Improved resolution in single-molecule localization microscopy using QD-PAINT

Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM.Subject terms: Fluorescence imaging, Quantum dots, Oligonucleotide probes, Fluorescent dyes, Super-resolution microscopy     

In Situ Visualization of Block Copolymer Self‐Assembly in Organic Media by Super‐Resolution Fluorescence Microscopy

Analytical methods that enable visualization of nanomaterials derived from solution self‐assembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization‐driven block copolymer (BCP) self‐assembly in organic media at the sub‐diffraction scale. Four different dyes were successfully used for single‐colour super‐resolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dual‐colour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although well‐established for aqueous systems, the results highlight the potential of super‐resolution microscopy techniques for the interrogation of self‐assembly processes in organic media.     

Spontaneously Blinking Rhodamine Dyes for Single-Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) has found extensive applications in various fields of biology and chemistry. As a vital component of SMLM, fluorophores play an essential role in obtaining super-resolution fluorescence images. Recent research on spontaneously blinking fluorophores has greatly simplified the experimental setups and extended the imaging duration of SMLM. To support this crucial development, this review provides a comprehensive overview of the development of spontaneously blinking rhodamines from 2014 to 2023, as well as the key mechanistic aspects of intramolecular spirocyclization reactions. We hope that by offering insightful design guidelines, this review will contribute to accelerating the advancement of super-resolution imaging technologies.     

Make them Blink: Probes for Super‐Resolution Microscopy

In recent years, a number of approaches have emerged that enable far‐field fluorescence imaging beyond the diffraction limit of light, namely super‐resolution microscopy. These techniques are beginning to profoundly alter our abilities to look at biological structures and dynamics and are bound to spread into conventional biological laboratories. Nowadays these approaches can be divided into two categories, one based on targeted switching and readout, and the other based on stochastic switching and readout of the fluorescence information. The main prerequisite for a successful implementation of both categories is the ability to prepare the fluorescent emitters in two distinct states, a bright and a dark state. Herein, we provide an overview of recent developments in super‐resolution microscopy techniques and outline the special requirements for the fluorescent probes used. In combination with the advances in understanding the photophysics and photochemistry of single fluorophores, we demonstrate how essentially any single‐molecule compatible fluorophore can be used for super‐resolution microscopy. We present examples for super‐resolution microscopy with standard organic fluorophores, discuss factors that influence resolution and present approaches for calibration samples for super‐resolution microscopes including AFM‐based single‐molecule assembly and DNA origami.     

Self-quenched Fluorophore Dimers for DNA-PAINT and STED Microscopy

Super-resolution techniques like single-molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non-covalent, weak affinity-based transient labelling systems. DNA-based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi-target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore-labelled single-stranded oligonucleotides. Here, we introduce short-distance self-quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2-fold. We characterise the optical and thermodynamic properties of fluorophore-dimer single-stranded DNA, and show super-resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background.     

Far‐Red Organic Fluorophores Contain a Fluorescent Impurity

Far‐red organic fluorophores commonly used in traditional and super‐resolution localization microscopy are found to contain a fluorescent impurity with green excitation and near‐red emission. This near‐red fluorescent impurity can interfere with some multicolor stochastic optical reconstruction microscopy/photoactivated localization microscopy measurements in live cells and produce subtle artifacts in chemically fixed cells. We additionally describe alternatives to avoid artifacts in super‐resolution localization microscopy.     

Super‐Chelators for Advanced Protein Labeling in Living Cells

Live‐cell labeling, super‐resolution microscopy, single‐molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N‐nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA‐based small interaction pairs described so far. Coupled to bright organic fluorophores with fine‐tuned photophysical properties, the super‐chelator probes were delivered into human cells by chemically gated nanopores. These super‐chelators permit kinetic profiling, multiplexed labeling of His₆‐ and His₁₂‐tagged proteins as well as single‐molecule‐based super‐resolution imaging.     

Long-Term,Single-Molecule Imaging of Proteins in Live Cells with Photoregulated Fluxional Fluorophores

Single-molecule localization microscopy (SMLM) can reveal nanometric details of biological samples, but its high phototoxicity hampers long-term imaging in live specimens. A significant part of this phototoxicity stems from repeated irradiations that are necessary for controlled switching of fluorophores to maintain the sparse labeling of the sample. Lower phototoxicity can be obtained using fluorophores that blink spontaneously, but controlling the density of single-molecule emitters is challenging. We recently developed photoregulated fluxional fluorophores (PFFs) that combine the benefits of spontaneously blinking dyes with photocontrol of emitter density. These dyes, however, were limited to imaging acidic organelles in live cells. Herein, we report a systematic study of PFFs that culminates in probes that are functional at physiological pH and operate at longer wavelengths than their predecessors. Moreover, these probes are compatible with HaloTag labeling, thus enabling timelapse, single-molecule imaging of specific protein targets for exceptionally long times.     

