Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging

Abstract

We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quantification of proteins in the inner mitochondrial membrane with approximately 20 nm optical resolution.

Origin and compensation of imaging artefacts in localization-based super-resolution microscopy

“…Multicolour localization microscopy requires multiple labelling and sequential [34] or simultaneous [35] excitation and localization of fluorescent molecules. In case of simultaneous excitation a dichroic mirror is applied to separate fluorescent light emitted by different dyes.…”

Fluorescence nanoscopy. Methods and applications

“…Nanoscopic imaging of multiple fluorophores in the same sample (multicolour PALM or STORM) based on the different emission or activation wavelength was readily achieved after these techniques had been first demonstrated with either two or a single imaging wavelength [45][46][47][48][49]; up to four fluorophores can be discriminated with these strategies [50,51]. Combinatorial pairing of photoswitchable tags with fluorophores that enhance photoactivation enabled subdiffraction multiparameter detection of six different probes [52].…”

Single-molecule Photoswitching and Localization

“…[31,41,72] In dSTORM millimolar concentrations of thiols such as b-mercaptoethylamine, glutathione (GSH), or dithithreitol in aqueous solvents at pH ,7-8 are used to reduce the triplet state of commercially available ATTO and Alexa Fluor fluorophores and generate stable non-fluorescent dark states, e.g., radical ions, with lifetimes of several hundred milliseconds to seconds. [31,72,73] The fluorescent state of the rhodamine and oxazine fluorophores is quantitatively recovered upon oxidation by molecular oxygen naturally present in aqueous solvents at concentrations of 200-250 mM at room temperature, [31] or photoinduced in an oxygen depleted solution for cyanine dyes. [32] The Role of Stable Non-fluorescent Off-states of Fluorophores…”