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. As the chemical buffer composition controls the transitions between non-fluorescent off- and fluorescent on-states, it is ultimately linked to the maximum achievable resolution. Here, we describe a workflow for screening optimal buffer conditions for single-molecule photoswitching. This is achieved by creating an intensity gradient within the field of view using a single micro-electromechanical systems (MEMS) mirror in the excitation path of the wide-field setup. Photon budget, on-state and off-state lifetimes are studied for different concentrations of mercatoethylamine (MEA) and buffer pH values. Both of MEA concentration and buffer pH determine the amount of thiolate, which is the main requirement for carbocyanine dye photoswitching. We show that thiolate acts as a concentration bandpass filter for the maximum achievable resolution and determine a minimum thiolate concentration 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.

An Optimized Buffer for Repeatable Multicolor STORM

“…Our initial assumption was that the ingredients of the buffer were chosen to ensure good blinking of Alexa-647, but we had to confirm this. We therefore immunostained αtubulin once again and tested three popular far-red dyes, Alexa-647, Dylight-649, and CF-647, and obtained high-quality images for all three with FRC resolutions in the 20−30 nm range, in line with values published in the literature with other buffers 21,25 as shown in Figures 4 and S3.…”

“…DABCO was previously used to improve the blinking of Cy3 in Vectashield and does not prevent the blinking of Alexa-647 when added to the canonical buffer To vary the pH, which is known to affect the blinking of dyes, − which is made simple by the use of sodium sulfite that does not lead to acidification and the possibility of preparing large volumes of buffers to accurately measure pH using a pH meter. To use water as the main solvent. While higher index media are very useful for 3D imaging, ,, they make TIRF imaging more difficult, and the majority of STORM imaging is performed in TIRF or grazing incidence with TIRF-based z -stabilization. …”

“…To vary the pH, which is known to affect the blinking of dyes, − which is made simple by the use of sodium sulfite that does not lead to acidification and the possibility of preparing large volumes of buffers to accurately measure pH using a pH meter.…”