노동연 (POSTECH)

서종철 (POSTECH)

노동연 (POSTECH)

장성재 (POSTECH)

서종철 (POSTECH)

 Cyanine dyes have unique molecular arrangement that give them special properties such as electronic structure. Also, cyanine dyes are key fluorescent markers in modern science and biology. They are widely used in single-molecule research and high-resolution imaging. Cationic cyanine dyes, the most well-known group of methine dyes, features a structure in which two heterocyclic nitrogen-containing rings are connected by a polymethine chain at both ends of the chromophore. The length of the chain primarily determines the optical properties of the dyes. A recent remarkable study demonstrated that the conversion of Cy5 to Cy3 can occur through an intermolecular reaction using a 642 nm visible laser.1 In this study, we used an instrument optimized for direct irradiation of the nano electrospray source with a 300mW continuous wave laser. This setup was coupled with nano Electrospray Ionization – Ion Mobility Spectrometry – Mass Spectrometry (nESI – IMS – MS). This combination allowed us to analyze photoconversion in situ and detect intermediates with short lifetimes. We found that gas-phase Cy5 ions exhibit multiple conformations, as separated by different arrival time distributions (ATDs) with varying drift times (DTs) in IMS-MS. We also discovered that only one specific conformation uniquely participates in the reaction. Additionally, by analyzing the real-time conversion between reactants, intermediates, and products, we provide experimental evidence for a promising mechanism. Understanding this mechanism helps make fluorescent dyes more reliable, improves data accuracy, and aids in developing new imaging techniques.

1. Cho, Yoonjung, et al. "Mechanism of cyanine5 to cyanine3 photoconversion and its application for high-density single-particle tracking in a living cell." Journal of the American Chemical Society 143.35 (2021): 14125-14135.