Methylene Blue- and Thiol-Based Oxygen Depletion for Super-Resolution Imaging

Abstract

Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions with the aim of designing an oxygen scavenger system as an alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

“…For d STORM imaging with Cy5, the sample was embedded in photoswitching buffer, that is, 100 mM mercaptoethylamine, pH 8.0, enzymatic oxygen scavenger system (5% (wt/vol) glucose, 5 U ml −1 glucose oxidase and 100 U ml −1 catalase59) and mounted on an inverted microscope (Olympus IX-71) equipped with an oil-immersion objective (60 × , numerical aperture 1.45, Olympus) and a nosepiece stage (IX2-NPS, Olympus)22. For excitation of Cy5, a 641-nm diode laser (Cube 640–100C, Coherent) was used.…”

Twinkle, twinkle little star: Photoswitchable fluorophores for super‐resolution imaging

“…The use of various additives to form an 'imaging cocktail' is an important part of many of these techniques, but often the effects are difficult to interpret since the additives may function in multiple ways. For example, thiols are often added to imaging cocktails and may function as a: triplet state quencher [107]; ligand that forms a non-fluorescent adduct with a dye [91]; reductant which can produce a long-lived, non-fluorescent radical species [92]; deoxygenation agent by reacting with oxygen to form disulfides and water [108]; or perhaps a mixture of these or other yet unidentified roles. The introduction of an oxygen scavenging system can lower oxygen concentrations substantially, but can lead to different outcomes.…”

The bright future of single-molecule fluorescence imaging

“…Although molecular oxygen is a strong triplet state quencher that tends to suppress triplet-induced blinking events, the oxidation of the fluorophore by molecular oxygen is particularly detrimental to single-molecule imaging [35,36], as it generates a highly reactive superoxide radical (O 2 - ) along with a cationic radical form of the fluorophore that are susceptible to photobleaching The common practice of removing molecular oxygen from the imaging buffer through enzymatic oxygen scavenging systems [37-40] therefore has the ambivalent result of improving overall fluorophore lifetimes at the expense of increased triplet-induced blinking.…”