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Long-term single-molecule fluorescence measurements are widely used powerful tools to study the conformational dynamics of biomolecules in real time to further elucidate their conformational dynamics. Typically, thousands or even more single-molecule traces are analyzed to provide statistically meaningful information, which is labor-intensive and can introduce user bias. Recently, several deep-learning models have been developed to automatically classify single-molecule traces. In this study, we introduce DEBRIS (Deep lEarning Based fRagmentatIon approach for Single-molecule fluorescence event identification), a deep-learning model focusing on classifying local features and capable of automatically identifying steady fluorescence signals and dynamically emerging signals of different patterns. DEBRIS efficiently and accurately identifies both one-color and two-color single-molecule events, including their start and end points. By adjusting user-defined criteria, DEBRIS becomes the pioneer in using a deep learning model to accurately classify four different types of single-molecule fluorescence events using the same trained model, demonstrating its universality and ability to enrich the current toolbox. DEBRIS is a deep learning-based model that can classify local features and identify steady events and dynamically emerging events within two-color single-molecule traces of varying lengths based on user-defined criteria. [ABSTRACT FROM AUTHOR]

Here we report the synthesis and luminescent properties of the modified polyhedral oligomeric silsesquioxane (POSS) that possesses two types of luminophores, cyanobenzene and N,N‐diethylaniline for inducing significant emission not only from each dye but also from molecular interactions on the POSS core. From the optical measurements in the diluted solution, we observed both locally‐excited emission and excimer emission originating from intramolecular interactions assisted by POSS. Moreover, highly‐efficient exciplex emission was observed from the thin film, indicating that the interaction between luminophores easily occurs in the solid state by accumulating two kinds of molecules in the single POSS molecule. Finally, we also found that the modified POSS showed a unique acid responsiveness which can be caused by protonation at two types of amino moieties. In this manuscript, it is shown that POSS is able to play various roles at the same time: recruiting excited molecules to form exciplexes, suppressing concentration quenching in the solid state, and imparting stimulus responsiveness to exciplexes. [ABSTRACT FROM AUTHOR]

A regular method has been proposed to quantitatively describe the fluorescence of molecules randomly moving in a nanowell. Correlation functions of the fluorescence of single molecules depending on the geometry of the nanowell and the penetration depth of an exciting field into it have been determined. The obtained results can be used to quantitatively analyze the properties of molecules and to extract information on the parameters of the nanowell. [ABSTRACT FROM AUTHOR]

Fluorescence spectroscopy serves as an ultrasensitive sophisticated tool where background noises which serve as a major impediment to the detection of the desired signals can be safely avoided for detections down to the single-molecule levels. One such way of bypassing background noise is plasmon-enhanced fluorescence (PEF), where the interactions of fluorophores at the surface of metals or plasmonic nanoparticles are probed. The underlying condition is a significant spectral overlap between the localized surface plasmon resonance (LSPR) of the nanoparticle and the absorption or emission spectra of the fluorophore. The rationale being the coupling of the excited state of the fluorophore with the localized surface plasmon leads to an augmented emission, owing to local field enhancement. It is manifested in enhanced quantum yields concurrent with a decrease in fluorescence lifetimes, owing to an increase in radiative rate constants. This improvement in detection provided by PEF allows a significant scope of expansion in the domain of weakly emitting fluorophores which otherwise would have remained unperceivable. The concept of coupling of weak emitters with plasmons can bypass the problems of photobleaching, opening up avenues of imaging with significantly higher sensitivity and improved resolution. Furthermore, amplification of the emission signal by the coupling of free electrons of the metal nanoparticles with the electrons of the fluorophore provides ample opportunities for achieving lower detection limits that are involved in biological imaging and molecular sensing. One avenue that has attracted significant attraction in the last few years is the fast, label-free detection of bio-analytes under physiological conditions using plasmonic nanoparticles for point-of-care analysis. This review focusses on the applications of plasmonic nanomaterials in the field of biosensing, imaging with a brief introduction on the different aspects of LSPR and fabrication techniques. [ABSTRACT FROM AUTHOR]

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MINFLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging. [ABSTRACT FROM AUTHOR]

Water molecules play an important role in the structure, function, and dynamics of (bio‐) materials. A direct access to the number of water molecules in nanoscopic volumes can thus give new molecular insights into materials and allow for fine‐tuning their properties in sophisticated applications. The determination of the local water content has become possible by the finding that H2O quenches the fluorescence of red‐emitting dyes. Since deuterated water, D2O, does not induce significant fluorescence quenching, fluorescence lifetime measurements performed in different H2O/D2O‐ratios yield the local water concentration. We combined this effect with the recently developed fluorescence lifetime single molecule localization microscopy imaging (FL‐SMLM) in order to nanoscopically determine the local water content in microgels, i.e. soft hydrogel particles consisting of a cross‐linked polymer swollen in water. The change in water content of thermo‐responsive microgels when changing from their swollen state at room temperature to a collapsed state at elevated temperature could be analyzed. A clear decrease in water content was found that was, to our surprise, rather uniform throughout the entire microgel volume. Only a slightly higher water content around the dye was found in the periphery with respect to the center of the swollen microgels. [ABSTRACT FROM AUTHOR]

Achieving high-contrast tricolor emissive regulation of a single-component molecule using a single type of external stimulus is highly desirable but challenging. In the present study, we report a symmetric acceptor-donor-acceptor (A-D-A)-type aggregation-induced emission-active luminogen, which displays a sequential high-contrast fluorescence switching just by anisotropic mechanical grinding. Specifically, upon light grinding, an orange-yellow-to-blue hypsochromic mechanofluorochromic response with a distinct color contrast (change in the maximum emission wavelength, Δλem,max = 122 nm) is noticed, and the slightly ground solid exhibits a blue-to-red high-contrast (Δλem,max = 185 nm) bathochromic mechanofluorochromic conversion upon vigorous grinding. Thus, using a single luminogen developed here, we can realize wide-range (Δλem,max > 100 nm) hypso-and bathochromic fluorescence mechanochromisms simultaneously. The tricolored mechanofluorochromic phenomenon is attributed to two different morphological transitions involving crystalline-to-crystalline and crystalline-to-amorphous states. Furthermore, three information anticounterfeiting systems are developed using the luminogen presented here. [ABSTRACT FROM AUTHOR]

