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.     

Cyclo-Ketal Xanthene Dyes: A New Class of Near-Infrared Fluorophores for Super-Resolution Imaging of Live Cells

Newly emerging super-resolution imaging techniques provide opportunities for precise observations on cellular microstructures. However, they also impose severe demands on fluorophores. Here, we develop a new series of NIR xanthene dyes, named as KRh s, by replacing the 10-position O of rhodamines with a cyclo-ketal. KRh s display an intense NIR emission peak at 700 nm with fluorescence quantum yields up to 0.64. More importantly, they, without the aid of enhancing buffer, exhibit stochastic fluorescence off–on switches to support time-resolved localization of single fluorophore. KRh s are functionalized into KRh-MitoFix , KRh-Mem and KRh-Halo that demonstrate mitochondria, plasma membrane and fusion protein targeting ability, respectively. Consequently, these KRh probes demonstrate straightforward usage for super-resolution imaging of these targets in live cells. Therefore, KRh s merit future development for fluorescence labeling and super-resolution imaging in the NIR region.     

Quantum-yield-optimized fluorophores for site-specific labeling and super-resolution imaging

Single-molecule applications, saturated pattern excitation microscopy, and stimulated emission depletion (STED) microscopy demand bright as well as highly stable fluorescent dyes. Here we describe the synthesis of quantum-yield-optimized fluorophores for reversible, site-specific labeling of proteins or macromolecular complexes. We used polyproline-II (PPII) helices as sufficiently rigid spacers with various lengths to improve the fluorescence signals of a set of different trisNTA-fluorophores. The improved quantum yields were demonstrated by steady-state and fluorescence lifetime analyses. As a proof of principle, we characterized the trisNTA-PPII-fluorophores with respect to in vivo protein labeling and super-resolution imaging at synapses of living neurons. The distribution of His-tagged AMPA receptors (GluA1) in spatially restricted synaptic clefts was imaged by confocal and STED microscopy. The comparison of fluorescence intensity profiles revealed the superior resolution of STED microscopy. These results highlight the advantages of biocompatible and, in particular, small and photostable trisNTA-PPII-fluorophores in super-resolution microscopy.     

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     

Photo-isomerization of the Cyanine Dye Alexa-Fluor 647 (AF-647) in the Context of dSTORM Super-Resolution Microscopy

Cyanine dyes, as used in super-resolution fluorescence microscopy, undergo light-induced “blinking”, enabling localization of fluorophores with spatial resolution beyond the optical diffraction limit. Despite a plethora of studies, the molecular origins of this blinking are not well understood. Here, we examine the photophysical properties of a bio-conjugate cyanine dye (AF-647), used extensively in dSTORM imaging. In the absence of a potent sacrificial reductant, light-induced electron transfer and intermediates formed via the metastable, triplet excited state are considered unlikely to play a significant role in the blinking events. Instead, it is found that, under conditions appropriate to dSTORM microscopy, AF-647 undergoes reversible photo-induced isomerization to at least two long-lived dark species. These photo-isomers are characterized spectroscopically and their interconversion probed by computational means. The first-formed isomer is light sensitive and transforms to a longer-lived species in modest yield that could be involved in dSTORM related blinking. Permanent photobleaching of AF-647 occurs with very low quantum yield and is partially suppressed by the anaerobic redox buffer.     

A unique class of near-infrared functional fluorescent dyes with carboxylic-acid-modulated fluorescence ON/OFF switching: rational design, synthesis, optical properties, theoretical calculations, and applications for fluorescence imaging in living animals

Fluorescence imaging is one of the most powerful techniques for monitoring biomolecules in living systems. Fluorescent sensors with absorption and emission in the near-infrared (NIR) region are favorable for biological imaging applications in living animals, as NIR light leads to minimum photodamage, deep tissue penetration, and minimum background autofluorescence interference. Herein, we have introduced a new strategy to design NIR functional dyes with the carboxylic-acid-controlled fluorescence on-off switching mechanism by the spirocyclization. Based on the design strategy, we have developed a series of Changsha (CS1-6) NIR fluorophores, a unique new class of NIR functional fluorescent dyes, bearing excellent photophysical properties including large absorption extinction coefficients, high fluorescence quantum yields, high brightness, good photostability, and sufficient chemical stability. Significantly, the new CS1-6 NIR dyes are superior to the traditional rhodamine dyes with both absorption and emission in the NIR region while retaining the rhodamine-like fluorescence ON-OFF switching mechanism. In addition, we have performed quantum chemical calculations with the B3LYP exchange functional employing 6-31G* basis sets to shed light on the structure-optical properties of the new CS1-6 NIR dyes. Furthermore, using CS2 as a platform, we further constructed the novel NIR fluorescent TURN-ON sensor 7, which is capable of imaging endogenously produced HClO in the living animals, demonstrating the value of our new CS NIR functional fluorescent dyes. We expect that the design strategy may be extended for development of a wide variety of NIR functional dyes with a suitable fluorescence-controlled mechanism for many useful applications in biological studies.     

