Squaraine Dyes Exhibit Spontaneous Fluorescence Blinking That Enables Live-Cell Nanoscopy.

Hampered by their susceptibility to nucleophilic attack and chemical bleaching, electron-deficient squaraine dyes have long been considered unsuitable for biological imaging. This study unveils a surprising twist: in aqueous environments, bleaching is not irreversible but rather a reversible spontaneous quenching process. Leveraging this new discovery, we introduce a novel deep-red squaraine probe tailored for live-cell super-resolution imaging. This probe enables single-molecule localization microscopy (SMLM) under physiological conditions without harmful additives or intense lasers and exhibits spontaneous blinking orchestrated by biological nucleophiles, such as glutathione or hydroxide anion. With a low duty cycle (∼0.1%) and high-emission rate (∼6 × 104 photons/s under 400 W/cm2), the squaraine probe surpasses the benchmark Cy5 dye by 4-fold and Si-rhodamine by a factor of 1.7 times. Live-cell SMLM with the probe reveals intricate structural details of cell membranes, which demonstrates the high potential of squaraine dyes for next-generation super-resolution imaging.

Near-Infrared Spontaneously Blinking Fluorophores for Live Cell Super-Resolution Imaging with Minimized Phototoxicity.

Single-molecule localization microscopy (SMLM) requires high-intensity laser irradiation, typically exceeding kW/cm2, to yield a sufficient photon count. However, this intense visible light exposure incurs substantial cellular toxicity, hindering its use in living cells. Here, we developed a class of near-infrared (NIR) spontaneously blinking fluorophores for SMLM. These NIR fluorophores are a combination of rhodamine spirolactams and merocyanine derivatives, where the rhodamine spirolactam component converts between a bright and dark state based on pH-dependent spirocyclization and merocyanine derivatives shift the excitation wavelength into the infrared. Single-molecule characterizations demonstrated their potential for SMLM. At a moderate power density of 3.93 kW/cm2, these probes exhibit duty cycle as low as 0.18% and an emission rate as high as 26,700 photons/s. Phototoxicity assessment under single-molecule imaging conditions reveals that NIR illumination (721 nm) minimizes harm to living cells. Employing these NIR fluorophores, we successfully captured time-lapse super-resolution tracking of mitochondria at a Fourier ring correlation (FRC) resolution of 69.4 nm and reconstructed the ultrastructures of endoplasmic reticulum (ER) in living cells.

Effect of Surface Modification on the Luminescence of Individual Upconversion Nanoparticles.

Lanthanide-doped upconversion nanoparticles (UCNPs) hold promise for single-molecule imaging owing to their excellent photostability and minimal autofluorescence. However, their limited water dispersibility, often from the hydrophobic oleic acid ligand during synthesis, is a challenge. To address this, various surface modification strategies' impact on single-particle upconversion luminescence are studied. UCNPs are made hydrophilic through methods like ligand exchange with dye IR806, HCl or NOBF4 treatment, silica coating (SiO2 or mesoporous mSiO2), and self-assembly with polymer of DSPE-PEG or F127. The studies revealed that UCNPs modified with NOBF4 and DSPE-PEG exhibited notably higher single-particle brightness with minimal quenching (3% and 8%, respectively), followed by SiO2, F127, IR806, mSiO2, and HCl (84% quenching). HCl disrupted UCNPs's crystal lattice, weakening luminescence, while mSiO2 absorbed solvent molecules, causing luminescence quenching. Energy transfer to IR806 also reduced the brightness. Additionally, a prevalence of upconversion red emission over green is observed, with the red-to-green ratio increasing with irradiance. UCNPs coated with DSPE-PEG exhibited the brightest single-particle luminescence in water, retaining 48% of its original emission due to a lower critical micelle concentration and superior water protection. In summary, the investigation provides valuable insights into the role of surface chemistry on UCNPs at the single-particle level.

Thiolation for Enhancing Photostability of Fluorophores at the Single-Molecule Level.

