The development of a compact and affordable fluorescence microscope can be a formidable challenge for growing needs in on-site testing and detection of fluorescent labeled biological systems, especially for those who specialize in biology rather than in engineering. In response to such a situation, we present an open-source miniature fluorescence microscope using Raspberry Pi. Our fluorescence microscope, with dimensions of 19.2 × 13.6 × 8.2 cm3 (including the display, computer, light-blocking case, and other operational requirements), not only offers cost-effectiveness (costing less than $500) but is also highly customizable to meet specific application needs. The 12.3-megapixel Raspberry Pi HQ Camera captures high-resolution imagery, while the equipped wide-angle lens provides a field of view measuring 21 × 15 mm2. The integrated wireless LAN in the Raspberry Pi, along with software-controllable high-powered fluorescence LEDs, holds potential for a wide range of applications. This open-source fluorescence microscope offers biohybrid sensor developers a versatile tool to streamline unfamiliar mechanical design tasks and open new opportunities for on-site fluorescence detections.

An intricate interplay of complex spatio-temporal events underlies brain functions. Therefore, clarifying these dynamic processes is indispensable for revealing the mechanisms of brain functions. Fluorescence imaging is a powerful technique for visualizing cellular and molecular dynamics in the brain. Recent developments in fluorescent indicators and specialized optics have advanced research in the field of neuroscience. In this review, I will exemplify the power and beauty of fluorescence imaging by discussing my work focusing on the molecular dynamics of metabotropic glutamate receptor (mGluR) signaling at the synapse. By developing novel fluorescent indicators for glutamate, inositol 1,4,5-trisphosphate and Ca2+ within the endoplasmic reticulum, I succeeded in imaging the spatio-temporal dynamics of synaptic mGluR signaling, which led to the identification of novel mechanisms of mGluR-mediated glutamatergic neurotransmission. These discoveries highlight the importance of the development and application of novel fluorescence imaging techniques for the investigation of brain functions.

Digital counting assays, that quantify targets by counting individual signal entities, provide a promising way for the sensitive analysis of biomarkers even at the single-molecule level. Considering the requirements of complex enzyme-catalyzed amplification techniques and specialized instruments in traditional digital counting biosensors, herein, a simple digital counting platform for microRNA (miRNA) analysis is developed by employing the miRNA-templated click chemical ligation to hinge ultrabright quantum dot-doped nanoparticles (QDNPs) on the bottom of microplate well. Compared with the traditional short miRNA-mediated sandwich hybridization mechanism, the click chemistry-mediated ligation featured enhanced stability, achieving higher sensitivity by directly counting the number of QDNPs with a common wide-field fluorescence microscope. Furthermore, enzyme-free cycling click ligation strategy is adopted to push the detection limit of miRNA down to a low level of 8 fM. What is more, taking advantages of the tunable emission wavelength and narrow emission spectra of fluorescent nanoparticles, the platform enables simultaneous detection of multiplex miRNA targets without cross interference. Benefiting from the simple operation, high sensitivity, and good generality, miRNA analysis in complex samples is successfully achieved. This method not only pioneers a new route for digital counting assays but also holds great potential in miRNA-related biological researches.

Background Ixora coccinea is a medicinal plant with many active constituents that are responsible for wound healing and have anticancer properties. Herbal extracts increase the mechanisms related to wound healing, like blood clotting, fighting infection, and epithelialization. The effect responsible for this property may be the presence of phytoconstituents like flavonoids, polyphenols, and alkaloids. Many researchers have evaluated the wound-healing effect of I. coccinea leaf extract in aqueous methanol. This study aimed to determine the in vitro wound healing and anticancer efficacy of I. coccinea leaf ethyl acetate extract and evaluate the in silico docking of the selected phytoconstituents of I. coccinea in the 2vcj protein. Materials and methods The human dermal fibroblast cell line was used to determine the rates of cell migration and proliferation for evaluating the wound-healing effect of the I. coccinea leaf ethyl acetate fraction. 4\',6-diamidino-2-phenylindole (DAPI) fluorescence labeling was used to estimate the rate of cell migration. The one-step TUNEL (TdT-mediated dUTP Nick-End Labeling) in situ apoptosis kit and the annexin V-FITC/7-AAD apoptosis kit were used to perform DNA damage assays in the malignant melanoma cell line. The ethyl acetate fraction of I. coccinea leaves was analyzed for its impact on wound healing markers, including keratin-10, keratin-14, type IV collagen, and α-SMA. Results The wound-healing nature was interesting in the ethyl acetate fraction at doses of 50 µg/mL and 100 µg/mL. Both studies involved in the DNA damage study against malignant melanoma cell lines showed the cleavage of apoptotic cancer cells, which was detected using a fluorescence microscope. When compared with the control, a dose of 100 μg/ml of ethyl acetate fraction from the leaves of I. coccinea showed fibroblast migration of cells into the wound area. The statistical values were considered significant at the level of P < 0.05. An in silico docking study on the 2vcj protein revealed that selected phytoconstituents of I. coccinea resulted in good docking scores to inhibit Hsp90. Conclusion I. coccinea ethyl acetate leaf extract can inhibit the growth of malignant melanoma cell lines and promote wound healing, as shown by the study results. It might be a viable therapeutic modality for skin cancer.

