Click chemical ligation-enabled digital particle counting for multiplexed microRNA analysis.

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.

In Vitro Wound Healing and Anticancer Effects of Ixora coccinea in Malignant Melanoma Cell Lines.

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.

Adsorption of CMIT/MIT on the Model Pulmonary Surfactant Monolayers.

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.

Penetration rates into the stratum corneum layer: A novel quantitative indicator for assessing skin barrier function.

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. MATERIALS AND 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. CONCLUSION: 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.

A Simple Immunofluorescence Method to Characterize Neurodegeneration and Tyrosine Hydroxylase Reduction in Whole Brain of a Drosophila Model of Parkinson's Disease.

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.

Evaluation of the Thermal Stability and Micro-Modification Mechanism of SBR/PP-Modified Asphalt.

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.

An actively stabilized, miniaturized epi-fluorescence widefield microscope for real-time observation in vivo.

Recent developments in real-time, in vivo micro-imaging have allowed for the visualization of tissue pathological changes, facilitating rapid diagnosis. However, miniaturization, magnification, the field of view, and in vivo image stabilization remain challenging factors to reconcile. A key issue for this technology is ensuring it is user friendly for surgeons, enabling them to use the device manually and obtain instantaneous information necessary for surgical decision-making. This descriptive study introduces a handheld, actively stabilized, miniaturized epi-fluorescence widefield microscope (MEW-M) for real-time observation in vivo with high resolution. The methodology of MEW-M system includes high resolution microscopy miniaturization technology, thousandfold shaking suppression (actively stabilized), ultra-photosensitivity, and tailored image signal processing cell image capture and processing technology, which support for the excellent real-time imaging performance of MEW-M system in brain, mammary, liver, lung, and kidney tissue imaging of rats in vivo. With a single-objective and high-frame-rate imaging, the MEW-M system facilitates roving image acquisition, enabling contiguous analysis of large tissue areas. RESEARCH HIGHLIGHTS: A handheld, actively stabilized MEW-M system was introduced. Excellent real-time, in vivo imaging with high resolution and active stabilization in brain, mammary, liver, lung, and kidney tissue of rats.

Does Aggregation of Therapeutic IgGs in PBS Offer a True Picture of What Happens in Models Derived from Human Body Fluids?

Therapeutic proteins such as monoclonal antibodies (mAb) are known to form aggregates due to various factors. Phosphate buffered saline (PBS), human serum, and human serum filtrate (HSF) are some of the models used to analyze mAb stability in physiologically relevant in-vitro conditions. In this study, aggregation of mAb in PBS and models derived from body fluids seeded with mAb samples subjected to various stresses were compared. Samples containing mAb subjected to pH, temperature, UV light, stirring, and interfacial agitation stress were seeded into different models for 2 case studies. In the first case study, %HMW (high molecular weight species) of mAb in PBS and HSF were compared using size exclusion chromatography. It was found that change in %HMW was higher in PBS compared to HSF. For example, PBS containing mAb that was subjected to UV light stress showed change in HMW by >10 % over 72 h, but the change was <5 % in HSF. In second case study, aggregates particles of FITC tagged mAb were monitored in PBS and serum using fluorescence microscope image processing. It was found that PBS and serum containing mAb subjected to stirring and interfacial agitation resulted in aggregates of >2 µm size, and average size and percentage number of particles having >10 µm size was higher in serum compared to PBS at all analysis time point. Overall, it was found that aggregation of mAb in PBS was different from that in human body fluids. Second case study also showed the importance of advanced strategies for further characterization of mAb in serum.

Identification and physico-chemical characterization of microplastics in marine aerosols over the northeast Arabian Sea.

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.

Starch Assisted the ZnS Buffer Layer in Enhancing the Photoluminescence of ZnSe/ZnS:Mn/ZnS Quantum Dots for Detecting E. Coli and MRSA Bacteria Quickly.

