Photoswitchable Fluorophores for Single-Molecule Localization Microscopy

Abstract

Over the past decade, fluorescence microscopy has been revolutionized by the development of novel techniques that allow near-molecular resolution. Many such methods-collectively referred to as "single-molecule localization microscopy" (SMLM)-are based upon the repeated imaging of sparse stochastic subsets of the fluorophores in a sample. Active fluorophores are localized by finding the centers of their point spread functions, and a super-resolution image is constructed.Key to this strategy is the use of fluorophores that can be switched "on" and "off" in a controllable manner. Here we review the strengths and weaknesses of the wide variety of SMLM-compatible photoswitchable fluorophores and labeling strategies currently available. We also discuss their suitability for live-cell and multicolor imaging, as well as molecular counting.

Controlling Photoswitching via pcFRET in Conjugated Polymer Nanoparticles

“…Likewise, light is used to control the molecular form of photochromic sensors, which may then exhibit analyte-dependent output fluorescence properties . Imaging and sensing applications such as these have prompted increased interest in fluorophores with photoswitchable emission properties. − …”

Super-resolution imaging in glycoscience: New developments and challenges

“…Super-Resolution Imaging 39,47 Overall,°u orescent proteins are much smaller (a barrel with dimensions 2.4 nm  4.2 nm) than°uorophore-linked antibodies (the size of an IgG antibody is $ 10 nm) which are typically used in dSTORM and STORM imaging. 48 Besides, psFPs are genetically fused to target proteins and endogenously expressed in cells and organelles.…”

Assembly Characterization of Human Equilibrium Nucleoside Transporter 1 (hENT1) by Inhibitor Probe-Based dSTORM Imaging

“…Owing to its nanoscale imaging resolution, direct stochastic optical reconstruction microscopy (dSTORM) has been a powerful tool to reveal the spatially distributed details of biomolecules in the cell. − According to the imaging principle, for high-quality dSTORM imaging, the fluorescence labeling method should be carefully chosen considering several key factors, such as photophysical properties, probe labeling characteristics, the influence on the target, etc. , In the current fluorescence labeling methods for NTs, fluorescence transfection technology or immunofluorescence labeling is commonly used. ,, Despite that these fluorescence labeling methods can also be applied in dSTORM imaging of NTs, the final dSTORM imaging will suffer from some disadvantages. For example, transfection labeling may affect the true structure and function of the target protein, and the presence of overexpression or underexpression of the fluorescent protein or fluorescent tag does not represent the original true protein expression level; furthermore, the weak photophysical properties of the probe also affect the localization accuracy.…”

“…10−14 According to the imaging principle, for high-quality dSTORM imaging, the fluorescence labeling method should be carefully chosen considering several key factors, such as photophysical properties, probe labeling characteristics, the influence on the target, etc. 15,16 In the current fluorescence labeling methods for NTs, fluorescence transfection technology or immunofluorescence labeling is commonly used. 4,17,18 Despite that these fluorescence labeling methods can also be applied in dSTORM imaging of NTs, the final dSTORM imaging will suffer from some disadvantages.…”