Energy transfer leaves fingerprints in cyanine photoswitching behavior

Super-resolution microscopy resolves molecular structures in biological systems but is limited by properties of the fluorescent labels. dSTORM experiments with multiple Cy5 fluorophores in sub-10 nm spaced labelling positions have shown distinct cumulative localization counts over time, depending on the distances between the fluorophores, that suggest Förster resonance energy transfer between ON states and long living OFF states. This photophysical effect hinders precise localization of the individual emitters but produces a photoswitching fingerprint that can be used to draw conclusions about sub-10 nm spatial conformations in molecular structures that are not spatially resolvable. Here we present a theoretical framework for analysing the photophysical systems that yield distinct fingerprint signatures. We developed a Python-based continuous-time Markov chain simulation software package that reproduces the photophysical processes of organic fluorophores. We show that the established photophysical models of Cy5 extended by Förster resonance energy transfer between the excited singlet state and the long living OFF state explain experimental fingerprint signatures as seen in dSTORM experiments. Matching experimental signatures including fluorescence lifetimes provides evidence for an additional energy transfer to the radical anion of cyanine dyes like Cy5. This work contributes to the understanding of proximity-based photophysical processes and paves the way for future development of sub-10 nm dSTORM imaging.Author summary: The spatial resolution in fluorescence microscopy is physically limited at a few hundred nanometres. State-of-the-art super-resolution microscopy techniques provide means to resolve smaller molecular structures at the nanometre length scale. However, superior image resolution requires dense labelling schemes such that fluorescent labels are often spaced at distances that allow for non-radiative energy transfer. In single-molecule localization microscopy (SMLM), a super-resolution technique that builds on stochastic activation of single-molecules for precise spatial localization, Förster resonance energy transfer can report on sub-10 nm distances and influence single-molecule fluorescence blinking. The notion that Förster resonance energy transfer between the excited singlet state of organic fluorophores and long living OFF states that are required for SMLM, can explain altered blinking dynamics is fundamental for understanding experimental observations and improving SMLM imaging capabilities. Here we present a photophysical model for the organic fluorophore Cy5 in different conditions including those for SMLM to quantify sub-10 nm interactions. Precise knowledge about the photophysical model with proximity-based photophysical processes is used to gain information about the local densities of fluorescent labels in sub-10 nm SMLM imaging and thus improve SMLM imaging at the molecular scale.