Fluorescent probes are essential for single-molecule imaging. However, their application in biological systems is often limited by the short photobleaching lifetime. To overcome this, we developed a novel thiolation strategy for squaraine dyes. By introducing thiolation of the central cyclobutene of squaraine (thio-squaraine), we observed a ≈5-fold increase in photobleaching lifetime. Our single-molecule data analysis attributes this improvement to improved photostability resulting from thiolation. Interestingly, bulk measurements show rapid oxidation of thio-squaraine to its oxo-analogue under irradiation, giving the perception of inferior photostability. This discrepancy between bulk and single-molecule environments can be ascribed to the factors in the latter, including larger intermolecular distances and restricted mobility, which reduce the interactions between a fluorophore and reactive oxygen species produced by other fluorophores, ultimately impacting photobleaching and photoconversion rate. We demonstrate the remarkable performance of thio-squaraine probes in various imaging buffers, such as glucose oxidase with catalase (GLOX) and GLOX+trolox. We successfully employed these photostable probes for single-molecule tracking of CD56 membrane protein and monitoring mitochondria movements in live neurons. CD56 tracking revealed distinct motion states and the corresponding protein fractions. This investigation is expected to propel the development of single-molecule imaging probes, particularly in scenarios where bulk measurements show suboptimal performance.

A Universal Photoactivatable Tag Attached to Fluorophores Enables Their Use for Single-Molecule Imaging.

Single molecule localization microscopy based on photoactivation is a powerful tool for investigating the ultrastructure of cells. We developed a general strategy for photoactivatable fluorophores, using 2,3-dihydro-1,4-oxathiine group (SO) as a tag to attach to various skeletal structures, including coumarin, BODIPY, rhodamine, and cyanine. The conjugation of SO resulted in a significant loss of fluorescence due to photoinduced electron transfer (PeT). Under the irradiation of excitation light, singlet oxygen generated by the fluorophores converted the SO moiety into its ester derivative, terminated the PeT process, and restored the fluorescence. Single molecule localization imaging was achieved using a dual functional illuminating beam in the visible, acting as both the activating and the exciting source. We successfully applied these photoactivatable probes for time-lapse super-resolution tracking in living cells and super-resolution imaging of microtubule structures in neurons.

Spatial Abundance, Diversity, and Activity of Ammonia-Oxidizing Bacteria in Coastal Sediments of the Liaohe Estuary.

Ammonia-oxidizing bacteria (AOB) play an important role in nitrification in estuaries. The aim of this study was to examine the spatial abundance, diversity, and activity of AOB in coastal sediments of the Liaohe Estuary using quantitative PCR, high-throughput sequencing of the amoA gene coding the ammonia monooxygenase enzyme active subunit, and sediment slurry incubation experiments. AOB abundance ranged from 8.54 × 104 to 5.85 × 106 copies g-1 of wet sediment weight and exhibited an increasing trend from the Liaohe Estuary to the open coastal zone. Potential nitrification rates (PNRs) ranged from 0.1 to 336.8 nmol N g-1 day-1 along the estuary to the coastal zone. Log AOB abundance and PNRs were significantly positively correlated. AOB richness decreased from the estuary to the coastal zone. High-throughput sequencing analysis indicated that the majority of amoA gene sequences fell within the Nitrosomonas and Nitrosomonas-like clade, and only a few sequences were clustered within the Nitrosospira clade. This finding indicates that the Nitrosomonas-related lineage may be more adaptable to the specific conditions in this estuary than the Nitrosospira lineage. Sites with high nitrification rates were located in the southern open region and were dominated by the Nitrosomonas-like lineage, whereas the Nitrosospira lineage was found primarily in the northern estuary mouth sites with low nitrification rates. Thus, nitrification potentials in Liaohe estuarine sediments in the southern open region were greater than those in the northern estuary mouth, and the Nitrosomonas-related lineage might play a more important role than the Nitrosospira lineage in nitrification in this estuary.

Boosting Dye-Sensitized Luminescence by Enhanced Short-Range Triplet Energy Transfer.

