Benchmarking Thiolate-Driven Photoswitching of Cyanine Dyes

Abstract

Carbocyanines are among the best performing dyes in single-molecule localization microscopy (SMLM), but their performance critically relies on optimized photoswitching buffers. Here, we study the versatile role of thiols in cyanine photoswitching at varying intensities generated in a single acquisition by a microelectro­mechanical systems (MEMS) mirror placed in the excitation path. The key metrics we have analyzed as a function of the thiolate concentration are photon budget, on-state and off-state lifetimes and the corresponding impact on image resolution. We show that thiolate acts as a concentration bandpass filter for the maximum achievable resolution and determine a minimum of ∼1 mM is necessary to facilitate SMLM measurements. We also identify a concentration bandwidth of 1–16 mM in which the photoswitching performance can be balanced between high molecular brightness and high off-time to on-time ratios. Furthermore, we monitor the performance of the popular oxygen scavenger system based on glucose and glucose oxidase over time and show simple measures to avoid acidification during prolonged measurements. Finally, the impact of buffer settings is quantitatively tested on the distribution of the glucose transporter protein 4 within the plasma membrane of adipocytes. Our work provides a general strategy for achieving optimal resolution in SMLM with relevance for the development of novel buffers and dyes.

FRET Imaging of Nonuniformly Distributed DNA SAMs on Gold Reveals the Role Played by the Donor/Acceptor Ratio and the Local Environment in Measuring the Rate of Hybridization

“…The AF488 fluorophore excited state can be quenched via FRET with AF647 in addition to a distance dependent quenching by the gold surface, so a correlation to coverage is limited to cases where the FRET efficiency is low. On the other hand, AF647 intensities are only quenched by the gold surface and should in principle be directly correlated to coverage, but study of the photophysics of AF647 has shown the formation of dark states , which limits its use as a measure of coverage. This is clear when analyzing the AF647 ssDNA SAM image which shows a darker than usual central portion of the bead where the 111 facet has a significantly lower intensity when compared to the 111 facet on the side of the bead.…”

“…It appears that FRET intensities do not suffer from the decrease in AF647 fluorescence due to this dark state. This can be explained recognizing that for FRET imaging, a 480 nm excitation is used which has been reported to facilitate the depopulation of the AF647 dark state back into its ground state. , The same effect is apparently occurring in these FRET images. Separate measurements of a 100% AF488 modified DNA were performed and showed a very similar pattern to the AF488 fluorescence images in the mixed composition layers suggesting that the FRET quenching of AF488 intensity may not be as significant (see Figure S10).…”

GLUT4 dispersal at the plasma membrane of adipocytes: a super-resolved journey

“…The first methods for achieving SMLM were shown with photo-activated localisation microscopy (PALM), where the specimen was imaged and subsequently bleached through depth [ 50 ], and stochastic orientation reconstruction microscopy (STORM), which used photo-switchable fluorophores to image single molecules over several thousand image frames [ 51 ]. Subsequently, the use of conventional cyanine dyes with specialised buffer solutions became increasingly popular [ 52 ], allowing for direct STORM (dSTORM), the direct nature coming from the fact that fluorescent antibody pairs are not required [ 53 , 54 ]. More recently, fluorescent transiently-binding single DNA strands have re-emerged as a versatile tool, allowing for improved single molecule localisation with the development of point accumulation in nanoscale topography (DNA-PAINT) [ 55 ].…”

Photoswitching Reagent for Super‐Resolution Fluorescence Microscopy

“…However, these studies focused only on thiol-induced photoswitching, particularly during the switchingoff step. [20][21][22][23][24] For example, a reversible transition occurs between the fluorescent and dark transient states, in which the formation of a nonemissive cyanine dye-thiolate (CyÀ SR) adduct is mainly responsible for the switching-off step. However, the detailed mechanism by which the fluorescent state is recovered remains unclear.…”

“…To reconcile the seemingly divergent results reported by several groups, efforts have recently been made to unify the different mechanisms for thiol‐induced photoswitching of cyanine dyes, which are the most utilized dyes for SMLM. However, these studies focused only on thiol‐induced photoswitching, particularly during the switching‐off step [20–24] . For example, a reversible transition occurs between the fluorescent and dark transient states, in which the formation of a nonemissive cyanine dye‐thiolate (Cy−SR) adduct is mainly responsible for the switching‐off step.…”