In this study, we used a starch paste stabilizer to synthesize ZnSe: Mn/ZnS- Starch and ZnSe/ZnS: Mn/ZnS-starch quantum dot (QDs) in a non-toxic aqueous solvent. The -CH2-OH group of the starch paste promotes dispersibility and improves the compatibility of quantum dots with antibodies, its bonding is observed in the FTIR spectrum. Besides, the Mn-doped ZnS buffer shell with various concentrations (1, 3, 5, 7, and 9%) influence structure, optical, and photoluminescence of QDs properties were investigated in detail. The greatest luminescence intensity is achieved at a molar ratio of 3% Mn2+/Zn2+. Moreover, the ZnS: Mn buffer shell helps to enhance the fluorescence intensity and quantum yield (QY) of the ZnSe/ZnS: Mn/ZnS QDs, which are higher than ZnSe: Mn/ZnS-starch QDs. Through protein A and EDC bridging, ZnSe/ZnS:3%Mn/ZnS- Starch resulted in good signal and sensitivity, with no toxicity to E. coli O157:H7 and MRSA strains.

Three filters for the enhancement of the images acquired from fluorescence microscope and weak-light-sources and the image compression.

Fluorescence images enhancement is important as it can provide more information for medical diagnosis. In this work, we design three simple yet useful filters based on the combinations of mathematical functions, which are proved to be effective in strengthening the images acquired from the fluorescence microscope. Using these filters, detailed objects can be found in the dark sections of the fluorescence images. In addition, these filters can be used to enhance the low-light image, which provide satisfactory visual information and marginal profile for the blurred objects in the image. Moreover, these filters have been used to enhance the image with high degradation by the Gaussian noise, where clear edge profile can be extracted. Finally, we have shown that these filters can be utilized for the image compression. Compression ratio can be obtained to be 0.9688. This study shows the making of the filters with dual functions for the image enhancement and the image compression. Our designed filters are showing the potentials in the field of biomedical imaging and pattern identification.

Cerebral cortex and hippocampus neural interaction during vagus nerve stimulation under in vivo large-scale imaging.

Objective: The purpose of this study was to study mechanisms of VNS modulation from a single neuron perspective utilizing a practical observation platform with single neuron resolution and widefield, real-time imaging coupled with an animal model simultaneously exposing the cerebral cortex and the hippocampus. Methods: We utilized the observation platform characterized of widefield of view, real-time imaging, and high spatiotemporal resolution to obtain the neuronal activities in the cerebral cortex and the hippocampus during VNS in awake states and under anesthesia. Results: Some neurons in the hippocampus were tightly related to VNS modulation, and varied types of neurons showed distinct responses to VNS modulation. Conclusion: We utilized such an observation platform coupled with a novel animal model to obtain more information on neuron activities in the cerebral cortex and the hippocampus, providing an effective method to further study the mechanisms of therapeutic effects modulated by VNS.

Sensitive SARS-CoV-2 salivary antibody assays for clinical saline gargle samples using smartphone-based competitive particle immunoassay platforms.

Antibody assay for SARS-CoV-2 has become increasingly important to track latent and asymptomatic infections, check the individual's immune status, and confirm vaccine efficacy and durability. However, current SARS-CoV-2 antibody assays require invasive blood collection, requiring a remote laboratory and a trained phlebotomist. Direct detection of SARS-CoV-2 antibodies from clinical saline gargle samples has been considered challenging due to the smaller number of antibodies in such specimens and the high limit of detection of currently available rapid tests. This work demonstrates simple and non-invasive methods for detecting SARS-CoV-2 salivary antibodies. Competitive particle immunoassays were developed on a paper microfluidic chip using the receptor-binding domain (RBD) antigens on spike proteins. Using a smartphone, they were monitored by counting the captured fluorescent particles or evaluating the capillary flow velocities. The limit of detection (LOD), cross-binding between alpha- and omicron-strains, and the effect of angiotensin-converting enzyme 2 (ACE2) presence were investigated. LODs were 1-5 ng/mL in both 10% and 1% saliva. Clinical saline gargle samples were assayed using both methods, showing a statistical difference between virus-negative and virus-positive samples, although the assays targeted antibodies. Only a small number of virus-positive samples were antibody-negative. The high assay sensitivity detected a small number of antibodies developed even during the early phase of infections. Overall, this work demonstrates the ability to detect SARS-CoV-2 salivary IgG antibodies on simple, cost-effective, portable platforms towards mitigating SARS-CoV-2 and potentially other respiratory viruses.

eXtreme gradient boosting-based classification of bacterial mixtures in water and milk using wireless microscopic imaging of quorum sensing peptide-conjugated particles.