Dye-sensitization can enhance lanthanide-based upconversion luminescence, but is hindered by interfacial energy transfer from organic dye to lanthanide ion Yb3+ . To overcome these limitations, modifying coordination sites on dye conjugated structures and minimizing the distance between fluorescence cores and Yb3+ in upconversion nanoparticles (UCNPs) are proposed. The specially designed near-infrared (NIR) dye, disulfo-indocyanine green (disulfo-ICG), acts as the antenna molecule and exhibits a 2413-fold increase in luminescence under 808 nm excitation compared to UCNPs alone using 980 nm irradiation. The significant improvement is attributed to the high energy transfer efficiency of 72.1% from disulfo-ICG to Yb3+ in UCNPs, with majority of energy originating from triplet state (T1 ) of disulfo-ICG. Shortening the distance between the dye and lanthanide ions increases the probability of energy transfer and strengthens the heavy atom effect, leading to enhanced T1 generation and improved dye-triplet sensitization upconversion. Importantly, this approach also applies to 730 nm excitation Cy7-SO3 sensitization system, overcoming the spectral mismatch between Cy7 and Yb3+ and achieving a 52-fold enhancement in luminescence. Furthermore, the enhancement of upconversion at single particle level through dye-sensitization is demonstrated. This strategy expands the range of NIR dyes for sensitization and opens new avenues for highly efficient dye-sensitized upconversion systems.

Enhancement of single upconversion nanoparticle imaging by topologically segregated core-shell structure with inward energy migration.

Manipulating topological arrangement is a powerful tool for tuning energy migration in natural photosynthetic proteins and artificial polymers. Here, we report an inorganic optical nanosystem composed of NaErF4 and NaYbF4, in which topological arrangement enhanced upconversion luminescence. Three architectures are designed for considerations pertaining to energy migration and energy transfer within nanoparticles: outside-in, inside-out, and local energy transfer. The outside-in architecture produces the maximum upconversion luminescence, around 6-times brighter than that of the inside-out at the single-particle level. Monte Carlo simulation suggests a topology-dependent energy migration favoring the upconversion luminescence of outside-in structure. The optimized outside-in structure shows more than an order of magnitude enhancement of upconversion brightness compared to the conventional core-shell structure at the single-particle level and is used for long-term single-particle tracking in living cells. Our findings enable rational nanoprobe engineering for single-molecule imaging and also reveal counter-intuitive relationships between upconversion nanoparticle structure and optical properties.

Polybrominated diphenyl ethers in sediments of the Daliao River Estuary, China: levels, distribution and their influencing factors.

The concentrations, compositional profiles, possible sources of polybrominated diphenyl ethers (PBDEs) in sediments of the Daliao River Estuary as well as the factors influencing the distribution of PBDEs were investigated. The total concentrations of PBDEs ranged from 0.13 to 1.98 ng g(-1)d.w. BDE209 was the dominating congener in all sediment samples, indicating the pollution of PBDEs in the Daliao River Estuary mainly came from the use of deca-BDE commercial mixtures. The intrusion of sea waters promoted the deposition of the colloid-associated PBDEs in the estuary. There were significantly negative correlations between PBDE concentration in sediment with pH value and salinity in the bottom water. The higher river flow in the flood season (summer) obviously accelerated the transport of PBDEs, and thereby increased the risk of PBDE contamination to the deep ocean. Moreover, a positive correlation between TOC and PBDE distributions was observed, suggesting that TOC regulated the distributions of PBDEs in sediments of Daliao River Estuary.

Dioxin-like compounds in sediments from the Daliao River Estuary of Bohai Sea: distribution and their influencing factors.

The concentrations, compositional profiles, and potential ecological risk of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs) and polychlorinated naphthalenes (PCNs) in sediments of the Daliao River Estuary were investigated. Total concentrations of PCDD/Fs, PCBs, and PCNs were in the range of 11.3-133.2 ng/kg dry weight (dw), 1 971-37 632 ng/kg dw and 33.1-284.4ng/kg dw, respectively. The total TEQ values varied from 0.37 to 4.08ng/kg dw, with the dominant contributions by PCDD/Fs, then by PCBs and PCNs. The spatial distributions of PCDD/Fs, PCBs and PCNs in the river estuary were much related to hydrodynamic conditions. The risk of contamination to the deeper sea was increased in the flood seasons. Moreover, our data confirmed that both organic matter in sediments and molecular properties of dioxin-like compounds were the factors which strongly influenced the partition behavior of these dioxin-like compounds between sediments and water phase in the estuarine zone.