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The current methods for detecting myelin changes in ischemic stroke are indirect and cannot accurately reflect their status. This study aimed to develop a novel fluorescent-magnetic resonance dual-modal molecular imaging probe for direct imaging of myelin. Compounds 7a and 7b were synthesized by linking the MeDAS group and Gadolinium (III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate. Compound 7a was selected for characterization and further study. Cell uptake, cytotoxicity, and magnetic resonance imaging scans were performed on cells. In vitro experiments on frozen brain sections from 7-day-old, 8-week-old, and ischemic stroke rats were compared with commercially available Luxol Fast Blue staining. After HPLC and MR scanning, brain tissue was soaked in 7a and scanned using T 1 WI and T 1 maps sequences. Spectrophotometer results showed that compounds 7a and 7b had fluorescent properties. MR scans indicated that the compounds had contrast agent properties. Cells could uptake 7a and exhibited high signals in imaging scans. Compound 7a brain tissue staining showed more fluorescence in myelin-rich regions and identified injury sites in ischemic stroke rats. MR scanning of brain sections provided clear myelin contrast. A novel fluorescent-magnetic resonance dual-modal molecular imaging probe for direct imaging of myelin was successfully developed and tested in rats with ischemic stroke. These findings provide new insights for the clinical diagnosis of demyelinating diseases. • Compounds 7a and 7b have fluorescent properties from spectrophotometer results. • Compounds 7a and 7b have MRI contrast agent properties. • Our novel dual-mode probes effectively visualized the rat infarction model. • Our study may provide new ideas for diagnosing demyelinating diseases. [ABSTRACT FROM AUTHOR]

Ultrasound-targeted microbubble cavitation (UTMC)-mediated blood–brain barrier (BBB) opening is being explored as a method to increase drug delivery to the brain. This strategy has progressed to clinical trials for various neurological disorders, but the underlying cellular mechanisms are incompletely understood. In the study described here, a contact co-culture transwell model of the BBB was developed that can be used to determine the signaling cascade leading to increased BBB permeability. This BBB model consists of bEnd.3 cells and C8-D1A astrocytes seeded on opposite sides of a transwell membrane. Pulsed ultrasound (US) is applied to lipid microbubbles (MBs), and the change in barrier permeability is measured via transendothelial electrical resistance and dextran flux. Live cell calcium imaging (Fluo-4 AM) is performed during UTMC treatment. This model exhibits important features of the BBB, including endothelial tight junctions, and is more restrictive than the endothelial cell (EC) monolayer alone. When US is applied to MBs in contact with the ECs, BBB permeability increases in this model by two mechanisms: UTMC induces pore formation in the EC membrane (sonoporation), leading to increased transcellular permeability, and UTMC causes formation of reversible inter-endothelial gaps, which increases paracellular permeability. Additionally, this study determines that calcium influx into ECs mediates the increase in BBB permeability after UTMC in this model. Both transcellular and paracellular permeability can be used to increase drug delivery to the brain. Future studies can use this model to determine how UTMC-induced calcium-mediated signaling increases BBB permeability. [ABSTRACT FROM AUTHOR]

Autologous cell-based therapy using dermal sheath cup (DSC) cells was reported as a new treatment for male and female pattern hair loss. However, the mechanisms underlying its action remain unclear. We investigated the mechanisms underlying the efficacy of DSC cells in cell-based therapy. We conducted multivariate analysis to categorize individuals based on treatment response as responders and non-responders. The differentially expressed genes in DSC cells from the two groups were evaluated using bulk transcriptome, quantitative polymerase chain reaction, and single-cell transcriptome analyses. We performed live cell imaging combined with immunostaining to characterize the DSC subpopulation associated with responders. We identified nine and three genes as high efficacy (HE) and low efficacy (LE) marker genes, respectively. The HE subpopulations were enriched for cell migration-related genes in single-cell analysis. In contrast, the LE subpopulation was enriched for basement membrane and vasculature-related genes. Moreover, DSC cells in culture were immunocytochemically and morphologically heterogeneous, expressing characteristic factors. Furthermore, live cell imaging showed that DSC cells expressing integrin subunit alpha 6 (ITGA6), an HE subpopulation gene, had markedly higher mobility than those expressing the LE subpopulation genes collagen type IV or CD36. ITGA6-positive DSC cells, with superior migratory activity, may contribute to cell-based therapy by promoting cell migration into nearby hair follicles. • Analyzed clinical results of cell therapy for PHL with multivariate analysis. • Identified differentially expressed genes between responders and non-responders. • Identified clinical efficacy-associated subpopulation by single cell analysis. • This subpopulation expressed integrin subunit alpha 6 (ITGA6). • ITGA6 + subpopulation had high migratory activity contributing to PHL cell therapy. [ABSTRACT FROM AUTHOR]

