Immunohistochemistry of Brain Tissues.

Immunohistochemistry (IHC) is the basis of histological or pathological analysis and is widely used to enable the detection and characterization of proteins in various organ tissues, including brain tissues. IHC is commonly performed on formalin-fixed paraffin-embedded (FFPE) tissues because of their easy storage and versatility. IHC is a key method for providing more accurate analysis of localization and function of neurons, neuroendocrine cells, and neural stem cells in the brain and other nervous systems. The related cells such as glial cells and neurovascular units have also been analyzed by IHC. Visualization of antibody-antigen interactions can be performed primarily in one of the following ways: chromogenically stained IHC and fluorescently stained IHC. In chromogenically stained IHC, an antibody is chemically conjugated to an enzyme, such as peroxidase, that can be reacted with a suitable substrate to give a colored product. In fluorescently stained IHC, the antibodies are finally tagged with fluorescent chemicals such as fluorescein isothiocyanate (FITC) or rhodamine. Here, we describe the standard methods of IHC applied to brain slice sections. Furthermore, an automated immunostainer is presented as another option for standardized immunohistochemistry.

Live Imaging of Migrating Neurons and Glial Progenitors Visualized by in Utero Electroporation.

During cortical development, both neurons and glial cells are generated in the germinal zone near the lateral ventricle, migrate in the correct direction, and settle in their appropriate locations. This developmental process can be clearly visualized by introducing fluorescent protein-expression vectors via in utero electroporation. In this chapter, we describe labeling methods for migrating neurons and glial progenitors, as well as methods for slice culture, and time-lapse imaging.

Nanoremediation and Antioxidant Potential of Biogenic Silver Nanoparticles Synthesized Using Leucena's Leaves, Stem, and Fruits.

Synthetic dyes are persistent organic environmental pollutants that can cause extensive damage to living beings and to the ecosystem as a whole. Cost-effective, sustainable, and efficient strategies to deal with this type of pollution are necessary as it commonly resists conventional water treatment methods. Silver nanoparticles (AgNPs) synthesized using the aqueous extract from the leaves, stem, and fruits of Leucaena leucocephala (Leucena) were produced and characterized through UV-vis, TEM, EDS, SDL, XPS, XRD, and zeta potential, and they proved to be able to promote adsorption to remediate methylene blue and tartrazine pollution in water. The nanoremediation was performed and did not require direct exposure to sunlight or any special lamp or a specific reduction agent. The AgNPs produced using the extract from the leaves exhibited the best performance in nanoremediation and also presented antioxidant activity that surpassed the one from butylated hydroxytoluene (BHT). Consequently, it is an interesting nanotool to use in dye nanoremediation and/or as an antioxidant nanostructure.

The Discovery of New Inhibitors of Insulin-Regulated Aminopeptidase by a High-Throughput Screening of 400,000 Drug-like Compounds.

With the ambition to identify novel chemical starting points that can be further optimized into small drug-like inhibitors of insulin-regulated aminopeptidase (IRAP) and serve as potential future cognitive enhancers in the clinic, we conducted an ultra-high-throughput screening campaign of a chemically diverse compound library of approximately 400,000 drug-like small molecules. Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 µM. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.

Functional Plasmonic Microscope: Characterizing the Metabolic Activity of Single Cells via Sub-nm Membrane Fluctuations.

Metabolic abnormalities are at the center of many diseases, and the capability to film and quantify the metabolic activities of a single cell is important for understanding the heterogeneities in these abnormalities. In this paper, a functional plasmonic microscope (FPM) is used to image and measure metabolic activities without fluorescent labels at a single-cell level. The FPM can accurately image and quantify the subnanometer membrane fluctuations with a spatial resolution of 0.5 µm in real time. These active cell membrane fluctuations are caused by metabolic activities across the cell membrane. A three-dimensional (3D) morphology of the bottom cell membrane was imaged and reconstructed with FPM to illustrate the capability of the microscope for cell membrane characterization. Then, the subnanometer cell membrane fluctuations of single cells were imaged and quantified with the FPM using HeLa cells. Cell metabolic heterogeneity is analyzed based on membrane fluctuations of each individual cell that is exposed to similar environmental conditions. In addition, we demonstrated that the FPM could be used to evaluate the therapeutic responses of metabolic inhibitors (glycolysis pathway inhibitor STF 31) on a single-cell level. The result showed that the metabolic activities significantly decrease over time, but the nature of this response varies, depicting cell heterogeneity. A low-concentration dose showed a reduced fluctuation frequency with consistent fluctuation amplitudes, while the high-concentration dose showcased a decreasing trend in both cases. These results have demonstrated the capabilities of the functional plasmonic microscope to measure and quantify metabolic activities for drug discovery.