We report a general molecular design strategy of spatial proximity, which allows intramolecular [2+2] photocycloaddition reaction to take place in both single molecules and molecular aggregates. Sharply contrasting photoinduced fluorescence changes in solution and in the solid state were found and attributed to the aggregation-induced quenching property of the monomers and the aggregation-induced emission nature of the photodimers. [ABSTRACT FROM AUTHOR]

A single layer of a dyad made of a cobalt(II) bis‐terpyridine complex functionalized with a Boron‐dipyrromethene (BODIPY) fluorophore on transparent Indium Tin Oxide (ITO) electrode is prepared. A modulation of the photoluminescence intensity of the fluorophore upon the application of an electric stimulus on the ITO electrode that supports 10 picomoles of molecules is demonstrated. The intensity of the fluorescence intensity decreases when the cobalt is at the oxidation state +III, due to CoIII oxidative quenching of BODIPY emission that has a higher driving force than the CoII reductive quenching initially responsible for the low fluorescence intensity of the chemisorbed dyad. [ABSTRACT FROM AUTHOR]

Monitoring the dynamics of a single molecule provides unique insights into the fundamental physical and chemical properties of individual molecules. Over the past few decades, various approaches have been developed to enable the real-time and high-resolution detection at the single-molecule level. Among them, electrical and optical methods are the most promising tools, for the reason that electrical detection offers high resolution while optical technology provides non-invasive, targeted measurement with the added benefit of visualization. In this review, we summarized the current state-of-the-art electrical and optical techniques for single-molecule measurement and discussed their applications in detecting dynamic events such as conformational isomerizations, intermolecular interactions, chemical reactions, and biomolecular activities. In addition, we discussed the challenges and opportunities in this area and proposed possible directions for future development. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: It has been a mystery that the detected fluorescence intensity of single MEH‐PPV molecules is much lower than expected based on their chain length. In this review, after re‐evaluating of the literature data in the light of new specially designed experiments, we present a comprehensive explanation of this issue: The actual size of MEH‐PPV molecules at single molecule level is much smaller than expected due to de‐aggregation and chain scission, while static quenching ("dark matter") also exists for large molecules, further reducing their brightness. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: It has been a mystery that the detected fluorescence intensity of single MEH‐PPV molecules is much lower than expected based on their chain length. In this review, after re‐evaluating of the literature data in the light of new specially designed experiments, we present a comprehensive explanation of this issue: The actual size of MEH‐PPV molecules at single molecule level is much smaller than expected due to de‐aggregation and chain scission, while static quenching ("dark matter") also exists for large molecules, further reducing their brightness. [ABSTRACT FROM AUTHOR]

Live-cell imaging is a powerful technique to study the dynamics and mechanics of various biological molecules like proteins, organelles, DNA, and RNA. With the rapid evolution of optical microscopy, our understanding of how these molecules are implicated in the cells' most critical physiological roles deepens. In this review, we focus on how spatiotemporal nanoscale live-cell imaging at the single molecule level allows for profound contributions towards new discoveries in life science. This review will start by summarizing how single-molecule tracking has been used to analyze membrane dynamics, receptor–ligand interactions, protein–protein interactions, inner- and extra-cellular transport, gene expression/transcription, and whole organelle tracking. We then move on to how current authors are trying to improve single-molecule tracking and overcome current limitations by offering new ways of labeling proteins of interest, multi-channel/color detection, improvements in time-lapse imaging, and new methods and programs to analyze the colocalization and movement of targets. We later discuss how single-molecule tracking can be a beneficial tool used for medical diagnosis. Finally, we wrap up with the limitations and future perspectives of single-molecule tracking and total internal reflection microscopy. [ABSTRACT FROM AUTHOR]

The pursuit of a hybrid spectroscopy that combines the superb sensitivity of fluorescence and the high chemical specificity of Raman scattering has lasted for 40 years, with multiple experimental and theoretical attempts in the literature. It was only recently that the stimulated Raman excited fluorescence (SREF) process was successfully observed in a broad range of fluorophores. SREF allows single-molecule vibrational spectroscopy and imaging in the optical far field without relying on plasmonic enhancement. In this perspective, we will first review the historical efforts that lead to the successful excitation and detection of SREF, followed by the underlying physical principles, then the remaining technical challenges will be discussed, and, at last, the future opportunities in this old but yet newly emerged spectroscopy are outlined. [ABSTRACT FROM AUTHOR]

Single‐molecule fluorescence techniques are essential for investigating the molecular mechanisms in biological processes. However, achieving sub‐millisecond temporal resolution to monitor fast molecular dynamics remains a significant challenge. The fluorescence brightness is the key parameter that generally defines the temporal resolution for these techniques. Conventional microscopes and standard fluorescent emitters fall short in achieving the high brightness required for sub‐millisecond monitoring. Plasmonic nanoantennas are proposed as a solution, but despite huge fluorescence enhancement having been obtained with these structures, the brightness generally remains below 1 million photons/s/molecule. Therefore, the improvement of temporal resolution is overlooked. This article presents a method for achieving high temporal resolution in single‐molecule fluorescence techniques using plasmonic nanoantennas, specifically optical horn antennas. This work demonstrates about 90% collection efficiency of the total emitted light, reaching a high fluorescence brightness of 2 million photons/s/molecule in the saturation regime. This enables observations of single molecules with microsecond binning time and fast fluorescence correlation spectroscopy measurements. This work expands the applications of plasmonic antennas and zero‐mode waveguides in the fluorescence saturation regime toward brighter single‐molecule signal, faster temporal resolutions, and improved detection rates to advance fluorescence sensing, DNA sequencing, and dynamic studies of molecular interactions. [ABSTRACT FROM AUTHOR]