Polyhexamethylene guanidine (PHMG) is a guanidine-based chemical that has long been used as an antimicrobial agent. However, recently raised concerns regarding the pulmonary toxicity of PHMG in humans and aquatic organisms have led to research in this area. Along with PHMG, there are concerns about the safety of non-guanidine 5-chloro-2-methylisothiazol-3(2H)-one/2-methylisothiazol-3(2H)-one (CMIT/MIT) in human lungs; however, the safety of such chemicals can be affected by many factors, and it is difficult to rationalize their toxicity. In this study, we investigated the adsorption characteristics of CMIT/ MIT on a model pulmonary surfactant (lung surfactant, LS) using a Langmuir trough attached to a fluorescence microscope. Analysis of the π-A isotherms and lipid raft morphology revealed that CMIT/MIT exhibited minimal adsorption onto the LS monolayer deposited at the air/water interface. Meanwhile, PHMG showed clear signs of adsorption to LS, as manifested by the acceleration of the L o phase growth with increasing surface pressure. Consequently, in the presence of CMIT/MIT, the interfacial properties of the model LS monolayer exhibited significantly fewer changes than PHMG.

BACKGROUND: The stratum corneum (SC), the outermost layer of the skin epidermis, acts as an effective bi-directional barrier, preventing water loss (inside-outside barrier) and entry of foreign substances (outside-inside barrier). Although transepidermal water loss (TEWL) is a widely-used measure of barrier function, it represents only inside-outside protection. Therefore, we aimed to establish a non-invasive method for quantitative evaluation of the outside-inside barrier function and visually present a skin barrier model.
METHODS: Skin barrier damage was induced by applying a closed patch of 1% sodium dodecyl sulfate to the forearms of eight participants; they were instructed to apply a barrier cream on a designated damaged area twice daily for 5 days. The SC barrier was evaluated by measuring TEWL and fluorescein sodium salt penetration rate before, immediately after, and 5 days after damage. The penetration rate was assessed using tape-stripping (TS) technique and fluorescence microscopy.
RESULTS: The rates of fluorescein sodium salt penetration into the lower layers of SC differed significantly based on the degree of skin barrier damage. The correlation between penetration rate and TEWL was weak after two rounds of TS and became stronger after subsequent rounds. Five days after skin barrier damage, the penetration rate of all layers differed significantly between areas with and without the barrier cream application.
CONCLUSIONS: Our findings demonstrated that the penetration rate was dependent on skin barrier conditions. The penetration rate and corresponding fluorescence images are suitable quantitative indicators that can visually represent skin barrier conditions.

Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson\'s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories. Key features • Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body. • Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.

To evaluate the thermal stability of composite polymer-modified asphalt, thermoplastic elastomer styrene-butadiene rubber (SBR)/polypropylene (PP) pellets were prepared using a hot-melt blending technique, with butyl rubber powder and waste polypropylene pellets as raw materials. The effects of different evaluation indexes on the thermal stability of SBR/PP-modified asphalt were investigated using a frequency scan test and a multi-stress creep recovery (MSCR) test, and the compatibility of SBR/PP particles with asphalt was studied using the Cole-Cole diagram and microstructure images. The tests show that, firstly, the performance grade (PG) classification of asphalt can be improved by adding an SBR/PP thermoplastic elastomer to enhance the adaptability of asphalt in high- and low-temperature environments, and the evaluation separation index can reflect the high-temperature storage stability of composite-modified asphalt more reasonably. Additionally, the larger the rubber-to-plastic ratio the worse the high-temperature thermal stability of composite-modified asphalt. Moreover, the addition of additives to the composite particles can promote the SBR/PP particles in the asphalt to be more uniformly dispersed, forming a more desirable microstructure and improving the thermal stability of composite-modified asphalt. Ultimately, the semicircular curve of the Cole-Cole diagram can reflect the compatibility characteristics of the two-phase structure of SBR/PP-modified asphalt, which can be used as an auxiliary index to evaluate the compatibility of polymer-modified asphalt.

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Microplastics (MPs) in the atmosphere can undergo long-range transport from emission regions to pristine terrestrial and oceanic ecosystems. Due to their inherent toxic and hazardous characteristics, MPs pose serious risks to both human well-being and the equilibrium of ecosystem. The present study outlines the comprehensive characterization, spanning physical and chemical attributes of MPs associated with atmospheric aerosols. Total suspended particulates (TSPs) were collected on a quartz fibre filter by operating a high-volume sampler for 24 h during distinct years (March, 2016 and November, 2020) at a coastal location in the northeast Arabian Sea. Subsequent to the sampling, a series of techniques were applied including density separation. The assessment and scrutiny of the MPs was carried out using stereo-zoom microscopy with supplementary validation using advanced fluorescence microscopy for enhanced precision in identification. Our comparative assessment suggests peroxide treatment followed by density separation could be a robust procedure for the definitive identification and characterization of MPs in the atmosphere. Average total abundance of MPs was found to be 1.30 ± 0.14 n/m3 in 2016 and 1.46 ± 0.12 n/m3 in 2020 with fibres, fragments and films having similar relative contributions (41 %, 31 %, 28 % in 2016 and 40 %, 35 %, 25 % in 2020). Fibres were found to be dominant morphotype followed by fragments and films over the coastal region of the Arabian Sea. In order to unravel the detailed chemical nature of these MPs, spectral analysis using μ-FTIR was carried out. The outcome of the analysis showed prevailing polymers as polyvinyl chloride and polymethyl methacrylate (50545 %) as dominant polymers followed by polyester (15 %), styrene butyl methacrylate (11 %), and polyacetal (9 %). MPs present in the vicinity of the Arabian Sea have potential to supply nutrients and toxicants, consequently can contribute to the modulation of the surface water biogeochemical processes.