs) at the α ‐carbon of the central polymethine chain are a few examples. [ 15 ] Although these approaches improve photostability, their photophysical properties, such as blue‐shifted maximum absorption wavelength, are always compromised, which is incompatible with clinical translation.…”