The Feulgen reaction at the electron microscopy level.

The Feulgen reaction has been the first specific method for detecting DNA available at light microscopy since 1924. However, a similar specific method was proposed for electron microscopy only 50 years later. Here, we discuss the problems encountered in finding the electrondense reagent capable of taking advantage of the extremely high resolution offered by electron microscopy as well as some applications of the method.

An Innovative and Efficient Fluorescent Detection Technique for Salmonella in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.

Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 101-2 × 109 CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.

Protein Corona-Mediated Inhibition of Nanozyme Activity: Impact of Protein Shape.

During biomedical applications, nanozymes, exhibiting enzyme-like characteristics, inevitably come into contact with biological fluids in living systems, leading to the formation of a protein corona on their surface. Although it is acknowledged that molecular adsorption can influence the catalytic activity of nanozymes, there is a dearth of understanding regarding the impact of the protein corona on nanozyme activity and its determinant factors. In order to address this gap, we employed the AuNR@Pt@PDDAC [PDDAC, poly(diallyldimethylammonium chloride)] nanorod (NR) as a model nanozyme with multiple activities, including peroxidase, oxidase, and catalase-mimetic activities, to investigate the inhibitory effects of the protein corona on the catalytic activity. After the identification of major components in the plasma protein corona on the NR, we observed that spherical proteins and fibrous proteins induced distinct inhibitory effects on the catalytic activity of nanozymes. To elucidate the underlying mechanism, we uncovered that the adsorbed proteins assembled on the surface of the nanozymes, forming protein networks (PNs). Notably, the PNs derived from fibrous proteins exhibited a screen mesh-like structure with smaller pore sizes compared to those formed by spherical proteins. This structural disparity resulted in a reduced efficiency for the permeation of substrate molecules, leading to a more robust inhibition in activity. These findings underscore the significance of the protein shape as a crucial factor influencing nanozyme activity. This revelation provides valuable insights for the rational design and application of nanozymes in the biomedical fields.

Biomimetic NIR-II fluorescent proteins created from chemogenic protein-seeking dyes for multicolor deep-tissue bioimaging.

Near-infrared-I/II fluorescent proteins (NIR-I/II FPs) are crucial for in vivo imaging, yet the current NIR-I/II FPs face challenges including scarcity, the requirement for chromophore maturation, and limited emission wavelengths (typically < 800 nm). Here, we utilize synthetic protein-seeking NIR-II dyes as chromophores, which covalently bind to tag proteins (e.g., human serum albumin, HSA) through a site-specific nucleophilic substitution reaction, thereby creating proof-of-concept biomimetic NIR-II FPs. This chemogenic protein-seeking strategy can be accomplished under gentle physiological conditions without catalysis. Proteomics analysis identifies specific binding site (Cys 477 on DIII). NIR-II FPs significantly enhance chromophore brightness and photostability, while improving biocompatibility, allowing for high-performance NIR-II lymphography and angiography. This strategy is universal and applicable in creating a wide range of spectrally separated NIR-I/II FPs for real-time visualization of multiple biological events. Overall, this straightforward biomimetic approach holds the potential to transform fluorescent protein-based bioimaging and enables in-situ albumin targeting to create NIR-I/II FPs for deep-tissue imaging in live organisms.

Ultra-sensitive fluorescent biosensor for multiple bacteria detection based on CDs/QDs@ZIF-8 and microfluidic fluidized bed.

