Self-healing dyes for super-resolution fluorescence microscopy

Abstract

In recent years, optical microscopy techniques have emerged that allow optical imaging at unprecedented resolution beyond the diffraction limit. These techniques exploit photostabilizing buffers to enable photoswitching and/or the enhancement of fluorophore brightness and stability. A major drawback with the use of photostabilizing buffers, however, is that they cannot be used in live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores, which undergo intramolecular photostabilization, in super-resolution microscopy examining both targeted (stimulated emission depletion (STED) microscopy) and stochastic readout (stochastic optical reconstruction microscopy (STORM)). The overall goal of the study was to identify dyes and conditions that lead to improved spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. As a result of previously shown superior performance, we identified an ATTO647N-photostabilizer conjugate as a potential candidate for STED microscopy. We have here characterized the photostability and resulting performance of this nitrophenylalanine (NPA) conjugate of ATTO647N on oligonucleotides in STED microscopy. We found that the superior photophysical performance resulted in optimal STED imaging and demonstrated that single-molecule fluorescent transients of individual fluorophores can be obtained with both the excitation-and STED-laser. In similar experiments, we also tested a nitrophenylacetic acid conjugate of STAR635P, another frequently used dye in STED microscopy, and present a characterization of its photophysical properties. Finally, we performed an analysis of the photoswitching kinetics of self-healing Cy5 dyes (containing trolox, cyclooctatetraene and NPA-based stabilizers) in the presence of Tris(2-carboxyethyl) phosphine and cysteamine, which are typically used in STORM microscopy. In line with previous work, we found that intramolecular photostabilization strongly influences Original content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence.

Ultrasmall, Bright, and Photostable Fluorescent Core–Shell Aluminosilicate Nanoparticles for Live‐Cell Optical Super‐Resolution Microscopy

“…[11] Previous work on STORM systems has produced simpler and less toxic protocols, typically involving self-healing dyes as well as heavy-atom-containing quantum dots. [12][13][14] Additionally, it has been shown that certain probes can be used for live-cell STORM, but only in specific organelles, such as the mitochondria, that have increased levels of thiol compound glutathione. [15] The Tinnefeld group described an experimental strategy to induce blinking with only one excitation light source by using an imaging buffer that contains both oxidizing and reducing agents.…”

Unraveling the Nanoscopic Organization and Function of Central Mammalian Presynapses With Super-Resolution Microscopy

“…By harnessing the resolving power of super-resolution optical microscopy, our understanding of synapse structure and function has taken big leaps in recent years. With the incessant quest for further refinements in terms of better hardware, increasingly powerful processing algorithms (e.g., Xu et al, 2017), or deep learning strategies (Ouyang et al, 2018;Wang et al, 2019;Jin et al, 2020), fluorophores with improved or tailored photophysical properties (e.g., Minoshima and Kikuchi, 2017;Thiel and Rivera-Fuentes, 2018;Halabi et al, 2019;Kozma and Kele, 2019;Velde et al, 2019;Xu et al, 2020), and, quite likely, yet new and revolutionary technical approaches altogether, the limits of what can be resolved today are bound to keep shrinking. In parallel, the evolution of strategies for high-throughput SRM are allowing faster imaging of increasing numbers of simultaneous targets over larger sample areas (Guo et al, 2019;Mahecic et al, 2019).…”

Nanoscopic Stoichiometry and Single‐Molecule Counting

“…What we seem to be observing is the effect of slight variations in buffer conditions on the dye photophysics, which may be due to diffusion-limited reactions between the thiol and the dyes or slight variations in oxygen concentration. Improved buffer conditions [32] or self-healing dyes [33,34] could mitigate this variability, but the message is clearly that one must be cautious both in performing and applying this calibration to a molecular counting experiment.…”