Photoinduced off‐switching of organic fluorophores is used in super‐resolution microscopy to separate and localize single fluorescent molecules. However, this method typically relies on the use of complex imaging buffers that make use of enzymatic oxygen scavengers to remove oxygen and primary thiols to reversibly reduce excited fluorophores to a non‐fluorescent state. Here, we show that the interactions between sodium sulfite and glycerol can also be used for efficient switching of common organic fluorophores. The addition of glycerol to the imaging buffer allows us to tune the dye switching from intermittent fluorescence to the off‐state. We additionally show that sodium sulfite‐glycerol‐based buffers are especially useful for imaging at low laser illumination intensities in high‐refractive index solutions and samples embedded in agarose gel. This significantly simplifies the implementation of different super‐resolution microscopy modalities such as direct STochastic Optical Reconstruction Microscopy (dSTORM) and Super‐resolution Optical Fluctuation Imaging (SOFI). [ABSTRACT FROM AUTHOR]

The flavin derivatives 10-methyl-isoalloxazine (MIA) and 6-fluoro-10-methyl-isoalloxazine (6F-MIA) were incorporated in two alternative metal-organic frameworks, (MOFs) MIL-53(Al) and MOF-5. We used a post-synthetic, diffusion-based incorporation into microcrystalline MIL-53 powders with one-dimensional (1D) pores and an in-situ approach during the synthesis of MOF-5 with its 3D channel network. The maximum amount of flavin dye incorporation is 3.9 wt% for MIA@MIL-53(Al) and 1.5 wt% for 6F-MIA@MIL-53(Al), 0.85 wt% for MIA@MOF-5 and 5.2 wt% for 6F-MIA@MOF-5. For the high incorporation yields the probability to have more than one dye molecule in a pore volume is significant. As compared to the flavins in solution, the fluorescence spectrum of these flavin@MOF composites is broadened at the bathocromic side especially for MIA. Time-resolved spectroscopy showed that multi-exponential fluorescence lifetimes were needed to describe the decays. The fluorescence-weighted lifetime of flavin@MOF of 4 ± 1 ns also corresponds to those in solution but is significantly prolonged compared to the solid flavin dyes with less than 1 ns, thereby confirming the concept of "solid solutions" for dye@MOF composites. The fluorescence quantum yield (ΦF) of the flavin@MOF composites is about half of the solution but is significantly higher compared to the solid flavin dyes. Both the fluorescence lifetime and quantum yield of flavin@MOF decrease with the flavin loading in MIL-53 due to the formation of various J-aggregates. Theoretical calculations using plane-wave and QM/MM methods are in good correspondence with the experimental results and explain the electronic structures as well as the photophysical properties of crystalline MIA and the flavin@MOF composites. In the solid flavins, π-stacking interactions of the molecules lead to a charge transfer state with low oscillator strength resulting in aggregation-caused quenching (ACQ) with low lifetimes and quantum yields. In the MOF pores, single flavin molecules represent a major population and the computed MIA@MOF structures do not find π-stacking interactions with the pore walls but only weak van-der-Waals contacts which reasons the enhanced fluorescence lifetime and quantum yield of the flavins in the composites compared to their neat solid state. To analyze the orientation of flavins in MOFs, we measured fluorescence anisotropy images of single flavin@MOF-5 crystals and a static ensemble flavin@MIL53 microcrystals, respectively. Based on image information, anisotropy distributions and overall curve of the time-resolved anisotropy curves combined with theoretical calculations, we can prove that all fluorescent flavins species have a defined and rather homogeneous orientation in the MOF framework. In MIL-53, the transition dipole moments of flavins are orientated along the 1D channel axis, whereas in MOF-5 we resolved an average orientation that is tilted with respect to the cubic crystal lattice. Notably, the more hydrophobic 6F-MIA exhibits a higher degree order than MIA. The flexible MOF MIL-53(Al) was optimized essentially to the experimental large-pore form in the guest-free state with QuantumEspresso (QE) and with MIA molecules in the pores the structure contracted to close to the experimental narrow-pore form which was also confirmed by PXRD. In summary, the incorporation of flavins in MOFs yields solid-state materials with enhanced rigidity, stabilized conformation, defined orientation and reduced aggregations of the flavins, leading to increased fluorescence lifetime and quantum yield as controllable photo-luminescent and photo-physical properties. [ABSTRACT FROM AUTHOR]

We develop a supersensitive "turn-on format" fluorescence sandwich immunoassay for detecting small single molecules. Gold nanoplate-based biotin antibodies and streptavidin–fluorophores were used instead of streptavidin–horseradish peroxidase reacting with a biotin tracer in a microplate-based competitive enzyme-linked immunosorbent assay (ELISA). Our platform showed a low detection limit of 5 zeptomolar (5 × 10–21 M), 5.4 × 1010 times higher detection sensitivity than the conventional tune-off format ELISA. [ABSTRACT FROM AUTHOR]

Second near‐infrared (NIR‐II, 1000–1700 nm) window fluorescence imaging‐guided photothermal therapy probes are promising for precise cancer phototheranostics. However, most of the currently reported probes do not demonstrate high NIR‐II fluorescent brightness (molar absorption coefficient (ε) × quantum yield (QY)) and photothermal performance (ε × photothermal conversion efficiency (PCE)) in a single molecule. Herein, a versatile strategy to solve this challenge is reported by fabricating a large π‐conjugated molecule (BNDI‐Me) with a rigid molecular skeleton and flexible side groups. The proposed BNDI‐Me nanoprobe boosts the ε and simultaneously optimizes its QY and PCE. Therefore, high NIR‐II fluorescent brightness (ε × QY = 2296 m−1 cm−1) and strong photothermal performance (ε × PCE = 82 000) are successfully incorporated in a single small molecule, and, to the best of knowledge, either of these two parameters is better than the best currently available fluorescent or photothermal probes. Thus, superior NIR‐II imaging effect in vivo and high photothermal tumor inhibition rate (81.2%) at low systemic injection doses are obtained. The work provides further insights into the relationship of photophysical mechanisms and structures, and presents promising molecular design guidelines for the integration of more efficient multiple theranostic functions in a single molecule. [ABSTRACT FROM AUTHOR]