As an important posttranslational modification, SUMOylation plays critical roles in almost all biological processes. Although it has been well-documented that SUMOylated proteins are mainly localized in the nucleus and have roles in chromatin-related processes, we showed recently that the SUMOylation machinery is actually enriched in the nuclear matrix rather than chromatin. Here, we provide compelling biochemical, cellular imaging and proteomic evidence that SUMOylated proteins are highly enriched in the nuclear matrix. We demonstrated that inactivation of SUMOylation by inhibiting SUMO-activating E1 enzyme or KO of SUMOconjugating E2 enzyme UBC9 have only mild effect on nuclear matrix composition, indicating that SUMOylation is neither required for nuclear matrix formation nor for targeting proteins to nuclear matrix. Further characterization of UBC9 KO cells revealed that loss of SUMOylation did not result in significant DNA damage, but led to mitotic arrest and chromosome missegregation. Altogether, our study demonstrates that SUMOylated proteins are selectively enriched in the nuclear matrix and suggests a role of nuclear matrix in mediating SUMOylation and its regulated biological processes. [ABSTRACT FROM AUTHOR]

Cell therapy is a promising treatment method that uses living cells to address a variety of diseases and conditions, including cardiovascular diseases, neurologic disorders and certain cancers. As interest in cell therapy grows, there is a need to shift to a more efficient, scalable and automated manufacturing process that can produce high-quality products at a lower cost. One way to achieve this is using non-invasive imaging and real-time image analysis techniques to monitor and control the manufacturing process. This work presents a machine learning-based image analysis pipeline that includes semantic segmentation and anomaly detection capabilities. This method can be easily implemented even when given a limited dataset of annotated images, is able to segment cells and debris and can identify anomalies such as contamination or hardware failure. [ABSTRACT FROM AUTHOR]

Macrophages must respond appropriately to pathogens and other pro-inflammatory stimuli in order to perform their roles in fighting infection. One way in which inflammatory stimuli can vary is in their dynamics--that is, the amplitude and duration of stimulus experienced by the cell. In this study, we performed long-term live cell imaging in a microfluidic device to investigate how the pro-inflammatory genes IRF1, CXCL10, and CXCL9 respond to dynamic interferon-gamma (IFN) stimulation. We found that IRF1 responds to low concentration or short duration IFN stimulation, whereas CXCL10 and CXCL9 require longer or higherconcentration stimulation to be expressed. We also investigated the heterogeneity in the expression of each gene and found that CXCL10 and CXCL9 have substantial cell-to-cell variability. In particular, the expression of CXCL10 appears to be largely stochastic with a subpopulation of nonresponding cells across all the stimulation conditions tested. We developed both deterministic and stochastic models for the expression of each gene. Our modeling analysis revealed that the heterogeneity in CXCL10 can be attributed to a slow chromatin-opening step that is on a similar timescale to that of adaptation of the upstream signal. In this way, CXCL10 expression in individual cells can remain stochastic in response to each pulse of repeated stimulation, which we also validated by experiments. Together, we conclude that pro-inflammatory genes in the same signaling pathway can respond to dynamic IFN stimulus with very different response features and that upstream signal adaptation can contribute to shaping heterogeneous gene expression. [ABSTRACT FROM AUTHOR]