Numerous bacteria can cause water- and foodborne diseases and are often found in bacterial mixtures, making their detection challenging. Specific bioreceptors or selective growth media are necessary for most bacterial detection methods. In this work, we collectively used five quorum sensing-based peptides identified from bacterial biofilms to identify 10 different bacterial species (Bacillus subtilis, Campylobacter jejuni, Enterococcus faecium, Escherichia coli, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium, Staphylococcus aureus, Vibrio parahaemolyticus) and their mixtures in water and milk. Four different machine learning classification methods were used: k-nearest neighbors (k-NN), decision tree (DT), support vector machine (SVM), and eXtreme Gradient Boosting (XGBoost). Peptides were crosslinked to submicron particles, and peptide-bacteria interactions on paper microfluidic chips caused the particle aggregation. A wireless, pocket fluorescence microscope (interfaced with a smartphone) counted such particle aggregations. XGBoost showed the best accuracy of 83.75% in identifying bacterial species from water samples using 320 different datasets and 91.67% from milk samples using 140 different datasets (5 peptide features per dataset). Each peptide's contribution to correct classification was evaluated. The results were concentration-dependent, allowing the identification of a dominant species from bacterial mixtures. Using XGBoost and the previous milk database, we tested 14 blind samples of various bacterial mixtures in milk samples, with an accuracy of 81.55% to predict the dominant species. The entire process could be completed within a half hour. The demonstrated system can provide a handheld, low-cost, easy-to-operate tool for potential hygiene spot-checks, public health, or personal healthcare.

Smartphone-microfluidic fluorescence imaging system for studying islet physiology.

Smartphone technology has been recently applied for biomedical image acquisition and data analysis due to its high-quality imaging capability, and flexibility to customize multi-purpose apps. In this work, we developed and characterized a smartphone-microfluidic fluorescence imaging system for studying the physiology of pancreatic islets. We further evaluated the system capability by performing real-time fluorescence imaging on mouse islets labeled with either chemical fluorescence dyes or genetically encoded fluorescent protein indicators (GEFPIs). Our results showed that the system was capable of analyzing key beta-cell insulin stimulator-release coupling factors in response to various stimuli with high-resolution dynamics. Furthermore, the integration of a microfluidics allowed high-resolution detection of insulin secretion at single islet level. When compared to conventional fluorescence microscopes and macro islet perifusion apparatus, the system has the advantages of low cost, portable, and easy to operate. With all of these features, we envision that this smartphone-microfluidic fluorescence imaging system can be applied to study islet physiology and clinical applications.

i-scope: a compact automated fluorescence microscope for cell counting applications in low resource settings.

Fluorescence microscopy has widespread applications across biological sciences. It has been routinely used for cell counting, which provides a preliminary diagnostic test for many infectious diseases. Conventional fluorescence microscopes are bulky, expensive, time-intensive and laborious. They often require trained operators to acquire and analyze data. We report a compact automated digital fluorescence microscopy system,i-scope, for cell counting applications. Thei-scopeemploys a total internal reflection fluorescence (TIRF) mode of sample illumination, along with a brightfield mode. It has a magnification of 30X, an optical resolution of ∼0.2µm/pixel and offers sample scanning over 20 mm × 20 mm. A custom-written program enables automated image acquisition and analysis, thereby enhancing ease of operation. It has a compact form-factor and has been developed into a standalone system with a processing unit, screen, and other accessories to offer a portable and economic point-of-care diagnostic solution in low-resource settings. We analysed the performance of the i-scopefor milk somatic cell enumeration and benchmarked it against that of a conventional fluorescence microscope.

rTg(TauP301L)4510 mice exhibit increased VGlut1 in hippocampal presynaptic glutamatergic vesicles and increased extracellular glutamate release.