Advanced fluorescence imaging techniques such as single-molecule localization microscopy (SMLM) fundamentally rely on the photophysical behavior of the employed fluorophores. This behavior is generally complex and impacts data quality in a subtle manner. A simulation software named Single-Molecule Imaging Simulator (SMIS) is introduced that simulates a widefield microscope and incorporates fluorophores with their spectral and photophysical properties. With SMIS, data collection schemes combining 3D, multicolor, single-particle-tracking or quantitative SMLM can be implemented. The influence of advanced fluorophore characteristics, imaging conditions, and environmental parameters can be evaluated, facilitating the design of real experiments and their proper interpretation. Single Molecule Imaging Simulator (SMIS) simulates how the photophysical behavior of fluorophores in single-molecule localization experiments influences experimental data, as evidenced by applying SMIS to several imaging scenarios. [ABSTRACT FROM AUTHOR]

We exploit single‐molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore formation of the archetypical cholesterol‐dependent cytolysin nanopore, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 ms temporal resolution and 20 nm spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assembly dominated by monomer addition, but with infrequent assembly from larger partial complexes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms. [ABSTRACT FROM AUTHOR]

The fluorescent moieties coumarin and xanthene (R6GCP) combined in a single molecule was designed and synthesized. The colorimetric and fluorescent variation of the probe towards the copper and mercury ions sensing is examined. With the added copper/mercury ions to the solution of R6GCP in DMF:H2O (2:8, v/v), the probe showed deep red color from yellow color. The probe showed turn-off and turn-on fluorescence for copper and mercury ion respectively. In the presence of other competing metal ions, the probe showed better sensitivity towards copper and mercury ions. The probe's detection limit found to be 5.29 × 10–6 M and 1.24 × 10–5 M for Cu2+ and Hg2+ ion respectively by the UV–visible spectral measurement. Fluorescence measurement, the detection limit for the Cu2+ and Hg2+ ions detection by this probe is 1.91 × 10–7 M, and 1.32 × 10–8 M respectively. 1:1 binding stoichiometry was confirmed between the probe and Cu2+/Hg2+ ions from jobs plot by UV–visible spectral technique. Moreover, R6GCP combined filter paper were prepared. These test paper containing probe could detect Cu2+/Hg2+ ions in real-time with a spontaneous color change. [ABSTRACT FROM AUTHOR]

Though 2,5-dihydroxyterephthalic acid (DHT) is composed of a single benzene molecule, it is blue-emissive in common organic solvents and in the solid state. Like most organic fluorophores, DHT is not soluble in water, which limits its versatile use in metal ion detection in an aqueous medium. To improve the water solubility of DHT and its use as a molecular sensor in aqueous solutions, its deprotonated form, DHT-K, was synthesized through the simple one-pot reaction of DHT with KOH. Compared with DHT, DHT-K was highly soluble in water and emitted yellow fluorescence in the solution and the solid. In addition, DHT-K showed high selectivity for Al ions, exhibiting fluorescence wavelength changes from 540 to 495 nm depending on the Al ion concentration. A linear relationship between the fluorescence intensity of DHT-K and Al ion concentration was established ranging from 18.96 to 247 μM with a detection limit of 1.84 µM. The binding stoichiometry between DHT-K and Al ions was determined by Job's plot and found to be 1:2. Upon exposure to Al ions, DHT-K showed significant changes in fluorescence color and emission wavelength, whereas no fluorescence changes were observed by the addition of various metal ions such as Zn2+, Mg2+, Ca2+, Cu2+, Fe3+, and Co2+. Thus, DHT-K can be applied as a fluorescent sensor that can selectively detect Al ions in aqueous solutions. [ABSTRACT FROM AUTHOR]

Fluorescent properties of single Rhodamine molecules at different distances to the surface of a high‐quality polycrystalline gold film are investigated by the time‐correlated single‐photon counting technique. The fluorescence lifetime and the fluorescence intensity of the dye molecules as a function of their distance to the gold surface are modeled quantitatively by a classical model for a horizontal electric dipole. The analysis shown herein represents a useful tool to model and further to control molecular fluorescence near complex plasmonic structures, where delicate manipulation of fluorescence emission intensity is of paramount importance. [ABSTRACT FROM AUTHOR]

Resonance fluorescence as the emission of a resonantly-excited two-level quantum system promises indistinguishable single photons and coherent high-fidelity quantum-state manipulation of the matter qubit, which underpin many quantum information processing protocols. Real applications of the protocols demand high degrees of scalability and stability of the experimental platform, and thus favor quantum systems integrated on one chip. However, the on-chip solution confronts several formidable challenges compromising the scalability prospect, such as the randomness, spectral wandering and scattering background of the integrated quantum systems near heterogeneous and nanofabricated material interfaces. Here we report an organic-inorganic hybrid integrated quantum photonic platform that circuits background-free resonance fluorescence of single molecules with an ultrastable lifetime-limited transition. Our platform allows a collective alignment of the dipole orientations of many isolated molecules with the photonic waveguide. We demonstrate on-chip generation, beam splitting and routing of resonance-fluorescence single photons with a signal-to-background ratio over 3000 in the waveguide at the weak excitation limit. Crucially, we show the photonic-circuited single molecules possess a lifetime-limited-linewidth transition and exhibit inhomogeneous spectral broadenings of only about 5% over hours' measurements. These findings and the versatility of our platform pave the way for scalable quantum photonic networks. Single molecules can generate high-quality single photons for quantum technologies, but coupling to waveguides is difficult. Here, the authors show on-chip background-free resonance fluorescence generation and routing from single molecules with lifetime-limited transition and waveguide-aligned dipoles. [ABSTRACT FROM AUTHOR]

Oligomerization and complex formation play a key role for many membrane proteins and has been described to influence ion channel function in both neurons and the heart. In this study, we observed clustering of single KcsA channels in planar lipid bilayer using single molecule fluorescence, while simultaneously measuring single channel currents. Clustering coincided with cooperative opening of KcsA. We demonstrate that clustering was not caused by direct protein-protein interactions or hydrophobic mismatch with the lipid environment, as suggested earlier, but was mediated via microdomains induced by the channel in the lipid matrix. We found that single channel activity of KcsA requires conically-shaped lipids in the lamellar liquid-crystalline (Lα) phase, and the need for a negative spontaneous curvature seem to lead to the deformations in the membrane that cause the clustering. The method introduced here will be applicable to follow oligomerization of a wide range of membrane proteins. [ABSTRACT FROM AUTHOR]

