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.     

Nanographenes: Ultrastable,Switchable, and Bright Probes for Super‐Resolution Microscopy

Super‐resolution fluorescence microscopy has enabled important breakthroughs in biology and materials science. Implementations such as single‐molecule localization microscopy (SMLM) and minimal emission fluxes (MINFLUX) microscopy in the localization mode exploit fluorophores that blink, i.e., switch on and off, stochastically. Here, we introduce nanographenes, namely large polycyclic aromatic hydrocarbons that can also be regarded as atomically precise graphene quantum dots, as a new class of fluorophores for super‐resolution fluorescence microscopy. Nanographenes exhibit outstanding photophysical properties: intrinsic blinking even in air, excellent fluorescence recovery, and stability over several months. As a proof of concept for super‐resolution applications, we use nanographenes in SMLM to generate 3D super‐resolution images of silica nanocracks. Our findings open the door for the widespread application of nanographenes in super‐resolution fluorescence microscopy.     

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N‐substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si‐containing analogue of Q‐rhodamine. This probe is the first example of a “caged” Si‐rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red‐shifted to allow multicolor imaging. The dye is a useful label for super‐resolution imaging and constitutes a new scaffold for far‐red fluorogenic molecules.     

A Small‐Molecule FRET Reporter for the Real‐Time Visualization of Cell‐Surface Proteolytic Enzyme Functions

Real‐time imaging of cell‐surface‐associated proteolytic enzymes is critical to better understand their performances in both physiological and pathological processes. However, most current approaches are limited by their complexity and poor membrane‐anchoring properties. Herein, we have designed and synthesized a unique small‐molecule fluorescent probe, which combines the principles of passive exogenous membrane insertion and Förster resonance energy transfer (FRET) to image cell‐surface‐localized furin‐like convertase activities. The membrane‐associated furin‐like enzymatic cleavage of the peptide probe leads to an increased fluorescence intensity which was mainly localized on the plasma membrane of the furin‐expressed cells. This small‐molecule fluorescent probe may serve as a unique and reliable reporter for real‐time visualization of endogenous cell‐surfaceassociated proteolytic furin‐like enzyme functions in live cells and tissues using one‐photon and two‐photon microscopy.     

Gold Nanorod Enhanced Fluorescence Enables Single‐Molecule Electrochemistry of Methylene Blue

Redox reactions are central to energy conversion and life metabolism. Herein we present electrochemical measurements with fluorescent readout of the redox‐sensitive dye Methylene Blue (MB), at the single‐molecule (SM) level. To overcome the low fluorescence quantum yield of MB we enhanced fluorescence by using individual gold nanorods to achieve the required sensitivity. By measuring the same molecule at different electrochemical potentials we determined the mid‐point potential of each single molecule through its redox‐induced fluorescence blinking dynamics.     

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.     

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.     

Controllable Blinking‐to‐Nonblinking Behavior of Aqueous CdTeS Alloyed Quantum Dots

Semiconductor quantum dots (QDs) are very important optical nanomaterials with a wide range of potential applications. However, the blinking of single QDs is an intrinsic drawback for some biological and photoelectric applications based on single‐dot emission. In this work, we systematically investigated the effects of certain synthetic conditions on the blinking behavior of aqueous CdTeS alloyed QDs, and observed that blinking behaviors of QDs were able to be controlled by the structure and concentration of the thiol compounds that were used as surface ligands. In optimal conditions, completely nonblinking QDs were prepared using certain thiol ligands as stabilizers in aqueous phase. The suppressed blinking mechanism was mainly attributed to elimination of QDs surface traps by coordination of thiol ligands with vacant Cd atoms, formation of appropriate CdS coating on QDs, and controlling the growth dynamics of QDs. Nonblinking QDs show high quantum yield, small size, and good solubility, and will be applied to some fields that were previously limited by blinking of traditional QDs.     

Direct Measurement of Single‐Molecule DNA Hybridization Dynamics with Single‐Base Resolution

Herein, we report label‐free detection of single‐molecule DNA hybridization dynamics with single‐base resolution. By using an electronic circuit based on point‐decorated silicon nanowires as electrical probes, we directly record the folding/unfolding process of individual hairpin DNAs with sufficiently high signal‐to‐noise ratio and bandwidth. These measurements reveal two‐level current oscillations with strong temperature dependence, enabling us to determine the thermodynamic and kinetic properties of hairpin DNA hybridization. More importantly, successive, stepwise increases and decreases in device conductance at low temperature on a microsecond timescale are successfully observed, indicating a base‐by‐base unfolding/folding process. The process demonstrates a kinetic zipper model for DNA hybridization/dehybridization at the single base‐pair level. This measurement capability promises a label‐free single‐molecule approach to probe biomolecular interactions with fast dynamics.     

Addressing the Requirements of High‐Sensitivity Single‐Molecule Imaging of Low‐Copy‐Number Proteins in Bacteria

Single‐molecule fluorescence super‐resolution imaging and tracking provide nanometer‐scale information about subcellular protein positions and dynamics. These single‐molecule imaging experiments can be very powerful, but they are best suited to high‐copy number proteins where many measurements can be made sequentially in each cell. We describe artifacts associated with the challenge of imaging a protein expressed in only a few copies per cell. We image live Bacillus subtilis in a fluorescence microscope, and demonstrate that under standard single‐molecule imaging conditions, unlabeled B. subtilis cells display punctate red fluorescent spots indistinguishable from the few PAmCherry fluorescent protein single molecules under investigation. All Bacillus species investigated were strongly affected by this artifact, whereas we did not find a significant number of these background sources in two other species we investigated, Enterococcus faecalis and Escherichia coli. With single‐molecule resolution, we characterize the number, spatial distribution, and intensities of these impurity spots.     