Chemically induced photoswitching of fluorescent probes--a general concept for super-resolution microscopy

We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.     

Silicon-substituted rhodamines for stimulated emission depletion fluorescence nanoscopy

As an essential part in the toolbox of super-resolution microscopy, stimulated emission depletion(STED)nanoscopy has been widely explored in revealing the substructure and bioactivities in fluorescence imaging. Among the applied STED fluorophores, silicon-substituted rhodamines(SiRs) belong to one of the most extensively employed fluorophores. The carboxy-SiR was favored in STED bioimaging with many advantages, including reliable photostability, cell permeability, tunable fluorogenicity, feasibl...     

Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution.     

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。     

Super-Resolution Tension PAINT Imaging with a Molecular Beacon

DNA-PAINT enabled super-resolution imaging through the transient binding of fluorescently-labelled single-stranded DNA (ssDNA) imagers to target ssDNA. However, its performance is constrained by imager background fluorescence, resulting in relatively long image acquisition and potential artifacts. We designed a molecular beacon (MB) as the PAINT imager. Unbound MB in solution reduces the background fluorescence due to its natively quenched state. They are fluorogenic upon binding to target DNA to create individual fluorescence events. We demonstrate that MB-PAINT provides localization precision similar to traditional linear imager DNA-PAINT. We also show that MB-PAINT is ideally suited for fast super-resolution imaging of molecular tension probes in living cells, eliminating the potential of artifacts from free-diffusing imagers in traditional DNA-PAINT at the cell-substrate interface.     

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.     

Isolation and Characterization of Precise Dye/Dendrimer Ratios

Fluorescent dyes are commonly conjugated to nanomaterials for imaging applications using stochastic synthesis conditions that result in a Poisson distribution of dye/particle ratios and therefore a broad range of photophysical and biodistribution properties. We report the isolation and characterization of generation 5 poly(amidoamine) (G5 PAMAM) dendrimer samples containing 1, 2, 3, and 4 fluorescein (FC) or 6‐carboxytetramethylrhodamine succinimidyl ester (TAMRA) dyes per polymer particle. For the fluorescein case, this was achieved by stochastically functionalizing dendrimer with a cyclooctyne “click” ligand, separation into sample containing precisely defined “click” ligand/particle ratios using reverse‐phase high performance liquid chromatography (RP‐HPLC), followed by reaction with excess azide‐functionalized fluorescein dye. For the TAMRA samples, stochastically functionalized dendrimer was directly separated into precise dye/particle ratios using RP‐HPLC. These materials were characterized using ¹H and ¹⁹F NMR spectroscopy, RP‐HPLC, UV/Vis and fluorescence spectroscopy, lifetime measurements, and MALDI.     

Photoswitchable nanoparticles enable high-resolution cell imaging: PULSAR microscopy

Beyond-diffraction-limit optical imaging of cells will reveal biological mechanisms, cellular structures, and physiological processes in nanometer scale. Harnessing the photoswitching properties of spiropyran fluorophores, we achieved nanoresolution fluorescence imaging using photoactuated unimolecular logical switching attained reconstruction (PULSAR) microscopy. The PULSAR microscope successfully resolved nanostructures and subcellular organelles when the photoswitchable nanoparticles containing spiropyran dyes were used as fluorescent probes.     

Non-covalent interactions of coumarin dyes with cucurbit[7]uril macrocycle: modulation of ICT to TICT state conversion