Enhanced dSTORM imaging using fluorophores interacting with cucurbituril

Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes or proteins as fluorophores. The photophysical properties of fluorophores have been proven to be crucial for super-resolution microscopy’s localization precision and imaging resolution. Fluorophores TAMRA and Atto Rho6G, which can interact with macrocyclic host cucurbit[7]uril (CB7) to form host-guest compounds, were found to improve the fluorescence intensity and lifetimes of these dyes. We enhanced the localization precision of direct stochastic optical reconstruction microscopy (dSTORM) by introducing CB7 into the imaging buffer, and showed that the number of photons as well as localizations of both TAMRA and Atto Rho6G increase over 2 times.     

Multicolor Caged dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain

The precision of single‐molecule localization‐based super‐resolution microscopy, including dSTORM, critically depends on the number of detected photons per localization. Recently, reductive caging of fluorescent dyes followed by UV‐induced recovery in oxidative buffer systems was used to increase the photon yield and thereby the localization precision in single‐color dSTORM. By screening 39 dyes for their fluorescence caging and recovery kinetics, we identify novel dyes that are suitable for multicolor caged dSTORM. Using a dye pair suited for registration error‐free multicolor dSTORM based on spectral demixing (SD), a multicolor localization precision below 15 nm was achieved. Caged SD‐dSTORM can resolve the ultrastructure of single 40 nm synaptic vesicles in brain sections similar to images obtained by immuno‐electron microscopy, yet with much improved label density in two independent channels.     

Nanoscopic Visualization of Soft Matter Using Fluorescent Diarylethene Photoswitches

The in situ imaging of soft matter is of paramount importance for a detailed understanding of functionality on the nanoscopic scale. Although super‐resolution fluorescence microscopy methods with their unprecedented imaging capabilities have revolutionized research in the life sciences, this potential has been far less exploited in materials science. One of the main obstacles for a more universal application of super‐resolved fluorescence microscopy methods is the limitation of readily available suitable dyes to overcome the diffraction limit. Here, we report a novel diarylethene‐based photoswitch with a highly fluorescent closed and a nonfluorescent open form. Its photophysical properties, switching behavior, and high photostability make the dye an ideal candidate for photoactivation localization microscopy (PALM). It is capable of resolving apolar structures with an accuracy far beyond the diffraction limit of optical light in cylindrical micelles formed by amphiphilic block copolymers.     

Versatile Near-Infrared Super-Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD-2

CRANAD-2 is a fluorogenic curcumin derivative used for near-infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD-2 in two super-resolution imaging techniques, namely stimulated emission depletion (STED) and single-molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD-2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45–55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super-resolved image. The good performance and versatility of CRANAD-2 provides a powerful tool for near-infrared nanoscopic imaging of amyloids in vitro and in vivo.     

Electrochemical Modulation of the Fluorescence of Cyanine Dye Cy5

Organic fluorescent dyes are widely used in single molecule localization microscopy, where their performances are determined by the photophysical properties. Herein, we utilized a sensitive method to modulate the fluorescence of organic dyes by external potentials using a combination of electrochemical cell and super‐resolution fluorescent microscopy. Cy5 (cyanine dye) was chosen as a model molecule considering its wide application and commercial availability. We applied different potentials on the Au electrode to change the Coulombic charge microenvironment of Cy5. When the electrode potential was adjusted negatively, Cy5 displayed a better photostability. This method is proved effective in adjusting the fluorescence of organic dyes.     

Live‐Cell Localization Microscopy with a Fluorogenic and Self‐Blinking Tetrazine Probe

Recent developments in fluorescence microscopy call for novel small‐molecule‐based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live‐cell single‐molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live‐cell experiments. At the same time, quality of super‐resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present the design and synthesis of a small‐molecule label comprising both fluorogenic and self‐blinking features. Bioorthogonal click chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe improves quality and conditions for regular and single‐molecule localization microscopy on live‐cell samples.     

A Photoactivatable BODIPY Probe for Localization‐Based Super‐Resolution Cellular Imaging

The synthesis and application of a photoactivatable boron‐alkylated BODIPY probe for localization‐based super‐resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron‐photodealkylation reaction with a single low‐power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization‐based microscopy of biological systems. The probe can be easily linked to nucleophile‐containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super‐resolution imaging of microtubules is demonstrated. This is the first example of single‐molecule localization‐based super‐resolution microscopy using a visible‐light‐activated BODIPY compound as a fluorescent probe.     