Single-molecule techniques have shifted the paradigm of biological measurements from ensemble measurements to probing individual molecules and propelled a rapid revolution in related fields. Compared to ensemble measurements of biomolecules, single-molecule techniques provide a breadth of information with a high spatial and temporal resolution at the molecular level. Usually, optical and electrical methods are two commonly employed methods for probing single molecules, and some platforms even offer the integration of these two methods such as optofluidics. The recent spark in technological advancement and the tremendous leap in fabrication techniques, microfluidics, and integrated optofluidics are paving the way toward low cost, chip-scale, portable, and point-of-care diagnostic and single-molecule analysis tools. This review provides the fundamentals and overview of commonly employed single-molecule methods including optical methods, electrical methods, force-based methods, combinatorial integrated methods, etc. In most single-molecule experiments, the ability to manipulate and exercise precise control over individual molecules plays a vital role, which sometimes defines the capabilities and limits of the operation. This review discusses different manipulation techniques including sorting and trapping individual particles. An insight into the control of single molecules is provided that mainly discusses the recent development of electrical control over single molecules. Overall, this review is designed to provide the fundamentals and recent advancements in different single-molecule techniques and their applications, with a special focus on the detection, manipulation, and control of single molecules on chip-scale devices. [ABSTRACT FROM AUTHOR]

Accurate size measurements of nanosized polymer chains in dilute solutions is important for understanding polymer behavior, however, these measurements can be challenging to implement accurately, and are technique dependent. Here we explore the use of Fluorescence Correlation Spectroscopy (FCS) to determine the size of single polymer chains in dilute solutions (<2 wt%). FCS, a technique with single molecule sensitivity, generally requires the use of covalently labelled polymers which can distort physicochemical behavior. Here, FCS based size measurements were based on the non-covalent interaction of fluorophores (Alexa 405, Atto 390, and Atto 425) with PNIPAm in water at 25 °C (below the Lower Critical Solution Temperature). FCS estimated size (hydrodynamic radius) of three different MW PNIPAm samples in water were: 5.0 ± 1.0 nm (28.5 kDa), 5.0 ± 1.0 nm (38 kDa), 3.3 ± 0.5 nm (55.5 kDa), in reasonable agreement with theoretical calculations. Accuracy was directly related to the fraction of PNIPAm-bound fluorophore, which were, for 1 wt% PNIPAm solutions in water: ∼11.5 % (Atto 390), ∼8.1 % (Atto 425), and 4 % (Alexa 405). This method has several advantages in that it does not require a covalent labelling of PNIPAm, it can be implemented on very small sample volumes, and allows for in-situ measurements. submit a graphic in the actual size to be used for the TOC that will fit in an area 1.375 in. High and 3.5 in. wide (3.6 cm &∼8.9 cm). [Display omitted] • First use of FCS for PNIPAm polymer chain sizing by non-covalent interactions. • Compares the efficacy of Alexa 405, Atto 390, and Atto 425 fluorophores. • Measurement method uses 405 nm excitation. • Suitable for in-situ measurements on low volume samples. [ABSTRACT FROM AUTHOR]

The point spread function (PSF) is fundamental to any type of microscopy, most importantly so for single-molecule localization techniques, where the exact PSF shape is crucial for precise molecule localization at the nanoscale. Optical aberrations and fixed fluorophore dipoles often result in non-isotropic and distorted PSFs, impairing and biasing conventional fitting approaches. Further, PSF shapes are deliberately modified in PSF engineering approaches for providing improved sensitivity, e.g., for 3D localization or determination of dipole orientation. As this can lead to highly complex PSF shapes, a tool for visualizing expected PSFs would facilitate the interpretation of obtained data and the design of experimental approaches. To this end, we introduce a comprehensive and accessible computer application that allows for the simulation of realistic PSFs based on the full vectorial PSF model. Our tool incorporates a wide range of microscope and fluorophore parameters, including orientationally constrained fluorophores, as well as custom aberrations, transmission and phase masks, thus enabling an accurate representation of various imaging conditions. An additional feature is the simulation of crowded molecular environments with overlapping PSFs. Further, our app directly provides the Cramér–Rao bound for assessing the best achievable localization precision under given conditions. Finally, our software allows for the fitting of custom aberrations directly from experimental data, as well as the generation of a large dataset with randomized simulation parameters, effectively bridging the gap between simulated and experimental scenarios, and enhancing experimental design and result validation. • User-friendly MATLAB application for accurate point spread function (PSF) simulation. • Full vectorial PSF model for simulating fixed and (partially) rotating fluorophores. • Wide range of customizable parameters, optical elements, and visualization options. • Calculation of Cramér–Rao bound to assess the achievable localization precision. • Phase and apodization retrieval from an experimentally recorded PSF stack. [ABSTRACT FROM AUTHOR]

Detection of single molecules or particles avoids ensemble averaging and thus is able to provide a very local heterogeneous information which is not available from an ensemble measurement. Single molecules or particles can be detected based on their three characteristic optical properties, absorption, scattering and fluorescence/photoluminescence, in addition to their label-free detection. This short review focuses on our group’s research towards understanding and imaging the above three kinds of optical signals from single molecules and particles. [ABSTRACT FROM AUTHOR]

In this work, we aimed to apply fluorescence microscopy to image protein conjugation to Ni-NTA modified silver nanowires in real time via the His-tag attachment. First, a set of experiments was designed and performed for the mixtures of proteins and silver nanowires in order to demonstrate plasmon enhancement of mCherry protein fluorescence as well as the ability to image fluorescence of single molecules. The results indicated strong enhancement of single-protein fluorescence emission upon coupling with silver nanowires. This conclusion was supported by a decrease in the fluorescence decay time of mCherry proteins. Real-time imaging was carried out for a structure created by dropping protein solution onto a glass substrate with functionalized silver nanowires. We observed specific attachment of mCherry proteins to the nanowires, with the recognition time being much longer than in the case of streptavidin–biotin conjugation. This result indicated that it is possible to design a universal and efficient real-time sensing platform with plasmonically active functionalized silver nanowires. [ABSTRACT FROM AUTHOR]