Non-covalent interaction of coumarin laser dyes, namely coumarin-1 (C1), coumarin-481 (C481) and coumarin-6H (C6H), with a versatile macrocyclic host molecule cucurbit[7]uril (CB7), has been investigated in aqueous solution using photophysical methods. Steady-state and time-resolved fluorescence studies illustrate significant enhancements/modifications in the fluorescence yields, lifetimes and spectral features of C1, C481 and C6H on interaction with CB7, and are assigned to 1 : 1 complex formation between the dyes and the CB7 host. The complex formation is mainly driven by charge-dipole interaction, as evident from the binding constant values (K ~ 10(4)-10(5) M(-1)). The large changes in the excited state behaviour of C1 and C481 as compared to C6H in the presence of CB7 indicate that CB7 binds C1 and C481 through the encapsulation of the 7-N,N'-diethylamino group of the dyes and the structural rigidity imposed by this interaction dramatically alters the excited state properties of the dyes by preventing the conversion of their emissive intramolecular charge transfer (ICT) state to the non-radiative twisted intramolecular charge transfer (TICT) state. The present results direct towards the probable supramolecular approach using water soluble macrocyclic CB7, in the development of aqueous dye laser systems in the blue-green region.     

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.     

Strongly Emissive and Photostable Four‐Coordinate Organoboron N,C Chelates and Their Use in Fluorescence Microscopy

Six strongly fluorescent four‐coordinate organoboron N,C chelates containing an aryl isoquinoline skeleton were prepared. Remarkably, the fluorescence quantum yields reach values of up to 0.74 in oxygen‐free toluene. The strong B?N interaction was corroborated by the single‐crystal X‐ray analysis of two dyes. The intramolecular charge‐transfer character of the fluorophores was evidenced by solvatochromism studies and time‐dependent DFT calculations at the PCM(toluene)/CAM‐B3LYP/6‐311++G(2d,p)//PCM(toluene)/B3LYP/6‐311G(2d,p) level of theory. The compounds combine high chemical stability with high photostability, especially when equipped with electron‐donating substituents. The strong fluorescence and the large Stokes shifts predestine these compounds for use in confocal fluorescence microscopy. This was demonstrated for the imaging of the N13 mouse microglial cell line. Moreover, significant two‐photon absorption cross sections (up to 61 GM) allow the use of excitation wavelengths in the near‐infrared region (>800 nm).     

Cucurbituril Encapsulation of Fluorescent Dyes

The potential of cucurbiturils, water-soluble macrocyclic host molecules composed of glycoluril units, for tuning the properties of fluorescent dyes and advancing new applications is illustrated. Cucurbit[7]uril (CB7), which presents a particularly attractive derivative due to its intermediary size and high water solubility, has been shown to display a variety of advantageous effects on fluorescent dyes, which include increased fluorescence intensity and brightness, enhanced photostability, protection towards fluorescence quenchers, solubilization, and deaggregation. Particularly noteworthy is the prolongation of the fluorescence lifetimes of different dyes, which can be traced back to the low polarizability of the host cavity. In addition, the host serves as cation receptor, which causes a considerable shift of protonation equilibria and assists the protonation of fluorescent dyes. The latter effect can be exploited in the design of protolytic fluorophore displacement assays. The perspective of cucurbiturils as stabilizers for laser dyes, enhancement agents in time-resolved fluorescence (TRF) assays, contrast agents for fluorescence lifetime imaging (FLIM), and dyes for fluorescent collectors for solar cells is mentioned. Original experimental results for the effect of CB7 on the fluorescence properties of three dyes (Macrolex Yellow 10 GN, Dapoxyl, and 4-(dimethylamino)benzonitrile) are presented.     

Geminate Recombination as a Photoprotection Mechanism for Fluorescent Dyes

Despite common presumption due to fast photodestruction pathways through higher excited states, we show that further improvement of photostability is still achievable with diffusion‐limited photoprotection formulas. Single‐molecule fluorescence spectroscopy reveals that thiolate ions effectively quench triplet states of dyes by photoinduced electron transfer. Interestingly, this reaction rarely yields a radical anion of the dye, but direct return to the ground state is promoted by an almost instantaneous back electron transfer (geminate recombination). This type of mechanism is not detected for commonly used reductants such as ascorbic acid and trolox. The mechanism avoids the formation of radical cations and improves the photostability of single fluorophores. We find that a combination of β‐mercaptoethanol and classical reducing and oxidizing systems yields the best results for several dyes including Atto532 and Alexa568.     

Strongly fluorescent, switchable perylene bis(diimide) host-guest complexes with cucurbit[8]uril in water

Supramolecular complexation of perylene bis(diimide) (PDI) dyes with the macrocyclic host cucurbit[8]uril (CB[8]) prevents self-aggregation of the dye molecules and enables their use as highly (photo)chemically stable, strongly-emitting fluorophores in water. The complexes are stimuli-responsive to binders and can be electrochemically cycled, leading to reversible on-off fluorescence switching and access to noncovalent formation of higher-order architectures in water.