Xanthene, cyanine, oxazine and BODIPY: the four pillars of the fluorophore empire for super-resolution bioimaging.

In the realm of biological research, the invention of super-resolution microscopy (SRM) has enabled the visualization of ultrafine sub-cellular structures and their functions in live cells at the nano-scale level, beyond the diffraction limit, which has opened up a new window for advanced biomedical studies to unravel the complex unknown details of physiological disorders at the sub-cellular level with unprecedented resolution and clarity. However, most of the SRM techniques are highly reliant on the personalized special photophysical features of the fluorophores. In recent times, there has been an unprecedented surge in the development of robust new fluorophore systems with personalized features for various super-resolution imaging techniques. To date, xanthene, cyanine, oxazine and BODIPY cores have been authoritatively utilized as the basic fluorophore units in most of the small-molecule-based organic fluorescent probe designing strategies for SRM owing to their excellent photophysical characteristics and easy synthetic acquiescence. Since the future of next-generation SRM studies will be decided by the availability of advanced fluorescent probes and these four fluorescent building blocks will play an important role in progressive new fluorophore design, there is an urgent need to review the recent advancements in designing fluorophores for different SRM methods based on these fluorescent dye cores. This review article not only includes a comprehensive discussion about the recent developments in designing fluorescent probes for various SRM techniques based on these four important fluorophore building blocks with special emphasis on their effective integration into live cell super-resolution bio-imaging applications but also critically evaluates the background of each of the fluorescent dye cores to highlight their merits and demerits towards developing newer fluorescent probes for SRM.

General practitioners' attitudes towards and frequency of collaboration with pharmacists in China: a cross-sectional study.

BACKGROUND: Building interprofessional working relationships between general practitioners (GPs) and pharmacists is essential to ensure high-quality patient care. However, there is limited Chinese literature on GP-pharmacist collaboration, and few studies have explored GPs' experiences with pharmacist integration into general practices. This study aimed to investigate GPs' attitudes towards and frequency of collaboration with pharmacists in China. METHODS: This cross-sectional study used an online self-administered questionnaire integrating two scales, ATCI-GP and FICI-GP, which had been translated and validated to investigate 3,248 GPs from February 15 to March 15, 2023 across Zhejiang Province, China. Descriptive analyses were used, and the factors associated with GPs' frequency of collaboration with pharmacists were explored using logistic regression analysis. RESULTS: A total of 2,487 GPs (76.6%) responded and consented to participate in the survey; 52.3% were male and the mean age was 35.4 years. Most GPs agreed that they shared common goals and objectives with pharmacists when caring for patients (90.0%), and pharmacists were open to working with them on patients' medication management (80.8%). However, half of the GPs did not change or seldom changed the patient's medication on the pharmacist's advice (51.4%). Logistic regression analysis showed that GPs who were older and had more years of practice were more likely to agree that pharmacists were willing to collaborate, had common goals for treatment and that they would change the patient's medication on the advice of the pharmacist. GPs who had regular communication protocols (adjusted odds ratio1 [aOR1] = 1.88, 95% CI 1.45-2.45; aOR2 = 3.33, 95% CI 2.76-4.02), participated in joint continuing education (aOR1 = 1.87, 95% CI 1.44-2.43; aOR2 = 2.27, 95% CI 1.91-2.70), provided recommendations for medication review (aOR1 = 3.01, 95% CI 2.07-4.38; aOR2 = 3.50, 95% CI 2.51-4.86), and communicated with pharmacists during resident training (aOR1 = 2.15, 95% CI 1.78-2.60; aOR2 = 1.38, 95% CI 1.18-1.62) were associated with a more positive attitude towards and higher frequency of cooperation. CONCLUSIONS: GPs in China displayed a positive attitude towards cooperating with pharmacists, but they did not demonstrate a similar level of practice. As environmental determinants impact interdisciplinary collaboration, healthcare managers and policy-makers need to implement measures that foster a supportive environment conducive to interdisciplinary collaboration.

NIR-II multifocal structured illumination microscopy.