The rise of super‐resolution fluorescence microscopy (SRM) has revolutionized our understanding of the nanoscale world. SRM has recently been correlated with spectroscopy techniques to obtain rich spectral information of individual molecules in addition to localization information. This technique ultimately provides multidimensional and functional information of single molecules, which is generally masked in the ensemble averages of previous bulk measurements and creates exciting new opportunities and challenges for single‐molecule spectroscopic imaging. We review the recent developments in single‐molecule spectroscopic imaging technique and how the challenges are addressed to effectively obtain single‐molecule spectroscopic information. Particular attention has been devoted to the applications of this method, including single‐molecule chemical kinetic studies and single‐molecule polarity sensing. Finally, we address the possible future enhancements and applications. Thus, we demonstrate how a single‐molecule spectroscopic imaging technique adds new dimensions of information to the SRM, providing new opportunities in a wide range of research fields. [ABSTRACT FROM AUTHOR]

Impressive developments have been achieved with the use of zeolitic imidazolate framework-8 (ZIF-8) as nanocarriers for tumor theranostics in recent decades by incorporating imaging agents and therapeutic drugs within ZIF-8. However, the simultaneous immobilization of hydrophilic and hydrophobic functional molecules into ZIF-8 nanoparticles in water or organic solvents still presents a daunting challenge. Herein, we developed a new synthesis/encapsulation two-in-one (denoted as one-pot) approach to synthesize uniform dextran-modified Cy5.5&ICG@ZIF-8-Dex nanoparticles in DMSO/H 2 O solvent mixtures, which enabled the simultaneous encapsulation of hydrophilic indocyanine green (ICG) and hydrophobic cyanine-5.5 (Cy5.5) during the same step. It was confirmed that the one-pot approach in this mixed solvents facilitated the loading of ICG and Cy5.5 molecules. Moreover, the encapsulation of Cy5.5 and ICG within ZIF-8 nanoparticles endowed them with fluorescence imaging capability and photothermal conversion capacity, respectively. The in vivo near-infrared (NIR) fluorescent images of A549-bearing mice injected with Cy5.5&ICG@ZIF-8-Dex demonstrated sufficient accumulations of Cy5.5 at tumor sites due to the enhanced permeability and retention effect. Most impressively, the fluorescent intensity of Cy5.5&ICG@ZIF-8-Dex at tumor site was approximately 40-fold higher than that of free Cy5.5. Additionally, the results of in vivo infrared imaging and photothermal therapy of Cy5.5&ICG@ZIF-8-Dex showed enhanced therapeutic efficiency in comparison with free ICG, further confirming its tumor-targeting capability and photothermal capacity. Therefore, this multifunctional system based on ZIF-8 nanocarriers offered a potential nanoplatform for tumor-targeting theranostics, thus broadening the synthesis and applications of ZIF-8 composite nanoparticles for NIR fluorescence imaging and photothermal therapy in the biomedical field. Simultaneous immobilization of hydrophilic and hydrophobic molecules into ZIF-8 nanoparticles still remains a daunting challenge. Therefore, we have developed a new synthesis/encapsulation two-in-one approach to synthesize uniform Cy5.5&ICG@ZIF-8-Dex composite nanoparticles in DMSO/H 2 O solvent mixtures, which enabled the simultaneous encapsulation of hydrophilic indocyanine green (ICG) and hydrophobic cyanine-5.5 (Cy5.5) functional molecules during a single step. The results showed that the co-loading of Cy5.5 and ICG within the ZIF-8 nanoparticles endowed them with a remarkable fluorescence imaging capability and photothermal conversion capacity. Based on their enhanced convenience and efficacy to simultaneously encapsulate hydrophilic and hydrophobic molecules, the multifunctional nanocarriers that were prepared in the DMSO/H 2 O mixed solvents provide a potential nanoplatform toward fluorescence imaging and photothermal therapy for tumor theranostics. [Display omitted] [ABSTRACT FROM AUTHOR]

pH-responsive fluorescence molecules are interesting candidates for achieving tunable as well as white light emission from a single molecule. Herein, we have synthesized pyridine-integrated carbazole and triphenylamine donor–acceptor isomers, (Z)-3-(4-(9H-carbazol-9-yl)phenyl)-2-(pyridinyl)acrylonitrile (1–3) and (Z)-3-(4-(diphenylamino)phenyl)-2-(pyridinyl)acrylonitrile (4–6), and studied the effects of the structure and solvent on acid-responsive fluorescence modulation in the solution state. Both carbazole (1–3) and triphenylamine (4–6) isomers produced twisted intramolecular charge transfer (TICT) states and showed tunable fluorescence depending on the solvent polarity. Highly blue shifted fluorescence was observed in non-polar toluene, while polar DMF showed large red-shifted fluorescence. Further, 1–3 isomers exhibited relatively strong fluorescence compared to 4–6. Interestingly, 1–6 exhibited organic acid-dependent (trifluoroacetic acid (TFA) and p-toluene sulphonic acid (PTSA)) and solvent-dependent fluorescence tuning. Carbazole isomers (1–3) showed wide fluorescence colour tuning from blue to red as well as white light emission in toluene, whereas green to red transition was observed in CHCl3. Addition of TFA, a stronger acid, to 1 and 3 afforded blue, green and red emission along with white emission in toluene, whereas weak PTSA produced blue and red emission along with white emission. In contrast, addition of TFA or PTSA to the triphenylamine isomers (4–6) exhibited complete quenching of fluorescence in all solvents. The meta-isomers did not show significant fluorescence tuning with acids. The wide fluorescence tuning of 1 in toluene with TFA has also been successfully transformed into a PMMA polymer matrix, which also exhibited clear fluorescence tuning from blue to red. Density functional theoretical (DFT) studies indicated high molecular twisting in the excited state and clear separation of charge transfer from the donor carbazole to the acceptor unit compared to the propeller-shaped triphenylamine isomer; this may contribute to the wide fluorescence tuning in the carbazole isomers. The present studies could be useful for choosing appropriate molecular structures and solvent systems for achieving wide fluorescence tuning and white light emission from a single donor–acceptor molecule. [ABSTRACT FROM AUTHOR]