The innate immune system provides the first line of defense against pathogens and cellular insults and is activated by pattern recognition receptors sensing pathogen- or damageassociated molecular patterns. This activation can result in inflammation via cytokine release as well as the induction of lytic regulated cell death (RCD). Innate immune signaling can also induce the expression of interferon regulatory factor 1 (IRF1), an important molecule in regulating downstream inflammation and cell death. While IRF1 has been shown to modulate some RCD pathways, a comprehensive evaluation of its role in inflammatory cell death pathways is lacking. Here, we examined the role of IRF1 in cell death during inflammasome and PANoptosome activation using live cell imaging, Western blotting, and ELISA in primary murine macrophages. IRF1 contributed to the induction of ZBP1- (Z-DNA binding protein 1), AIM2- (absent in melanoma-2), RIPK1- (receptor interacting protein kinase 1), and NLRP12 (NOD-like receptor family, pyrin domain–containing 12)-PANoptosome activation and PANoptosis. Furthermore, IRF1 regulated the cell death under conditions where inflammasomes, along with caspase-8 and RIPK3, act as integral components of PANoptosomes to drive PANoptosis. However, it was dispensable for other inflammasomes that form independent of the PANoptosome to drive pyroptosis. Overall, these findings define IRF1 as an upstream regulator of PANoptosis and suggest that modulating the activation of molecules in the IRF1 pathway could be used as a strategy to treat inflammatory and infectious diseases associated with aberrant inflammatory cell death. [ABSTRACT FROM AUTHOR]

Radiotherapy is one of the mainstay treatments for hepatocellular carcinoma (HCC). However, a substantial number of patients with HCC develop radioresistance and eventually suffer from tumor progression or relapse, which is a major impediment to the use of radiotherapy. Therefore, elucidating the mechanisms underlying radioresistance and identifying novel therapeutic targets to improve patient prognosis are important in HCC management. In this study, using in vitro and in vivo models, laser microirradiation and live cell imaging methods, and coimmunoprecipitation assays, we report that a DNA repair enhancer, human positive cofactor 4 (PC4), promotes nonhomologous end joining-based DNA repair and renders HCC cells resistant to radiation. Mechanistically, PC4 interacts with poly (ADP-ribose) polymerase 1 and directs Ku complex PARylation, resulting in the successful recruitment of the Ku complex to damaged chromatin and increasing the efficiency of nonhomologous end joining repair. Clinically, PC4 is highly expressed in tumor tissues and is correlated with poor prognosis in patients with HCC. Taken together, our data suggest that PC4 is a DNA repair driver that can be targeted to radiosensitize HCC cells. [ABSTRACT FROM AUTHOR]

Mechanical stretching is experienced ubiquitously by human tissues and their constituent cells and impacts biochemical and (mechano)biological processes relevant to health and disease. Numerous cell-stretching devices (CSDs) have been developed and used to apply mechanical strain to cells and tissues in in vitro settings, however, few have shown compatibility with live-cell microscopy. In most microscopy-compatible CSDs, imaging is performed post-mechanical stretching while live-cell imaging during cyclic stretching is very rarely shown, especially at high stretching frequencies and for long experiments (i.e., hours long). Live-cell imaging during cell stretching has revealed distinct time-dependent biomechanical features of cells, such as cell fluidization at high amplitudes or frequencies, or cracking followed by recovery of cell monolayers. Basic human functions such as breathing and digestion require mechanical stretching of cells and tissues. However, when it comes to laboratory experiments, the mechanical stretching that cells experience in the body is not often replicated, limiting the biomimetic nature of the studies and the relevance of results. Herein, we establish the importance of mechanical stretching during in vitro investigations by reviewing seminal works performed using cell-stretching platforms, highlighting important outcomes of these works as well as the engineering characteristics of the platforms used. Emphasis is placed on the compatibility of cell-stretching devices (CSDs) with live-cell imaging as well as their limitations and on the research advancements that could arise from live-cell imaging performed during cell stretching. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is asymptomatic at an early stage which delays its timely diagnosis and treatment. Circulating tumor cells (CTCs), derived from a primary or secondary tumor, may help in the management of HCC. Here, we evaluate and characterize CTCs in liver disease patients. In total, 65 patients, categorized into liver cirrhosis (LC) (n = 30) and HCC (n = 35), were enrolled. Using ImagestreamX MkII imaging flow cytometer, CTCs were detected and characterized using biomarker expression of EpCAM, CK, AFP, CD45, and DRAQ5 in LC and HCC patients. CTCs were detected in 33/35 (94%) HCC patients and in 28/30 (93%) LC patients. In the HCC group, the number of biomarker-positive CTCs was higher in BCLC stage D when compared with others. EpCAM + CK was the most expressed biomarker on CTCs in LC versus HCC (83.3% vs. 77.14%), followed by AFP (80% vs. 65.71%), EpCAM (30% vs. 28.57%), and CK (16.6% vs. 14.28%). The EpCAM cell area was significantly associated (P value = 0.031) with the CTC-positive status. The combination biomarker expression of CTCs cell area (EpCAM, CK, and AFP) performed well with the area under the curve of 0.92, high sensitivity, and specificity in detecting early-stage and AFP-negative HCC as well as in AFP-negative LC cases. Enumeration and cell area of CTCs may be used as a biomarker for early detection of HCC and guiding treatment. [Display omitted] • CTCs were characterized using a biomarker panel (+EpCAM, +CK, +AFP, −CD45, +DRAQ) in LC and HCC patients using IFC. • Surface markers, EpCAM + CK, were the most expressed biomarkers on CTCs in LC vs. HCC followed by AFP, EpCAM and CK. • The cell area of AFP was significantly higher in the LC group when compared with the HCC group (P value = 0.035). • CTCs cell area (EpCAM+CK+ AFP) performed well with an AUC of 0.92, in detecting early-stage and AFP-negative HCC and LC cases. [ABSTRACT FROM AUTHOR]

