Single molecule localization microscopy (SMLM) is a powerful method to achieve high spatial resolution beyond the light’s diffraction limit, and its development sparked a revolution in biological imaging. Photoswitchable fluorescent organic dyes are required for SMLM and extant probes are currently divided into three categories: dSTORM dyes, photoactivatable or “caged” dyes, and spontaneously blinking dyes. dSTORM allows traditional dyes to photoswitch, but only under intense illumination in the presence of reducing agents and oxygen scavengers. This complicates their use in live-cells and makes SMLM multicolor imaging difficult as each fluorophore requires specific environmental conditions to switch. Caged dyes do not require specialized buffers and are better for live-cell single particle tracking experiments, but the requisite violet photoactivation light contributes to cellular phototoxicity and increased background signal. Finally, spontaneously blinking (SB) dyes undergo unassisted photoswitching under biologically relevant conditions. Here, we describe a universal method to incorporate spontaneous blinking behavior in the widely-used Alexa Fluor™ (AF) and Janelia Fluor® (JF) scaffolds, through a simple chemical modification. The blinking rate of these SB dyes can be further controlled through either light irradiation or slight pH changes. We have demonstrated the utility of these SB dyes in different types of imaging experiments: live-cell, fixed-cell, and multicolor SMLM, utilizing different labeling strategies: immunostaining, small-molecule ligands, and self-labeling enzymes.

Spontaneously blinking dyes for single molecule localization microscopy

2019 Spring National Meeting

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