Optical microscopy has been widely used as a versatile tool in biological research. However, its penetration depth and spatial resolution are desperately limited by light scattering during deep propagation in turbid medium. Here, we implement near-infrared second window (1000-1700 nm) multifocal structured illumination microscopy (NIR-II MSIM) capable of deep penetration, high contrast, and enhanced spatial resolution. Raster-scanning multifocal illumination patterns ensure homogeneous illumination of the sample. By integrating NIR-II photoemission into multifocal photoexcitation, NIR-II MSIM affords deep imaging with improved lateral resolution (∼1.49 µm) at a depth of 2.5 mm in an Intralipid/agar phantom and outstanding contrast. Additionally, imaging at longer wavelength in the NIR-II region shows superior performance. This NIR-II MSIM system will afford a promising platform for studying physiological phenomena in turbid specimens in the future.

A lysosome-targeted near-infrared fluorescent probe for imaging of acid phosphatase in living cells.

Fluorescent probes for the detection of acid phosphatases (ACP) are important in the investigation of the pathology and diagnosis of diseases. We reported a lysosome-targeted near-infrared (NIR) fluorescent probe SHCy-P based on a novel NIR-emitting thioxanthene-indolium dye for the detection of ACP. The probe showed a long wavelength fluorescence emission at λem = 765 nm. Due to the ACP-catalyzed cleavage of the phosphate group in SHCy-P, the probe exhibited high selectivity and sensitivity for the 'turn-on' detection of ACP with a limit of detection as low as 0.48 U L-1. The probe SHCy-P could also be used to detect and image endogenous ACP in lysosomes. In light of these prominent properties, we envision that SHCy-P will be an efficient optical imaging approach for investigating the ACP activity in disease diagnosis.

Novel near-infrared fluorescent probe with a large Stokes shift for sensing hypochlorous acid in mitochondria.

Hypochlorous acid (HOCl) plays a crucial role in various physiological and pathological processes. However, it is still a challenge to design a xanthene-based near-infrared (NIR) fluorescent probe with a large Stokes shift for sensing HOCl. In this work, a novel mitochondria-targeted fluorescent probe, MXS, with a large Stokes shift based on a xanthene-hemicyanine dyad structure, has been successfully designed and synthesized for the specific detection of HOCl. Gratifyingly, the peak-to-peak Stokes shift of MXS was found to be 130 nm, which was obviously larger than those of conventional rhodamine dyes and most reported xanthene-based hypochlorous acid probes. As expected, MXS exhibited high selectivity, high sensitivity, and fast response time (30 s) for the detection of HOCl via a specific HOCl-promoted intramolecular charge transfer process. The detection limit of MXS for HOCl is calculated to be as low as 72 nM, enabling its use within the physiological concentration range of HOCl (5-25 µM). Importantly, MXS is able to permeate cell membranes and accumulate in the mitochondria, which is convenient for monitoring the variation of hypochlorous acid concentration in the mitochondria of living cells.

Fluorescent Porous Carbazole-Decorated Copolymer Monodisperse Microspheres: Facile synthesis, Selective and Recyclable Detection of Iron†(III) in Aqueous Medium.

We demonstrate an environmentally friendly one-step soap-free emulsion polymerization strategy to develop fluorescent carbazole-based copolymer monodisperse microspheres for highly sensitive and selective detection of Fe3+ . The copolymer microspheres feature a stable spherical morphology with a narrow size distribution through regulating N-vinylcarbazole (NVCz) content (1.25-10.0 wt.%). Notably, the as-made microspheres exhibit a strong luminescence, tunable emission intensity and specific surface areas. Interestingly, the fluorescence of the copolymer microspheres can be selectively quenched by trace amounts of Fe3+ due to the oxidation of carbazole, and the quenching fluorescence can be facilely recovered by reduction with NaBH4 . Its excellent sensing performance is shown in terms of high sensitivity (low limit of detection, 1.3 µm), excellent selectivity, and rapid response rate, due to the porous nature of the copolymer microspheres. These results illustrate the copolymer microspheres obtained by simple preparative procedure without using expensive or toxic raw materials would serve as a high performance sensor for highly selective and recyclable detection of Fe3+ in aqueous medium.

Synthesis of near-infrared fluorescent rhodamines via an SNArH reaction and their biological applications.