An ultra-sensitive fluorescent biosensor based on CDs/QDs@ZIF-8 and microfluidic fluidized bed was developed for rapid and ultra-sensitive detection of multiple target bacteria. The zeolitic imidazolate frameworks (ZIF-8) act as the carrier to encapsulate three kinds of fluorescence signal molecules from the CDs/QDs@ZIF-8 signal amplification system. Besides, three kinds of target pathogenic bacteria were automatically, continuously, and circularly captured by the magnetic nanoparticles (MNPs) in the microfluidic fluidized bed. The neutral Na2EDTA solution was the first time reported to not only dissolve the ZIF-8 frameworks from the MNPs-bacteria-CDs/QDs@ZIF-8 sandwich complexes, but also release the CDs/QDs from sandwich complexes with no loss of fluorescence signal. Due to the advantages of signal amplification and automated sample pretreatment, the proposed fluorescent biosensor can simultaneously detect Escherichia coli O157:H7, Salmonella paratyphi A, and Salmonella paratyphi B as low as 101 CFU/mL within 1.5 h, respectively. The mean recovery in spiked milk samples can reach 99.18%, verifying the applicability of this biosensor in detecting multiple bacteria in real samples.

UFObow: A single-wavelength excitable Brainbow for simultaneous multicolor ex-vivo and in-vivo imaging of mammalian cells.

Brainbow is a genetic cell-labeling technique that allows random colorization of multiple cells and real-time visualization of cell fate within a tissue, providing valuable insights into understanding complex biological processes. However, fluorescent proteins (FPs) in Brainbow have distinct excitation spectra with peak difference greater than 35 nm, which requires sequential imaging under multiple excitations and thus leads to long acquisition times. In addition, they are not easily used together with other fluorophores due to severe spectral bleed-through. Here, we report the development of a single-wavelength excitable Brainbow, UFObow, incorporating three newly developed blue-excitable FPs. We have demonstrated that UFObow enables not only tracking the growth dynamics of tumor cells in vivo but also mapping spatial distribution of immune cells within a sub-cubic centimeter tissue, revealing cell heterogeneity. This provides a powerful means to explore complex biology in a simultaneous imaging manner at a single-cell resolution in organs or in vivo.

Molecularly imprinted ratiometric fluorescence sensor for visual detection of 17ß-estradiol in milk: A generalized strategy toward imprinted ratiometric fluorescence construction.

17ß-Estradiol (E2) is the typical endocrine disruptor of steroidal estrogens and is widely used in animal husbandry and dairy processing. In the environment, even lower concentrations of E2 can cause endocrine dysfunction in organisms. Herein, we have developed a novel molecularly imprinted ratiometric fluorescent sensor based on SiO2-coated CdTe quantum dots (CdTe@SiO2) and 7-hydroxycoumarin with a post-imprint mixing strategy. The sensor selectively detected E2 in aqueous environments due to its two fluorescent signals with a self-correction function. The sensor has been successfully used for spiking a wide range of real water and milk samples. The results showed that the sensor exhibited good linearity over the concentration range 0.011-50 µg/L, obtaining satisfactory recoveries of 92.4-110.6% with precisions (RSD) < 2.5%. Moreover, this sensor obtained an ultra-low detection limit of 3.3 ng/L and a higher imprinting factor of 13.66. By using estriol (E3), as a supporting model, it was confirmed that a simple and economical ratiometric fluorescent construction strategy was provided for other hydrophobic substances.

Noninvasive In Planta Live Measurements of H2O2 and Glutathione Redox Potential with Fluorescent roGFPs-Based Sensors.

In this protocol, we present a noninvasive in planta bioimaging technique for the analysis of hydrogen peroxide (H2O2) and glutathione redox potential in adult Arabidopsis thaliana plants. The technique is based on the use of stereo fluorescence microscopy to image A. thaliana plants expressing the two genetically encoded fluorescent sensors roGFP2-Orp1 and Grx1-roGFP2. We provide a detailed step-by-step protocol for performing low magnification imaging with mature plants grown in soil or hydroponic systems. This protocol aims to serve the scientific community by providing an accessible approach to noninvasive in planta bioimaging and data analysis.

Simple and rapid determination of tartrazine in fake saffron using the metal organic framework (Fe SA MOF@CNF) by HPLC/PDA.