Fluorescence in situ hybridization (FISH) is the gold standard for visualizing genomic DNA in fixed cells and tissues, but it is incompatible with live-cell imaging, and its combination with RNA imaging is challenging. Consequently, due to its capacity to bind double-stranded DNA (dsDNA) and design flexibility, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) technology has sparked enormous interest over the past decade. In this review, we describe various nucleic acid (NA)- and protein-based (amplified) signal generation methods that achieve imaging of repetitive and single-copy sequences, and even single-nucleotide variants (SNVs), next to highly multiplexed as well as dynamic imaging in live cells. With future progress in the field, the CRISPR-(d)Cas9-based technology promises to break through as a next-generation cell-imaging technique. Adoption of clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging requires consideration of (amplified) signal generation complexity, an appropriate cell delivery mechanism, and the capacity for single-copy sequence imaging. Single-copy sequence imaging is achieved both with and without signal amplification, whereas SNP or single-nucleotide variant (SNV) detection requires signal amplification. CRISPR-based imaging with specificity at the SNV and SNP levels was achieved in both live and fixed cells. The versatility of paired RNA aptamer-based signal generation enables highly multiplexed live-cell imaging of six repetitive sequences. CRISPR-based imaging was used in a clinical setting, allowing the detection of Patau syndrome (in the form of Chr13 trisomy) in patient-derived cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Novel ultrasmall organosilica nanoparticles (ONs) were synthesized by a one-step hydrothermal method. • Ultrasmall ONs with avoiding aggregation-induced quenching effect exhibit strong solid-state orange fluorescence. • Ultrasmall ONs can be used in fingerprints detection, white light-emitting diodes, and fluorescence imaging of lysosomes. Organosilica nanoparticles (ONs), which are a new type of photoluminescent nanomaterial (PM) with excellent biocompatibility, have caught more attention in recent years. However, their applications are significantly impeded by the complicated preparation process, poor photostability, and especially aggregation-induced quenching. The present study was aimed to design and prepare solid-state fluorescent ONs to avoid aggregation-induced quenching effect. In addition, the uses of ONs for fingerprint detection, white light-emitting diodes (WLEDs) and lysosome-targetable cellular imaging were demonstrated. Here, for the first time, we designed and prepared novel solid-state fluorescent ultrasmall ONs with orange-emitting photoluminescence via a one-step hydrothermal method. The prepared solid-state fluorescent ONs could be successfully employed in fingerprint detection, WLEDs fabrication and cellular imaging. Intriguingly, the ultrasmall ONs specifically localized to lysosomes rather than other subcellular organelles across distinct cell lines, including cancer cells and noncancerous cells. Collectively, these data showed that the new ONs presented in this study could be ideal candidates for PMs in biological and photoelectric applications. [ABSTRACT FROM AUTHOR]