Near-infrared (NIR) dyes are of great interest in biomedicine due to diminished interfering absorption and fluorescence from biological samples, reduced scattering, and enhanced tissue penetration depth. In this context, we report the synthesis of rectilinearly π-extended rhodamine dyes using a unique intramolecular nucleophilic substitution of aromatic hydrogen (SNArH) strategy. The strategy makes use of an SNArH reaction between a preorganized aromatic amino nitrogen and an electron-deficient carbon in the xanthylium ion. The SNArH reaction presented herein can be performed under mild conditions without a transition metal catalyst and can be expected to enable the preparation of a wide variety of π-extended near-infrared fluorescent rhodamine dyes. Using this strategy, seven rectilinearly π-extended rhodamines (RE1-RE7) that had fluorescence emission wavelengths in the near-infrared region were synthesized. RE1, RE3, and RE4 were lysosome targetable and showed good photostabilities. In addition, using dye RE1 as a precursor, we constructed a novel NIR fluorescent turn-on probe (RE1-Cu), which can be used for detecting Cu2+ in living cells, demonstrating the value of our NIR functional fluorescent dyes.

Deciphering the Decomposition Mechanisms of Ether and Fluorinated Ether Electrolytes on Lithium Metal Surfaces: Insights from CMD and AIMD Simulations.

The performance of lithium metal batteries can be significantly enhanced by incorporating fluorinated ether-based electrolytes, yet the solid electrolyte interphase (SEI) formation mechanism on lithium metal surfaces remains elusive. This study employs classical and ab initio molecular dynamics simulations to investigate the decomposition mechanisms of lithium bis(fluoromethanesulfonyl)imide (LiFSI) in 1,2-diethoxyethane (DEE) and its fluorinated analogues, F5DEE and F2DEE, when in contact with lithium metal. Our findings indicate that F5DEE-based electrolytes favor the formation of a FSI-rich primary solvation shell around Li+, while F2DEE-based electrolytes yield a solvent-rich environment. The normalized number density at the Li/electrolyte/Li interface shows a depletion of FSI anions in the electrochemical double layer (EDL) structure near the Li anode upon charging, with the distance between the first main peak of the FSI anion and Li anode following the order F5DEE < DEE < F2DEE. Analysis of the electronic projected density of states and charge transfer dynamics unveils the reductive dissociation pathways of FSI anions and fluorinated DEE solvents on the lithium metal surface, taking into account the influence of the EDL structure. DEE is identified as the most reduction-stable solvent, leading to the selective dissociation of FSI anions and the formation of an entirely inorganic SEI. In contrast, F2DEE displays a pronounced reduction tendency, forming an organic-rich SEI due to the solvent-dominated lowest unoccupied molecular orbital at the interface. F5DEE, competing with FSI anions for reduction, results in the formation of an inorganic-rich hybrid SEI with the highest LiF content. The simulation results correlate well with experimental observations and underscore the pivotal role of various fluorinated functional groups in the formation of EDL and SEI near the lithium metal surface.

Rhodamine-based fluorescent probe for dynamic STED imaging of mitochondria.

Stimulated emission depletion (STED) microscopy holds tremendous potential and practical implications in the field of biomedicine. However, the weak anti-bleaching performance remains a major challenge limiting the application of STED fluorescent probes. Meanwhile, the main excitation wavelengths of most reported STED fluorescent probes were below 500 nm or above 600 nm, and few of them were between 500-600 nm. Herein, we developed a new tetraphenyl ethylene-functionalized rhodamine dye (TPERh) for mitochondrial dynamic cristae imaging that was rhodamine-based with an excitation wavelength of 560 nm. The TPERh probe exhibits excellent anti-bleaching properties and low saturating stimulated radiation power in mitochondrial STED super-resolution imaging. Given these outstanding properties, the TPERh probe was used to measure mitochondrial deformation, which has positive implications for the study of mitochondria-related diseases.

WeChat official accounts' posts on medication use of 251 community healthcare centers in Shanghai, China: content analysis and quality assessment.

