Photophysics of Organic Fluorophore Photobluing and Its Applications in Fluorescence and Super-Resolution Microscopy

ConspectusPhotoconversion─the light-induced shift of a fluorophore's absorption and emission spectra─has attracted increasing attention across diverse fields, including single-molecule spectroscopy and super-resolution microscopy. Although blinking and photobleaching are well-established photophysical phenomena of organic dyes and have been extensively studied, photoconversion has historically received comparatively limited attention. First reported decades ago in rhodamine dyes, photoconversion has since been identified in a broad range of organic fluorophores and has become increasingly evident with advances in fluorescence imaging technologies. Unintended photoconversion can introduce spectral crosstalk and lead to misinterpretation in multicolor experiments, motivating intensified efforts to elucidate its underlying mechanisms and develop effective mitigation strategies. In this Account, we focus on the occurrence and molecular mechanisms of photoconversion in rhodamine- and cyanine-based dyes, which are widely used in commercial fluorescent probes, and discuss practical approaches to suppress its impact in fluorescence experiments. Beyond the challenge of controlling unwanted spectral shifts, we also highlight emerging opportunities to exploit photoconversion as an alternative form of "photo-switching" for biophysical applications. We first provide a historical overview of photoconversion phenomena observed in organic fluorophores, including rhodamine, cyanine, and other synthetic dye families. We then summarize the current state of research on photoconversion mechanisms. In rhodamine dyes, photobluing proceeds primarily through progressive N-dealkylation upon irradiation, which has motivated the development of synthetic strategies aimed at producing photoconversion-resistant rhodamine derivatives. Studies on cyanine dyes have revealed that photoconversion requires singlet oxygen and is strongly modulated by buffer pH. Notably, we have demonstrated by NMR spectroscopy that the photoconverted form of Cy5 is structurally identical to Cy3, a troubling observation in light of the widespread use of the Cy3-Cy5 pair as a fluorophore combination for single-molecule fluorescence resonance energy transfer (smFRET) without awareness of the underlying photoconversion. For the C₂H₂ excision reaction that converts Cy5 into Cy3, both intramolecular and intermolecular mechanisms have been proposed. Here, we present detailed experimental evidence supporting each photobluing pathway and offer mechanistic insights into why two distinct routes can emerge. Depending on the specific cyanine dye and experimental conditions, either pathway may dominate, or both pathways may operate concurrently within the same reaction system. We then focus on the consequences of photobluing for smFRET measurements. Building on our initial observation that even ambient light can induce Cy5 photobluing, we demonstrate that this process not only generates false donor signals but can also produce spurious FRET efficiencies. Finally, we highlight emerging applications that deliberately harness photoconversion as a new class of tools for biophysical research. By addressing its potential, we aim not only to facilitate the regulation of photoconversion in commercial dyes but also to accelerate its purposeful exploitation as a new asset in super-resolution microscopy and single-molecule spectroscopy.