Amplified adipose tissue (AT) lipolysis and suppressed lipogenesis characterize the periparturient period of dairy cows. The intensity of lipolysis recedes with the progression of lactation; however, when lipolysis is excessive and prolonged, disease risk is exacerbated and productivity compromised. Interventions that minimize lipolysis while maintaining adequate supply of energy and enhancing lipogenesis may improve periparturient cows' health and lactation performance. Cannabinoid-1 receptor (CB1R) activation in rodent AT enhances the lipogenic and adipogenic capacity of adipocytes, yet the effects in dairy cow AT remain unknown. Using a synthetic CB1R agonist and an antagonist, we determined the effects of CB1R stimulation on lipolysis, lipogenesis, and adipogenesis in the AT of dairy cows. Adipose tissue explants were collected from healthy, nonlactating and nongestating (NLNG; n = 6) or periparturient (n = 12) cows at 1 wk before parturition and at 2 and 3 wk postpartum (PP1 and PP2, respectively). Explants were treated with the β-adrenergic agonist isoproterenol (1 μ M) in the presence of the CB1R agonist arachidonyl-2′-chloroethylamide (ACEA) ± the CB1R antagonist rimonabant (RIM). Lipolysis was quantified based on glycerol release. We found that ACEA reduced lipolysis in NLNG cows; however, it did not exhibit a direct effect on AT lipolysis in periparturient cows. Inhibition of CB1R with RIM in postpartum cow AT did not alter lipolysis. To evaluate adipogenesis and lipogenesis, preadipocytes isolated from NLNG cows' AT were induced to differentiate in the presence or absence of ACEA ± RIM for 4 and 12 d. Live cell imaging, lipid accumulation, and expressions of key adipogenic and lipogenic markers were assessed. Preadipocytes treated with ACEA had higher adipogenesis, whereas ACEA+RIM reduced it. Adipocytes treated with ACEA and RIM for 12 d exhibited enhanced lipogenesis compared with untreated cells (control). Lipid content was reduced in ACEA+RIM but not with RIM alone. Collectively, our results support that lipolysis may be reduced by CB1R stimulation in NLNG cows but not in periparturient cows. In addition, our findings demonstrate that adipogenesis and lipogenesis are enhanced by activation of CB1R in the AT of NLNG dairy cows. In summary, we provide initial evidence which supports that the sensitivity of the AT endocannabinoid system to endocannabinoids, and its ability to modulate AT lipolysis, adipogenesis, and lipogenesis, vary based on dairy cows' lactation stage. [ABSTRACT FROM AUTHOR]

Synthesis of monoclinic Y 2 O 3 remains a challenging task due to complex formation conditions such as high pressure or high temperature environments. Monoclinic yttria exhibits better photoluminescence characteristics compared to the cubic phase and hence can be a better host for nanophosphors. Reports are available predicting the stability of monoclinic phase at low temperature in extremely fine particles. But experimental preparation of this phase at low temperature is not yet reported. Here we report the first-time preparation of monoclinic Y 2 O 3 at 90 °C using simple laboratory hot air oven. The synthesized nanoparticles exhibit intense PL emission in the blue region and hence can find applications in display industry. The band structure calculations were carried out using density functional theory and the excitonic Bohr radius was estimated using the electron as well as hole effective masses determined from band structure. The cytotoxicity studies reveal non-toxic nature the particles. The fluorescence imaging experiments show that the material is capable of emitting blue fluorescence in L6 skeletal cell lines and it ensures the applicability of this material in the biomedical field. [ABSTRACT FROM AUTHOR]