Background: The dissemination of online health information (OHI) on medication use via WeChat Official Accounts (WOAs) is an effective way to help primary care practitioners (PCPs) address drug-related problems (DRPs) in the community. Although an increasing number of primary care institutions in China have published WOA posts on medication use, their content and quality have not yet been assessed. Objective: This study aimed to explore the general features and content of WOA posts on medication use published by community healthcare centers (CHCs) in Shanghai, China and to assess their quality of content. It also aimed to explore the factors associated with the number of post views. Methods: From June 1 to October 31, 2022, two coauthors independently screened WOA posts on medication use published throughout 2021 by the CHCs in Shanghai. Content analysis was performed to analyze their general features (format, length, and source, etc.) and content (types of medicines and diseases). The QUEST tool was used to assess the quality of the posts. We compared the differences among posts published by CHCs in central urban areas and suburban areas, and used multiple linear regression to explore the factors associated with the number of post views. Results: A total of 236 WOAs of interest published 37,147 posts in 2021, and 275 (0.74%) of them were included in the study. The median number of post views was 152. Thirty percent of the posts were reviewed by the CHCs' staff before publication and only 6% provided information on PCPs' consultations. The most commonly mentioned medicines and diseases in the posts were Chinese patent medicines (37.1%) and respiratory diseases (29.5%). The posts frequently provided information on indications (77%) and usage (56%) but rarely on follow-up (13%) and storage (11%). Of the posts, 94.9% had a total QUEST score < 17 (full score = 28). The median number of post views and total post quality scores did not significantly differ among the CHCs in central urban and suburban areas. In the multiple linear regression model, the number of post views was associated with scores of complementarity (B = 56.47, 95% CI 3.05, 109.89) and conflict of interest (B = -46.40, 95% CI -56.21, -36.60). Conclusion: The quantity and quality of WOA posts on medication use published by CHCs in China need improvement. The quality of posts may partially impact the dissemination effect, but intrinsic causal associations merit further exploration.

A Digital Twin of the Coaxial Lamination Mixer for the Systematic Study of Mixing Performance and the Prediction of Precipitated Nanoparticle Properties.

The synthesis of nanoparticles in microchannels promises the advantages of small size, uniform shape and narrow size distribution. However, only with insights into the mixing processes can the most suitable designs and operating conditions be systematically determined. Coaxial lamination mixers (CLM) built by 2-photon polymerization can operate long-term stable nanoparticle precipitation without fouling issues. Contact of the organic phase with the microchannel walls is prevented while mixing with the aqueous phase is intensified. A coaxial nozzle allows 3D hydrodynamic focusing followed by a sequence of stretch-and-fold units. By means of a digital twin based on computational fluid dynamics (CFD) and numerical evaluation of mixing progression, the influences of operation conditions are now studied in detail. As a measure for homogenization, the mixing index (MI) was extracted as a function of microchannel position for different operating parameters such as the total flow rate and the share of solvent flow. As an exemplary result, behind a third stretch-and-fold unit, practically perfect mixing (MI>0.9) is predicted at total flow rates between 50 µL/min and 400 µL/min and up to 20% solvent flow share. Based on MI values, the mixing time, which is decisive for the size and dispersity of the nanoparticles, can be determined. Under the conditions considered, it ranges from 5 ms to 54 ms. A good correlation between the predicted mixing time and nanoparticle properties, as experimentally observed in earlier work, could be confirmed. The digital twin combining CFD with the MI methodology can in the future be used to adjust the design of a CLM or other micromixers to the desired total flow rates and flow rate ratios and to provide valuable predictions for the mixing time and even the properties of nanoparticles produced by microfluidic antisolvent precipitation.

Horseshoe lamination mixer (HLM) sets new standards in the production of monodisperse lipid nanoparticles.

Microfluidic mixers promise unique conditions for the controlled and continuous preparation of nanoparticles by antisolvent precipitation. Nanoparticles may enable encapsulation of drug or mRNA molecules in the form of carrier nanoparticles or can provide higher bioavailability in the form of drug nanoparticles. The ultimate goal in microfluidic approaches is the production of nanoparticles with narrow size distributions while avoiding contaminations and achieving sufficiently high throughput. To achieve this, a novel microfluidic precipitation device was developed and realized by two-photon polymerization: mixing elements were designed in such a way that the liquids undergo a repeated Smale horseshoe transformation resulting in an increased interfacial area and mixing times of less than 10 ms. These elements and an additional 3D flow focusing ensure that no organic phase is exposed to the channel walls. The integration of a fluidic shield layer in the flow focusing proved to be useful to delay the precipitation process until reaching a sufficient distance to the injection nozzle. Lipid nanoparticle preparation with different concentrations of castor oil or the hard fat Softisan® 100 were performed at different flow rates and mixing ratios with and without a shield layer. Flow rates of up to 800 µl min-1 and organic phase mixing ratios of up to 20% resulted in particle sizes ranging from 42 nm to 166 nm with polydispersity indices from 0.04 to 0.11, indicating very narrowly distributed, and in most cases even monodisperse, nanoparticles. The occurrence of fouling can be completely suppressed with this new type of mixing elements, as long as Dean vortices are prevented. Moreover, this parameter range in the horseshoe lamination mixer provided a stable and continuous process, which enables a scalable production.