The present study of a novel metal-organic framework containing Fe single atoms doped on electrospun carbon nanofibers (Fe SA-MOF@CNF) based on dispersive micro solid phase extraction (D-µ-SPE) using HPLC-PDA for detection tartrazine in fake saffron samples was designed. The Fe SA-MOF@CNF sorbent was extensively characterized through various techniques including N2 adsorption-desorption isotherms, X-ray diffraction (XRD), scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The specific area of surface of the sorbent was 577.384 m2/g. The study variables were optimized via the central composite design (CCD), which included a sorbent mass of 15 mg, a contact time of 6 min, a pH of 7.56, and a tartrazine concentration of 300 ng/ml. Under the optimum condition, the calibration curve of this method was linear in the range of 5-1000 ng/mL, with a correlation coefficient of 0.992. The LOD and LOQ values were ranged 0.38-0.74 and 1.34-2.42 ng/ml, respectively. This approach revealed significant improvements, including high extraction recovery (98.64), recovery rates (98.43-102.72%), and accuracy (RSDs < 0.75 to 3.6%). the enrichment factors were obtained in the range of 80.6-86.4 with preconcentration factor of 22.3. Consequently, the D-µ-SPE method based on synthesized Fe SA-MOF@CNF could be recommended as a sustainable sorbent for detecting tartrazine in saffron samples.

A fluorescent MOF and its synthesized MOF@cotton composite: Ratiometric sensing of vitamin B2 and antibiotic drug molecule.

Here, we demonstrated the synthesis of a zinc based luminescent MOF, 1 (NDC = 2,6- naphthalenedicarboxylate) for the ratiometric detection of biomarker riboflavin (RBF; vitamin B2) in water dispersed medium. Further, this MOF detected two other antibiotic drug molecules, nitrofurantoin (NFT) and nitrofurazone (NZF). The detection of these analytes is very quick (∼seconds), and the limit of detection (LOD) for RBF, NZF and NFT are calculated as 16.58 ppm, 47.63 ppb and 56.96 ppb, respectively. The detection of these analytes was also comprehended by solid, solution, cost-effective paper strip method i.e., triphasic identification capabilities. The sensor is reusable without losing its detection efficacy. The sensor further showed the recognition abilities of these antibiotics in real field samples (river water, urine and tablet) and RBF in vitamin B2 pills and food samples (milk and cold drinks). The sensing merit of 1 urged us to fabricate of 1@cotton fabric composite, which exhibited the colorimetric detection of these analytes. In-depth experimental analysis suggested that the occurrence of photo-induced electron transfer (PET), fluorescence resonance energy transfer (FRET), and the inner filter effect (IFE) are the possible sensing mechanisms for the recognition of the antibiotics drug. The FRET mechanism is responsible for the recognition of RBF. The sensing mechanism is further supported by the theoretical analysis and the excited lifetime measurement.

Development of a fluorescent ligand that can illuminate nuclear G-quadruplexes and modulate oncogene expression.

In this study, we developed a promising dual-function fluorescent ligand termed KS-1 by a slight structural modification on a reported carbazole-based scaffold. KS-1 was then found to mainly bind and illuminate the nuclear DNA G-quadruplexes (G4s) in a sandwich-like interacting mode, and also effectively modulate the oncogene expression through a G4-mediated manner. Furthermore, KS-1 was proved to inhibit cancer cell growth either in 2D monolayer cells or 3D multicellular tumor spheroids. To be noted, this ligand could overcome the shortcomings of other reported dual-function ligands that appeared to accumulate in the lysosomes or mitochondria, and may be used as a theranostic agent in the future.

Eye-Readable and Wearable Colorimetric Sensor Arrays for In Situ Monitoring of Volatile Organic Compounds.

Wearable sensors utilize changes in color as a response to physiological stimuli, making them easily recognizable by the naked eye. These colorimetric wearable sensors offer benefits such as easy readability, rapid responsiveness, cost-effectiveness, and straightforward manufacturing techniques. However, their applications in detecting volatile organic compounds (VOCs) in situ have been limited due to the low concentration of complex VOCs and complicated external interferences. Aiming to address these challenges, we introduced readable and wearable colorimetric sensing arrays with a microchannel structure and highly gas-sensitive materials for in situ detection of complex VOCs. The highly gas-sensitive materials were designed by loading gas-sensitive dyes into the porous metal-organic frameworks and further depositing the composites on the electrospun nanofiber membrane. The colorimetric sensor arrays were fabricated using various gas-sensitive composites, including eight dye/MOF composites that respond to various VOCs and two Pd2+/dye/MOF composites that respond to ethylene. This enables the specific recognition of multiple characteristic VOCs. A microfluidic channel made of polydimethylsiloxane (PDMS) was integrated with different colorimetric elements to create a wearable sensor array. It was attached to the surface of fruits to collect and monitor VOCs using the DenseNet classification method. As a proof of concept, we demonstrated the feasibility of the wearable sensing system in monitoring the ripening process of fruits by continuously measuring the VOC emissions from the skin of the fruit.