Fluorescent probes are a powerful tool in biological and biochemical fields. Herein, we prepared a new small molecule fluorescent probe PYQU as a robust tool that can explore the interaction between RNA and SO2 in biological environments. The probe PYQU could detect SO2 and RNA by a change in green and red fluorescence, respectively. Besides, the probe PYQU has excellent properties, such as low biological toxicity, good membrane permeability, and high selectivity. PYQU was used to detect SO2 in living cells and zebrafish in the green channel and visualize endogenous RNA of cells with a deep red fluorescence signal. Therefore, we believe that the probe PYQU is a valuable, robust, and reliable tool for studies of RNA and SO2. [ABSTRACT FROM AUTHOR]

This review covers recent progress in using single molecule fluorescence microscopy imaging to understand the nanoconfinement in porous materials. The single molecule approach unveils the static and dynamic heterogeneities from seemingly equal molecules by removing the ensemble averaging effect. Physicochemical processes including mass transport, surface adsorption/desorption, and chemical conversions within the confined space inside porous materials have been studied at nanometer spatial resolution, at the single nanopore level, with millisecond temporal resolution, and under real chemical reaction conditions. Understanding these physicochemical processes provides the ability to quantitatively measure the inhomogeneities of nanoconfinement effects from the confining properties, including morphologies, spatial arrangement, and trapping domains. Prospects and limitations of current single molecule imaging studies on nanoconfinement are also discussed. [ABSTRACT FROM AUTHOR]

In view of the fact that multivalent intermolecular noncovalent interactions are the main elements for the formation of phase separation, supramolecular entities are a type of multivalent system. However, the phase separation system based on single supramolecular molecules was limited. In this paper, two permethyl-β-cyclodextrin modified perylene monoamide derivatives (PMI-B-CD and PMI-N-CD) with modification of benzene and naphthalene groups at the bay position of perylene backbone were synthesized. Of interest, PMI-B-CD and PMI-N-CD showed a LCST effect due to a self-inclusion interaction between PMCD and the benzene or naphthalene groups in the bay position of perylene monoamide backbone. Dynamic confocal fluorescence imaging of PMI-B-CD and PMI-N-CD showed thermo-responsive phase separation process. It is surprised that PMI-B-CD and PMI-N-CD achieved thermo-responsive capture and release of aromatic guests. Dynamic phase separation and controllable capture and release of guests with thermo-responsive phase transition process are found, which opens a novel supramolecular principle of liquid-liquid transition. Dynamic phase separation and controllable capture and release of guests based on permethyl-β-cyclodextrin modified perylene monoamide derivatives with thermo-responsive phase transition process are found, which opens a novel supramolecular principle of phase separation. [Display omitted] • Dynamic fluorescence imaging for phase separation behaviors was investigated. • Controllable capture and release of guests with thermo-responsive phase transition process were studied. • A supramolecular principle of liquid-liquid transition was developed. • Supramolecular fluorescent systems to insight the molecular principles of the phase separation transition were developed. [ABSTRACT FROM AUTHOR]

Bifluorophores (5-FAM)2 and (6-FAM)2 were synthesized on a 3,5-diaminobenzoic acid framework, which allows the insertion of two fluorophore molecules into a single site. Despite the similarity of the structures of the 5- and 6-carboxyfluorescein isomers (5-FAM and 6-FAM), the photophysical behavior of the corresponding bifluorophores differs. The luminescence quantum yield of (6-FAM)2 in a buffer solution with pH 8.5 is 2.4 times lower than for (5-FAM)2. The structural relaxation of molecules in the excited state was shown to be slower with increasing solution viscosity when using a water–glycerol mixture and with greater microviscosity in the case of a micellar solution. The slower relaxation leads to an increase in the fluorescence intensity, lifetime of the excited states, and extent of polarization, i.e., the photophysical properties of the synthesized compounds are largely by the viscosity of the medium. [ABSTRACT FROM AUTHOR]

Fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules, respectively. Lateral diffusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger diffusion coefficient determined by FRAP, but the sources of the inconsistency have not been investigated thoroughly. Here, we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes–Einstein equation in aqueous solution. The combined i FRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP. Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis. Our i FRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices. [ABSTRACT FROM AUTHOR]

The design of ratiometric probes for imaging of carbon monoxide (CO) in subcellular organelles is critical to elucidate its biological and pathological functions. In this work, we establish a ratiometric fluorescent probe (Mito-NIB-CO) for imaging of CO in mitochondria. The mitochondria-targeting unit (triphenylphosphonium moiety) and CO-responsive unit (allyl ether moiety) are covalently linking into the single molecule (Mito-NIB-CO) to achieve the multifunctional effect. Upon being treated with CO, Mito-NIB-CO underwent the cleavage of allyl ether element in the presence of PdCl2, resulting in the structural and spectral conversion. This characteristic afforded Mito-NIB-CO to be a ratiometric probe for CO with two fluorescent emission bands. Additionally, the probe Mito-NIB-CO exhibited other distinct merits, including preeminent selectivity and sensitivity. What's more, profiting from triphenylphosphonium moiety, the probe Mito-NIB-CO can specifically target the mitochondria and realize quantitative detection of exogenous/endogenous CO in mitochondria. [ABSTRACT FROM AUTHOR]

Single molecule sequencing is imperative to overall genetic analysis in areas such as genomics, transcriptomics, clinical test, drug development, and cancer screening. In addition, fluorescence-based sequencing is primarily applied in single molecule sequencing besides other methods, precisely in the fields of DNA sequencing. Modern-day fluorescence labeling methods exploit a charge-coupled device camera to capture snapshots of a number of pixels on the single molecule sequencing. The method discussed in this article involves fluorescence labeling detection with a single pixel, outrivals in high accuracy and low resource requirement under low signal-to-noise ratio conditions, as well as benefits from higher throughput comparing with others. Through discussion in this article, we explore the single molecule synthesis process modeling using negative binomial distributions. Furthermore, incorporating the method of maximum likelihood and Viterbi algorithm in this modeling enhances the signal detection accuracy. The fluorescence-based model benefits in simulating actual experiment processes and assisting in understanding relations between the fluorescence emission and the signal receiving events. Last but not least, the model offers potential candidates on fluorescence dye selection that yields more accurate experiment results. [ABSTRACT FROM AUTHOR]