Water-soluble Mn2+ doped CsPbCl 3 nanocrystals coating by 6-aminocaproic acid (ACA) ligand were prepared by a simple ligand assisted reprecipitation method at room temperature. The CsPbCl 3 :Mn2+/ACA nanocrystals were almost cubic phase and had mean size of about 10 nm. Due to the coating and passivation of ACA, the CsPbCl 3 :Mn2+/ACA nanocrystals retained the initial fluorescence intensity of 20% after being dispersed in deionized water for 20 days. The orange LED device based on CsPbCl 3 :Mn2+/ACA nanocrystals were prepared with excellent luminescence properties. In the cytotoxicity test, even if the MCF-7 cells were cultured for 24 h at the concentration of CsPbCl 3 :Mn2+/ACA nanocrystals as high as 160 μg·ml−1, the cell survival rate was still 70%. Confocal fluorescent cell imaging results showed that the nanocrystals had remarkable fluorescent cell imaging effect even after 24 h. The synthesized CsPbCl 3 :Mn2+/ACA nanocrystals have nice water solubility, good water stability, low cytotoxicity and highly luminescence properties, and have great application potential in the field of orange LED and fluorescent probes. [ABSTRACT FROM AUTHOR]

Direct cleavage, a type of abnormal cleavage in which one zygote divides into three or more blastomeres, has been reported in mammals. The incidence of direct cleavage increases in zygotes with three or more pronuclei (multi-PN) and those showing abnormal pronuclei migration. However, there are few reports on the relationship between pronuclei and direct cleavage, and the effects of these relationships on subsequent embryogenesis have not been clarified. It is difficult to observe pronuclei under visible light, especially in bovine zygotes, because of abundant dark lipid droplets in the cytoplasm. We visualized pronuclei by removing lipid droplets from bovine zygotes and analyzed the relationship between the number of pronuclei and direct cleavage using time-lapse cinematography. The direct cleavage rate of multi-PN zygotes was 78.6%, which was significantly higher than that of zygotes with one pronucleus (1 PN, 0.0%) and two pronuclei (2 PN, 8.2%). Observation of pronuclei migration in 2 PN zygotes showed that 3.1% of 2 PN zygotes had non-apposed pronuclei. The direct cleavage rate of zygotes with non-apposed pronuclei was 66.7%, which was significantly higher than that of zygotes with apposed pronuclei (6.4%). Among multi-PN zygotes, the proportions of zygotes with apposed pronuclei and non-apposed pronuclei were 37.5% and 64.3%, respectively. The direct cleavage rate of multi-PN zygotes with non-apposed pronuclei was 100.0%, which was significantly higher than that of zygotes with apposed pronuclei (40.0%). Three-dimensional live-cell imaging of bovine zygotes injected with the mRNA-encoding histone H2B-mCherry showed that the direct cleavage rates of 2 PN and multi-PN zygotes bypassing syngamy were 63.2% and 75.5%, respectively. These rates were significantly higher than that of 2 PN and multi-PN zygotes that underwent syngamy (5.6% and 20.0%, respectively). Regardless of the number of pronuclei, a high frequency of direct cleavage was observed in zygotes in which the pronuclei did not migrate inward the cytoplasm and bypassed syngamy. These results suggest that abnormal fertilization such as multi-PN and migration error of pronuclei in cattle is the primary reason for direct cleavage during the first mitosis. Assessment of direct cleavage during the first mitosis allows exclusion of embryos with abnormal fertilization and may contribute to in vitro produced embryo transfer success. [ABSTRACT FROM AUTHOR]

Heparin can block pathological responses associated with diabetic nephropathy in animal models and human patients. Our previous studies showed that the interaction of heparin on the surface of rat mesangial cells (RMCs) entering G1 of cell division in hyperglycemic glucose: 1) blocked glucose uptake by glucose transporter 4; 2) inhibited cytosolic uridine diphosphate-glucose elevation that would occur within 6 h from G0/G1; and 3) prevented subsequent activation of hyaluronan synthesis in intracellular compartments and subsequent inflammatory responses. However, specific proteins that interact with heparin are unresolved. Here, we showed by live cell imaging that fluorescent heparin was rapidly internalized into the cytoplasm and then into the endoplasmic reticulum, Golgi, and nuclei compartments. Biotinylated-heparin was applied onto the surface of growth arrested G0/G1 RMCs in order to extract heparin-binding protein(s). SDS-PAGE gels showed two bands at -70 kDa in the extract that were absent when unlabeled heparin was used to compete. Trypsin digests of the bands were analyzed by MS and identified as calreticulin and prelamin A/C. Immunostaining with their antibodies identified the presence of calreticulin on the G0/G1 RMC cell surface. Previous studies have shown that calreticulin can be on the cell surface and can interact with the LDL receptor-related protein, which has been implicated in glucose transport by interaction with glucose transporter 4. Thus, cell surface calreticulin can act as a heparin receptor through a mechanism involving LRP1, which prevents the intracellular responses in high glucose and reprograms the cells to synthesize an extracellular hyaluronan matrix after division. [ABSTRACT FROM AUTHOR]