DCDHF Fluorophores for Single‐Molecule Imaging in Cells

There is a persistent need for small‐molecule fluorescent labels optimized for single‐molecule imaging in the cellular environment. Application of these labels comes with a set of strict requirements: strong absorption, efficient and stable emission, water solubility and membrane permeability, low background emission, and red‐shifted absorption to avoid cell autofluorescence. We have designed and characterized several fluorophores, termed “DCDHF” fluorophores, for use in live‐cell imaging based on the push–pull design: an amine donor group and a 2‐dicyanomethylene‐3‐cyano‐2,5‐dihydrofuran (DCDHF) acceptor group, separated by a π‐rich conjugated network. In general, the DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. Especially valuable are fluorophores with sophisticated photophysics for applications requiring additional facets of control, such as photoactivation. For example, we have reengineered a red‐emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low‐intensity violet light. This molecule and its relatives provide a new class of bright photoactivatable small‐molecule fluorophores, which are needed for super‐resolution imaging schemes that require active control (here turning‐on) of single‐molecule emission.     

SLAP: Small Labeling Pair for Single‐Molecule Super‐Resolution Imaging

Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small‐molecule recognition unit (Ni‐trisNTA) and the genetically encoded minimal protein His_6‐10‐tag. High‐density protein tracing by SLAP was demonstrated. This technique allows super‐resolution fluorescence imaging and fulfills the necessary sampling criteria for single‐molecule localization‐based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).     

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.     

Live‐Cell Stimulated Raman Scattering Imaging of Alkyne‐Tagged Biomolecules

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman‐silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label‐free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state‐of‐the‐art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non‐invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live‐cell imaging.     

Power-law blinking in the fluorescence of single organic molecules.

The blinking behavior of perylene diïmide molecules is investigated at the single‐molecule level. We observe long‐time scale blinking of individual multi‐chromophoric complexes embedded in a poly(methylmethacrylate) matrix, as well as for the monomeric dye absorbed on a glass substrate at ambient conditions. In both these different systems, the blinking of single molecules is found to obey analogous power‐law statistics for both the on and off periods. The observed range for single‐molecular power‐law blinking extends over the full experimental time window, covering four orders of magnitude in time and six orders of magnitude in probability density. From molecule to molecule, we observe a large spread in off‐time power‐law exponents. The distributions of off‐exponents in both systems are markedly different whereas both on‐exponent distributions appear similar. Our results are consistent with models that ascribe the power‐law behavior to charge separation and (environment‐dependent) recombination by electron tunneling to a dynamic distribution of charge acceptors. As a consequence of power‐law statistics, single molecule properties like the total number of emitted photons display non‐ergodicity.     

Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

Single‐molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around a fluorophore (rKACB), thereby enabling real‐time single‐molecule measurement of the structure‐changing dynamics of nucleic acids.     

A Vinyl Sulfone‐Based Fluorogenic Probe Capable of Selective Labeling of PHGDH in Live Mammalian Cells

Chemical probes are powerful tools for interrogating small molecule‐target interactions. With additional fluorescence Turn‐ON functionality, such probes might enable direct measurements of target engagement in live mammalian cells. DNS‐pE (and its terminal alkyne‐containing version DNS‐pE2) is the first small molecule that can selectively label endogenous 3‐phosphoglycerate dehydrogenase (PHGDH) from various mammalian cells. Endowed with an electrophilic vinyl sulfone moiety that possesses fluorescence‐quenching properties, DNS‐pE/DNS‐pE2 became highly fluorescent only upon irreversible covalent modification of PHGDH. With an inhibitory property (in vitro K_i=7.4 μm ) comparable to that of known PHGDH inhibitors, our probes thus offer a promising approach to simultaneously image endogenous PHGDH activities and study its target engagement in live‐cell settings.     

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.     

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.     

Controlled‐Atmosphere Flame Fusion Single‐Crystal Growth of Non‐Noble fcc,hcp, and bcc Metals Using Copper,Cobalt, and Iron

The growth of noble‐metal single crystals via the flame fusion method was developed in the 1980s. Since then, there have been no major advancements to the technique until the recent development of the controlled‐atmosphere flame fusion (CAFF) method to grow non‐noble Ni single crystals. Herein, we demonstrate the generality of this method with the first preparation of fcc Cu as well as the first hcp and bcc single crystals of Co and Fe, respectively. The high quality of the single crystals was verified using scanning electron microscopy and Laue X‐ray backscattering. Based on Wulff constructions, the equilibrium shapes of the single‐crystal particles were studied, confirming the symmetry of the fcc, hcp, and bcc single‐crystal lattices. The low cost of the CAFF method makes all kinds of high‐quality non‐noble single crystals independent of their lattice accessible for use in electrocatalysis, electrochemistry, surface science, and materials science.