Stacking‐induced fluorescence increase (SIFI) was introduced recently as a method to probe DNA structure and dynamics using only a single fluorescent label. Here we show that the same DNA hairpin dynamics can be recovered, at the single‐molecule level, using either SIFI (with Cy3 as the label) or FRET (with Cy3 as donor and Cy5 as acceptor). We also measured FRET using a donor that cannot undergo SIFI, Cy3B, in the presence and absence of a molecular crowding agent (PEG). Although crowding increases hairpin hybridisation to the same extent with either Cy3 or Cy3B as the donor, the absolute rates are affected by the choice of donor dye. This work shows that SIFI can be used to measure single‐molecule dynamics, which could offer advantages over FRET in some cases. It also illustrates how local dye interactions can influence biomolecular dynamics, which should be considered when designing experiments. [ABSTRACT FROM AUTHOR]

Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves singlemolecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ~30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with singlemolecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region. [ABSTRACT FROM AUTHOR]

Enhancement of optical emission on plasmonic nanostructures is intrinsically limited by the distance between the emitter and nanostructure surface, owing to a tightly-confined and exponentially-decaying electromagnetic field. This fundamental limitation prevents efficient application of plasmonic fluorescence enhancement for diversely-sized molecular assemblies. We demonstrate a three-dimensionally-tapered gap plasmon nanocavity that overcomes this fundamental limitation through near-homogeneous yet powerful volumetric confinement of electromagnetic field inside an open-access nanotip. The 3D-tapered device provides fluorescence enhancement factors close to 2200 uniformly for various molecular assemblies ranging from few angstroms to 20 nanometers in size. Furthermore, our nanostructure allows detection of low concentration (10 pM) biomarkers as well as specific capture of single antibody molecules at the nanocavity tip for high resolution molecular binding analysis. Overcoming molecule position-derived large variations in plasmonic enhancement can propel widespread application of this technique for sensitive detection and analysis of complex molecular assemblies at or near single molecule resolution. Plasmon-enhanced fluorescence is strictly dependent on molecule size and surface position, restricting application of diversely sized molecules. Here, the authors overcome this by introducing a 3D tapered gap plasmon nanocavity with fluidic access and near homogeneous confinement of the electromagnetic fields. [ABSTRACT FROM AUTHOR]

Single-Molecule Localization Microscopy (SMLM) has become one of the most important methods of super-resolution fluorescence microscopy. It is based on the precise localization of single molecules in wide-field microscopy images. It is well known that the localization accuracy can show a significant bias if the imaged molecules have a fixed orientation and are located either close to an interface or not exactly within the focal plane of the microscope. In this Letter, we propose a simple solution to this problem, which is based on polarization-resolved imaging. This method can be easily implemented into any existing SMLM setup, and we demonstrate its performance by imaging single dye molecules embedded into a polymer film, which fixes their orientation in space. [ABSTRACT FROM AUTHOR]

White light emitting (WLE) materials are of increasing interest owing to their promising applications in artificial lighting, display devices, molecular sensors, and switches. In this context, organic WLE materials cater to the interest of the scientific community owing to their promising features like color purity, long‐term stability, solution processability, cost‐effectiveness, and low toxicity. The typical method for the generation of white light is to combine three primary (red, green, and blue) or the two complementary (e.g., yellow and blue or red and cyan) emissive units covering the whole visible spectral window (400–800 nm). The judicious choice of molecular building blocks and connecting them through either strong covalent bonds or assembling through weak noncovalent interactions are the key to achieve enhanced emission spanning the entire visible region. In the present review article, molecular engineering approaches for the development of all‐organic WLE materials are analyzed in view of different photophysical processes like fluorescence resonance energy transfer (FRET), excited‐state intramolecular proton transfer (ESIPT), charge transfer (CT), monomer‐excimer emission, triplet‐state harvesting, etc. The key aspect of tuning the molecular fluorescence under the influence of pH, heat, and host–guest interactions is also discussed. The white light emission obtained from small organic molecules to supramolecular assemblies is presented, including polymers, micelles, and also employing covalent organic frameworks. The state‐of‐the‐art knowledge in the field of organic WLE materials, challenges, and future scope are delineated. [ABSTRACT FROM AUTHOR]

Synthetic host–guest complexes are inherently dynamic as they employ weak and reversible noncovalent interactions for their recognition processes. We strategically exploited dynamic supramolecular recognition between fluorescently labeled guest molecules to complementary cucurbit[7]uril hosts to obtain stochastic switching between fluorescence ON- and OFF-states, enabling PAINT-based nanoscopic imaging in cells and tissues. [ABSTRACT FROM AUTHOR]

[Display omitted] • Proteins located in cell membranes have a defined orientation that is established during their integration into the membrane. • Fluorescence-based methods have been developed to detect the topological orientation of proteins in the bacterial cytoplasmic membrane at up to single molecule sensitivity. • These methods show that a very small proportion of membrane proteins are inserted into the cytoplasmic membrane in the wrong orientation. • Low level mislocalization of a soluble protein across the cytoplasmic membrane is also detected. • This study demonstrates the power of fluorescence methods to uncover low frequency events in living cells. The cytoplasmic membrane compartmentalises the bacterial cell into cytoplasm and periplasm. Proteins located in this membrane have a defined topology that is established during their biogenesis. However, the accuracy of this fundamental biosynthetic process is unknown. We developed compartment-specific fluorescence labelling methods with up to single-molecule sensitivity. Application of these methods to the single and multi-spanning membrane proteins of the Tat protein transport system revealed rare topogenesis errors. This methodology also detected low level soluble protein mislocalization from the cytoplasm to the periplasm. This study shows that it is possible to uncover rare errors in protein localization by leveraging the high sensitivity of fluorescence methods. [ABSTRACT FROM AUTHOR]