Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single Molecule Localization Microscopy

Abstract

<p>The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multi-step sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.</p>

Cyanine Phototruncation Enables Cell Labeling with Spatiotemporal Control

“…Building on our prior observation that additives, pH and buffers dramatically impact phototruncation yield, we hypothesized that polar functional group on the cyanine chromophore might impact reaction conversion in neutral aqueous conditions. 36 We find that 3'-OMe-substitution on the polymethine chain leads to a dramatic increase (~ 8×) under physiological conditions. We then demonstrate that PACT can be implemented in vivo through intratumoral injection of a 3'-OMe-variant of the celltracking DiR dye to track immune cell migration into TDLNs.…”

“…35 This chemistry, mediated by singlet oxygen, involves the formal excision of the ethene diradical from the polymethine chain resulting in the two-carbon truncated homologue. 36 We demonstrated that cyanine phototruncation could be applied for single-molecule localization microscopy (SMLM) applications. 36 However, the modest yield of the cyanine photoconversion reaction in neutral buffer limited the scope of this photoconversion chemistry.…”

“…36 We demonstrated that cyanine phototruncation could be applied for single-molecule localization microscopy (SMLM) applications. 36 However, the modest yield of the cyanine photoconversion reaction in neutral buffer limited the scope of this photoconversion chemistry. Here we report the development of phototruncation-assisted cell tracking (PACT) (Figure 1).…”

“…In our prior report, we proposed a computationally supported mechanism involving an exothermic 4-membered-ring-driven elimination of oxidized ethene. 36 This involved the asynchronous attack of 1 O2 at C1' on the polymethine. However, the formation of the non-substituted pentamethine suggests that 1 O2 attacks at C3' on the polymethine, presumably due to the increased electrophilicity imposed by 3'-OMe substitution.…”

Mechanism of Cyanine5 to Cyanine3 Photoconversion and Its Application for High-Density Single-Particle Tracking in a Living Cell

“…An intramolecular pathway is also a possibility (Figure S16b), 45 particularly for the photoconversion of Cy5 singly labeled DNAs that has been performed with an exceedingly low concentration of Cy5. We tested if the enhanced photoconversion of the dual-labeled dT0 relative to the singly labeled DNAs was due primarily to the increased number of sensitizers, which led to a higher local concentration of singlet oxygen that served as the limiting factor of the photoconversion process.…”