The molecular pathways that contribute to the onset of symptoms in tauopathy models, including Alzheimer's disease (AD), are difficult to distinguish because multiple changes can happen simultaneously at different stages of disease progression. Understanding early synaptic alterations and their supporting molecular pathways is essential to develop better pharmacological targets to treat AD. Here, we focus on an early onset rTg(TauP301L )4510 tauopathy mouse model that exhibits hyperexcitability in hippocampal neurons of adult mice that is correlated with presynaptic changes and increased extracellular glutamate levels. However, it is not clear if increased extracellular glutamate is caused by presynaptic changes alone, or if presynaptic changes are a contributing factor among other factors. To determine whether pathogenic tau alters presynaptic function and glutamate release, we studied cultured hippocampal neurons at 14-18 days in vitro (DIV) from animals of both sexes to measure presynaptic changes in tauP301L positive mice. We observed that presynaptic vesicles exhibit increased vesicular glutamate transporter 1 (VGlut1) using immunohistochemistry of fixed cells and an established pH-sensitive green fluorescent protein approach. We show that tauP301L positive neurons exhibit a 40% increase in VGlut1 per vesicle compared to tauP301L negative littermates. Further, we use the extracellular glutamate reporter iGluSnFR to show that increased VGlut1 per vesicle directly translates into a 40% increase in extracellular glutamate. Together, these results show that increased extracellular glutamate levels observed in tauP301L mice are not caused by increased vesicle exocytosis probability but rather are directly related to increased VGlut1 transporters per synaptic vesicle.

Comparative study of methods for detecting extraterrestrial life in exploration mission of Mars and the solar system.

The detection and analysis of extraterrestrial life are important issues of space science. Mars is among the most important planets to explore for extraterrestrial life, owing both to its physical properties and to its ancient and present environments as revealed by previous exploration missions. In this paper, we present a comparative study of methods for detecting extraterrestrial life and life-related substances. To this end, we have classified and summarized the characteristics targeted for the detection of extraterrestrial life in solar system exploration mission and the methods used to evaluate them. A summary table is presented. We conclude that at this moment (i) there is no realistic single detection method capable of concluding the discovery of extraterrestrial life, (ii) no single method has an advantage over the others in all respects, and (iii) there is no single method capable of distinguishing extraterrestrial life from terrestrial life. Therefore, a combination of complementary methods is essential. We emphasize the importance of endeavoring to detect extraterrestrial life without overlooking possible alien life forms, even at the cost of tolerating false positives. Summaries of both the targets and the detection methods should be updated continuously, and comparative studies of both should be pursued. Although this study assumes Mars to be a model site for the primary environment for life searches, both the targets and detection methods described herein will also be useful for searching for extraterrestrial life in any celestial environment and for the initial inspection of returned samples.

Simultaneous Observation of Mouse Cortical and Hippocampal Neural Dynamics under Anesthesia through a Cranial Microprism Window.

The fluorescence microscope has been widely used to explore dynamic processes in vivo in mouse brains, with advantages of a large field-of-view and high spatiotemporal resolution. However, owing to background light and tissue scattering, the single-photon wide-field microscope fails to record dynamic neural activities in the deep brain. To achieve simultaneous imaging of deep-brain regions and the superficial cortex, we combined the extended-field-of-view microscopy previously proposed with a novel prism-based cranial window to provide a longitudinal view. As well as a right-angle microprism for imaging above 1 mm, we also designed a new rectangular-trapezoidal microprism cranial window to extend the depth of observation to 1.5 mm and to reduce brain injury. We validated our method with structural imaging of microglia cells in the superficial cortex and deep-brain regions. We also recorded neuronal activity from the mouse brains in awake and anesthesitized states. The results highlight the great potential of our methods for simultaneous dynamic imaging in the superficial and deep layers of mouse brains.