Detection and conversion of mechanical forces into biochemical signals is known as mechanotransduction. From cells to tissues, mechanotransduction regulates migration, proliferation, and differentiation in processes such as immune responses, development, and cancer progression. Mechanosensitive structures such as integrin adhesions, the actin cortex, ion channels, caveolae, and the nucleus sense and transmit forces. In vitro approaches showed that mechanosensing is based on force-dependent protein deformations and reorganizations. However, the mechanisms in cells remained unclear since cell imaging techniques lacked molecular resolution. Thanks to recent developments in super-resolution microscopy (SRM) and molecular force sensors, it is possible to obtain molecular insight of mechanosensing in live cells. We discuss how understanding of molecular mechanotransduction was revolutionized by these innovative approaches, focusing on integrin adhesions, actin structures, and the plasma membrane. Mechanotransduction is involved in a variety of cell and tissue processes. For this, mechanosensitive structures, such as integrin adhesions, the cytoskeleton, or the plasma membrane, are key to sense, integrate, and transmit mechanical forces. Novel imaging methods and molecular sensors have unveiled how molecular mechanosensing occurs in these structures within the context of live cells. These include super-resolution imaging, single particle tracking, and molecular force sensors. Super-resolution microscopy and single protein tracking have revealed the 3D nanoscale organization and dynamics of integrin adhesion structures in various cell types, which are divided into functional nanolayers with specific paths for protein diffusion. Forces on actin regulators and F-actin trigger conformational changes controlling actin regulator function and binding, and therefore F-actin assembly and architecture. In migrating cells, mechanical plasticity emerges from global geometrical reorganization of actin networks under loads, or forces from elongating actin filaments controlling locally actin regulators dynamics and functions. Cell surface mechanics are emerging as a key parameter controlling cell motility, division, and differentiation. Variations in cortical tension and membrane-to-cortex attachment regulate the timing of differentiation while the nanoscale architecture of the actin cortex is linked to cortical tension. [ABSTRACT FROM AUTHOR]

Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy. [ABSTRACT FROM AUTHOR]

Due to the damage during production, transportation and installation, some defects inevitably occur in the solar cells, which will reduce the power generation efficiency. Benefiting from the development of deep learning, the performance of solar cell defect detection has been improved by a considerable margin. However, a problem persists that a model trained on one batch of data may not work on another batch following different production schemes. Moreover, compared with monocrystalline cells, more impurities on the surface of polycrystalline cells make it more difficult to inspect defects and obtain labels. To address this problem, we formulate it as a monocrystalline to polycrystalline task based on an unsupervised domain adaptation task in transfer learning. A standard network with CNN architecture is adopted as our classifier, which can tell whether the solar cell is cracked. Based on that, we embed adversarial learning in the network, where the domain discriminator is added to distinguish data domains, so that the feature extractor can learn domain-invariant features. Considering the scenario of defect detection in Electroluminescence (EL) images, we further propose attention-based transfer learning and class-aware domain discriminator to enhance the effect of knowledge transfer. When detecting a batch of new polycrystalline products, our method can improve F1-score by 0.2631, and the recall and precision can reach 84.70% and 90.15%, respectively. • Using transfer learning to adapt defect detection model to various production lines. • Attention mechanism is introduced in knowledge transfer to optimize features. • Align distribution of channel attention to reduce feature distribution gap. • Proposing class-aware domain classification to combat the impact of class imbalance. [ABSTRACT FROM AUTHOR]