Photochemical Mechanisms of Fluorophores Employed in Single-Molecule Localization Microscopy

Decoding cellular processes requires visualization of the spatial distribution and dynamic interactions of biomolecules. It is therefore not surprising that innovations in imaging technologies have facilitated advances in biomedical research. The advent of super-resolution imaging technologies has empowered biomedical researchers with the ability to answer long-standing questions about cellular processes at an entirely new level. Fluorescent probes greatly enhance the specificity and resolution of super-resolution imaging experiments. Here, we introduce key super-resolution imaging technologies, with a brief discussion on single-molecule localization microscopy (SMLM). We evaluate the chemistry and photochemical mechanisms of fluorescent probes employed in SMLM. This Review provides guidance on the identification and adoption of fluorescent probes in single molecule localization microscopy to inspire the design of next-generation fluorescent probes amenable to single-molecule imaging.     

Single‐Molecule Spectroscopy,Imaging, and Photocontrol: Foundations for Super‐Resolution Microscopy (Nobel Lecture)

The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single‐molecule limit in 1989 using frequency‐modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super‐resolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super‐resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.     

多种超分辨荧光成像技术比较和进展评述

所见即所得是生命科学研究的中心哲学,贯穿在不断认识单个分子、分子复合体、分子动态行为和整个分子网络的历程中。活的动态的分子才是有功能的,这决定了荧光显微成像在生命科学研究中成为不可替代的工具。但是当荧光成像聚焦到分子水平的时候,所见并不能给出想要得到的。这个障碍是由于受光学衍射极限的限制,荧光显微镜无法在衍射受限的空间内分辨出目标物。超分辨荧光成像技术突破衍射极限的限制,在纳米尺度至单分子水平可视化生物分子,以前所未有的时空分辨率研究活细胞结构和动态过程,已成为生命科学研究的有力工具,并逐渐应用到材料科学、催化反应过程和光刻等领域。超分辨成像技术原理不同,其具有的技术性能各异,限制了各自特定的技术特色和应用范围。目前主流的超分辨成像技术包括3种:结构光照明显微镜技术(structured illumination microscopy, SIM)、受激发射损耗显微技术(stimulated emission depletion, STED)和单分子定位成像技术(single molecule localization microscopy, SMLM)。这些显微镜采用不同的复杂技术,但是策略却是相同和简单的,即通过牺牲时间分辨率来提升衍射受限的空间内相邻两个发光点的空间分辨。该文通过对这3种技术的原理比较和在生物研究中的应用进展介绍,明确了不同超分辨成像技术的技术优势和适用的应用方向,以方便研究者在未来研究中做合理的选择。     

Mechanism of Intramolecular Photostabilization in Self‐Healing Cyanine Fluorophores

Organic fluorophores, which are popular labels for microscopy applications, intrinsically suffer from transient and irreversible excursions to dark‐states. An alternative to adding photostabilizers at high concentrations to the imaging buffer relies on the direct linkage to the fluorophore. However, the working principles of this approach are not yet fully understood. In this contribution, we investigate the mechanism of intramolecular photostabilization in self‐healing cyanines, in which photodamage is automatically repaired. Experimental evidence is provided to demonstrate that a single photostabilizer, that is, the vitamin E derivative Trolox, efficiently heals the cyanine fluorophore Cy5 in the absence of any photostabilizers in solution. A plausible mechanism is that Trolox interacts with the fluorophore through intramolecular quenching of triplet‐related dark‐states, which is a mechanism that appears to be common for both triplet‐state quenchers (cyclooctatetraene) and redox‐active compounds (Trolox, ascorbic acid, methylviologen). Additionally, the influence of solution‐additives, such as cysteamine and procatechuic acid, on the self‐healing process are studied. The results suggest the potential applicability of self‐healing fluorophores in stochastic optical reconstruction microscopy (STORM) with optical super‐resolution. The presented data contributes to an improved understanding of the mechanism involved in intramolecular photostabilization and has high relevance for the future development of self‐healing fluorophores, including their applications in various research fields.     

Reductively Caged,Photoactivatable DNA‐PAINT for High‐Throughput Super‐resolution Microscopy

In DNA points accumulation in nanoscale topography (DNA‐PAINT), capable of single‐molecule localization microscopy with sub‐10‐nm resolution, the high background stemming from the unbound fluorescent probes in solution limits the imaging speed and throughput. Herein, we reductively cage the fluorescent DNA probes conjugated with a cyanine dye to hydrocyanine, acting as a photoactivatable dark state. The additional dark state from caging lowered the fluorescent background while enabling optically selective activation by total internal reflection (TIR) illumination at 405 nm. These benefits from “reductive caging” helped to increase the localization density or the imaging speed while preserving the image quality. With the aid of high‐density analysis, we could further increase the imaging speed of conventional DNA‐PAINT by two orders of magnitude, making DNA‐PAINT capable of high‐throughput super‐resolution imaging.