Prevalence and genotype distribution of human papillomavirus among 29 263 women from the Longgang community of Shenzhen.

BACKGROUND: Human papillomavirus (HPV) vaccination and screening have been demonstrated to be effective methods for controlling cervical cancer. To provide evidence for effective targeted interventions, we investigated the HPV prevalence and genotypic distribution in women of different age groups in the Longgang community of Shenzhen, China. METHODS: A total of 29 263 women were enrolled in the Longgang District Central Hospital from January 2018 to October 2020. Cervical specimens were collected at enrolment. We used a polymerase chain reaction diagnostic kit to detect the genotypes of HPV. RESULTS: The prevalence of overall, high-risk, probable high-risk (PHR) and low-risk HPV infection was 8.2% (95% confidence interval [CI] 7.9 to 8.5), 6.9% (95% CI 6.6 to 7.2), 0.9% (95% CI 0.8 to 1.1) and 1.6% (95% CI 1.5 to 1.8), respectively. High-risk HPV genotype infections accounted for 84.4% (95% CI 82.8 to 85.8) of the overall HPV infections. The five most predominant genotypes were HPV-52, HPV-16, HPV-58, HPV-53 and HPV-51. CONCLUSION: Our study found that the prevalence of HPV infection increased with age and women 55-59 y of age presented the highest HPV prevalence. As a PHR subtype, HPV-53 has a higher infection rate in women. HPV-52, HPV-16 and HPV-58 are common infection genotypes.

A new near-infrared fluorescent probe for sensing extreme acidity and bioimaging in lysosome.

Since the intracellular pH plays an important role in the physiological and pathological processes, however, the probes that can be used for monitoring pH fluctuation under extreme acidic conditions are currently rare, so it is necessary to construct fluorescent probes for sensing pH less than 4. In this work, we developed a new near-infrared (NIR) fluorescent probeCy-SNNfor sensing pH fluctuation under extremely acidic conditions. For the preparation of this probe, benzothiozolium moiety was chosen as lysosomal targeting unit and NIR fluorophore, and barbituric acid moiety was fused in the polymethine chain of probe to introduce protonation center. Surprisingly, on the basis of the balance of quaternary ammonium salts and free amines, the pkavalue ofCy-SNNwas calculated as low as 2.96, implying thatCy-SNNcan be used in acidic conditions with pH < 4. Moreover,Cy-SNNexhibited highly selective response to H+over diverse analytes in real-time with dependable reversibility. Importantly,Cy-SNNcan be used to specifically target lysosome, providing potential tools for monitoring the function of lysosome in autophagy process.

A highly sensitive sensor for colorimetric detection of palladium(II) in lysosomes and its applications.

Considering the scarcity of palladium ion probes with subcellular organelle targeting, especially probes with near-infrared (NIR) emission wavelength fluorophores, our group has been working to overcome this problem and looking forward to providing potential practical tools for exploring the toxicity of palladium ions at the subcellular level. In this paper, a novel colorimetric and NIR fluorescent probe, BHCy-Pd, for the specific detection of palladium ions (Pd2+) in lysosomes via an internal charge-transfer (ICT) mechanism was designed and synthesized. As expected, BHCy-Pd exhibited a rapid, selective, and sensitive response for palladium with an ultralow limit of detection at 5.9 nM, accompanied by a distinct color change from purple to blue. Furthermore, BHCy-Pd can be made into a simple test strip for rapid and easy detection of Pd2+ in practical applications. Importantly, BHCy-Pd is capable of specific distribution in lysosomes, and thus can detect Pd2+ in real-time, thereby providing a potential tool for studying the cytotoxicity of Pd2+ ions at the subcellular level.

