노동연 (POSTECH)

서종철 (POSTECH)

노동연 (POSTECH)

서종철 (POSTECH)

A Sulfo-Cyanine 5 (SCy5) is an indocarbocyanine fluorophore widely used in high-resolution fluorescence imaging. Upon light irradiation, SCy5 can undergo photoblueing through the apparent excision of C2H2, producing an SCy3-like chain-truncated product. Although this photoconversion can cause spectral crosstalk and signal loss in fluorescence imaging, the chemical processes underlying this phenomenon remain poorly understood. Conventional spectroscopic methods typically analyze samples after the reaction has been completed, making it difficult to capture short-lived intermediates and determine how individual reaction pathways evolve over time.

Here, we developed a laser-coupled system that irradiates a nanoelectrospray emitter with a continuous-wave laser during mass spectrometric analysis. By combining controlled photoexcitation with online nanoelectrospray ionization–mass spectrometry (nESI–MS), we directly intercepted transient intermediates and monitored changes in their relative ion abundances throughout SCy5 photoconversion.

Isotope-pattern analysis supported two coexisting oxygen-dependent pathways. A slow, non-radical oxygen-addition pathway became more prominent at later reaction times, whereas a distinct radical-mediated pathway emerged rapidly during the initial stage. Notably, an early oxygen-addition species was assigned to a superoxide radical intermediate, providing evidence for a previously unresolved radical-mediated pathway. This interpretation was further supported by pH-dependent tests, radical-quencher tests, and UV/H2O2 radical-generation tests. Together, these results demonstrate that laser-coupled in situ nESI–MS can distinguish parallel photochemical pathways and capture short-lived intermediates during SCy5 photoconversion.