Cellular Distribution and Intracellular Localization of Different Sizes of Fluorescent Thiol-Organosilica Particles in Mouse Lungs.

We investigated the distribution of intratracheally administered thiol-organosilica (thiol-OS) particles in mouse lungs. Toward this end, single doses of thiol-OS particles containing fluorescein (140 nm in diameter) (F140) and rhodamine B (Rh) (Rh160, Rh280, Rh420, Rh640, and Rh1630 with diameters of 160, 280, 420, 640, and 1630 nm, respectively) were administered. After 24 h, fluorescence imaging revealed homogeneous fluorescence with a patchier pattern on the lung surface and no difference among the six particle sizes. Simultaneous dual administration of Rh and F140 particles did not reveal any size-dependent differences in the lung surface fluorescence. Fluorescence microscopy of the lung sections revealed a similar tissue distribution in the fluorescent areas of Rhs and F140. Some fluorescent areas showed one type of particle fluorescence or only one fluorescence. Cellular distribution of particles was observed in bronchoalveolar lavage cells and lung sections under a high magnification, and correlative light and electron microscopy revealed large cells with fluorescence corresponding to both particle types and small cells with fluorescence of individual particle types, indicating a cell-subset-dependent particle size effect. Rh280, Rh420, and Rh640 exhibited significant size effects and were taken up by alveolar macrophages. Extracellular particles were observed, indicating that saturation exceeded the particle dose threshold in the alveoli. F140 taken up by small and large macrophages colocalized with CD68, CD11c, and CD11b and correlated with CD11c. The size effect, intracellular localization, and extracellular distribution of particles provide insights into lung and systemic drug delivery.

Comparing the Impact of Diluents and Injection Techniques on Patient Comfort During Indocyanine Green Lymphangiography: A Prospective Study.

Background: Indocyanine green (ICG) lymphography, a key diagnostic tool for lymphedema, is influenced by the dilution process of ICG dye, impacting patient experience. Methods and Results: In our study, we assessed three different ICG diluents-water for injection (WFI), normal saline (NS), and Dextrose® plus human albumin-in five healthy volunteer individuals undergoing superficial lymphography of the upper limb over 3 weeks. Results indicated that NS, as a diluent for ICG, caused the least discomfort during injection, in contrast to WFI, which led to the highest levels of discomfort. Transport time of ICG from the injection site to the axillary lymph nodes was notably shorter in intradermal injections than in subdermal injections. Conclusion: Our findings advocate for using NS as the optimal and cost-effective diluent for ICG, enhancing patient experience.

The role of the correlated motion(s) of the chromophore in photoswitching of green and red forms of the photoconvertible fluorescent protein mSAASoti.

Wild-type SAASoti and its monomeric variant mSAASoti can undergo phototransformations, including reversible photoswitching of the green form to a nonfluorescent state and irreversible green-to-red photoconversion. In this study, we extend the photochemistry of mSAASoti variants to enable reversible photoswitching of the red form. This result is achieved by rational and site-saturated mutagenesis of the M163 and F177 residues. In the case of mSAASoti it is M163T substitution that leads to the fastest switching and the most photostable variant, and reversible photoswitching can be observed for both green and red forms when expressed in eukaryotic cells. We obtained a 13-fold increase in the switching efficiency with the maximum switching contrast of the green form and the appearance of comparable switching of the red form for the C21N/M163T mSAASoti variant. The crystal structure of the C21N mSAASoti in its green on-state was obtained for the first time at 3.0 Å resolution, and it is in good agreement with previously calculated 3D-model. Dynamic network analysis reveals that efficient photoswitching occurs if motions of the 66H residue and phenyl fragment of chromophore are correlated and these moieties belong to the same community.