Rational design of a large Stokes shift xanthene-benzothiozolium dyad for probing cysteine in mitochondria.

Xanthene-modified cyanine dyes are considered to be an effective means to extend the emission wavelength and improve the photo-stability of cyanine dyes. However, the fluorophores obtained by this strategy generally have narrow Stokes shifts, which severely limits their application in biological imaging. Herein, a reasonable design strategy is adopted to provide an effective strategy to commendably improve the Stokes shift of xanthene-benzothiozolium fluorophores via the simultaneous expansion of a molecular π-conjugated system and heteroatomic substitution. Combined with density functional theory calculation guidance, the O atom is replaced with the S atom in the xanthene moiety and a π-conjugated benzene ring is introduced in the benzothiozolium moiety. Surprisingly, the results of the spectroscopic experiment showed that the fluorescence emission wavelength of PhCy-OH was extended to 803 nm, and the Stokes shift was improved to 68 nm. PhCy-Cys can effectively distinguish Cys from homocysteine (Hcy) and glutathione (GSH) with an extremely low detection limit of 0.166 µM. Importantly, PhCy-Cys has the ability to image endogenous Cys in mitochondria, providing the potential for exploring the specific function and mechanism of Cys in regulating oxidative stress in mitochondria.

Novel near-infrared spectroscopic probe for visualizing hydrogen sulfide in lysosomes.

Considering the scarcity of hydrogen sulfide (H2S) probes with subcellular organelle targeting, especially probes with near-infrared (NIR) emission wavelengths fluorophores, our group has been working to overcome this problem and looking forward to providing potential practical tools for exploring the relationship between the physiology and pathology of H2S at subcellular level. In this paper, a novel colorimetric and NIR fluorescent probe SHCy-H2S for the specific detection of H2S in lysosome over other biological thiols was designed and synthesized. The xanthene-benzothiozolium fluorophore was chosen to provide fluorescence emission maxima over 735 nm, and 2,4-dinitrophenyl group was chosen as fluorescence quenching group and specific H2S response site. Impressively, SHCy-H2S exhibited high selectivity, fast response and detection limit as low as 0.116 µM for H2S, marked obvious color changes in naked-eye and fluorescence. Specially, SHCy-H2S was capable of specifically imaging endogenous lysosomal hydrogen sulfide, providing a potential tool for exploring the function of H2S at subcellular level.

Mitochondrial structural variations in the process of mitophagy.

Mitochondrion is one of significant organelles inside cells because it serves as a hub for energy management and intracellular signaling. Internal/external damages on mitochondria would lead to mitochondrial stresses with the malfunctions, accompanying with the changes of morphological structure and abnormal local environments (pH values). Mitophagy is capable of degradation of damaged mitochondrial segments to restore its normal metabolism, dynamics, and biogenesis. The dynamic structural visualization and pH quantification can be helpful for the understanding of mitochondrial functions as well as the diagnosis of disorders linking with this process. In this work, we use confocal laser scanning microscopy, STED super-resolution nanoscopy and fluorescence lifetime imaging microscopy, in conjunction with a mitochondrial probe to image the dynamic changes in the mitochondrial morphology and microenvironmental pH values during mitophagy in live cells, in particular, the structural changes of mitochondrial cristae beyond optical diffraction can be distinguished by STED nanoscopy with/without treatment by CCCP, which will provide a new view for the diagnosis and personalized treatment of mitochondrial dysfunction-related diseases.

Halogen-doped phosphorescent carbon dots for grayscale patterning.

Flexible organic materials that exhibit dynamic ultralong room temperature phosphorescence (DURTP) via photoactivation have attracted increasing research interest for their fascinating functions of reversibly writing-reading-erasing graphic information in the form of a long afterglow. However, due to the existence of a nonnegligible activation threshold for the initial exposure dose, the display mode of these materials has thus far been limited to binary patterns. By resorting to halogen element doping of carbon dots (CDs) to enhance intersystem crossing and reduce the activation threshold, we were able to produce, for the first time, a transparent, flexible, and fully programmable DURTP composite film with a reliable grayscale display capacity. Examples of promising applications in UV photography and highly confidential steganography were constructed, partially demonstrating the broad future applications of this material as a programmable platform with a high optical information density.