Compared with visible fluorescence carbon dots (CDs), the multi-emission (visible and near infrared) fluorescence CDs based platform (V-N Platform) with blood-brain barrier penetration, real-time brain imaging and drug loading capacities, is of great significance. Herein, a novel type of multi-emission fluorescence CDs (GT) was created to penetrate BBB for brain imaging and local drug delivery, which was synthesized by hydrothermal method, using endogenous l -tryptophan and d -glucose. The capability for in vitro imaging was verified through the experiments on cell imaging. Moreover, the ability of in vivo imaging, especially for brain imaging, was confirmed by experiments in mice and zebrafish. In addition, the in vitro and in vivo experiments preliminarily confirmed its potential to act as a drug delivery platform. This V-N Platform (GT) will be expected to have a significant impact on the researches for CDs and the theranostics of brain diseases. A novel kind of multi-emission fluorescence carbon dots (GT) was created with NIR fluorescence to act as the "V-N Platform", using endogenous nutrients. With the BBB-penetrating ability and drug loading potential, GT could be expected to with the potential of theranostics for brain diseases. [Display omitted] • GT CDs was synthesized using two endogenous nutrients (d -glucose and l -tryptophan) as the precursor by hydrothermal method. • The retained bio-activity of d -glucose and l -tryptophan enabled GT to cross the BBB effectively. • Carbonization endowed GT with multi-emission fluorescence, especially the NIR fluorescence. • GT could be used as fluorescent probe at multi-level,especially the brain imaging in real-time. • Nanoscale ensured GT as a drug carrier for loading drugs with various polarity. [ABSTRACT FROM AUTHOR]

Fluorescence lifetimes and quantum yields featuring polycyclic aromatic hydrocarbons (PAHs) and other organics constituting pyrogenic carbon particulate matter (PM) are seldom measured. In this work, PM sampled in a fuel-rich ethylene flame was firstly separated in organic carbon (OC), soluble in dichloromethane, and refractory organic carbon (ROC), soluble in N -methyl pyrrolidinone, and then analyzed by size exclusion chromatography (SEC) coupled with online UV and fluorescence detection, and by offline fluorescence spectroscopy and mass spectrometry. It was found that three classes of differently light emitting carbon dots (CDs) could be bottom-up synthesized in the same flame system by selecting appropriately the residence time. Actually, OC presented blue fluorescence regardless the residence time, whereas ROC sampled at low and high residence time emitted fluorescence in the green (green CDs) and in the yellow (yellow CDs) region, respectively. The SEC molecular weight of all CDs presented similar trimodal distributions, centered around 300, 1000 and 10,000 u. For the first time fluorescence lifetimes and quantum yields of pyrogenic CD fractions were measured as additional parameters useful for discriminating the fluorescent components and inferring their structural properties, with the support of mass spectrometry. The different spectroscopic features of CDs could be associated to different compositional characteristics as the polydispersity of molecular components featuring blue CDs, opposed to the oligomer-like nature of green and yellow CDs. Pyrogenic CDs showed different fluorescence emission ranges, quantum yield and lifetimes, appealing for their possible applications in the fields of imaging, electronics and sensors. [Display omitted] [ABSTRACT FROM AUTHOR]

To investigate correlations between clinical and histopathologic characteristics of idiopathic epiretinal membrane (ERM). Retrospective interventional case series. In total, 87 eyes from 87 patients with idiopathic ERM who underwent pars plana vitrectomy with peeling of the ERM from 2019 to 2020 were included. The outcomes of clinical ophthalmic examination, including measurement of best-corrected visual acuity (BCVA) and spectral-domain OCT (SD-OCT), before and after surgery were reviewed. Surgical specimens were fixed in formalin and embedded in paraffin for histologic and immunohistochemical analysis. The association between morphological characteristics revealed on SD-OCT images and the cellular composition of the surgically excised ERM demonstrated with immunohistochemical staining were the main outcome measures. Changes in the BCVA and central macular thickness (CMT) were assessed through a comparison of preoperative and postoperative measurements. Based on SD-OCT morphological characteristics in the foveal area, 15 cases were classified into group 1A (mainly outer retinal thickening), 39 into group 1B (more tenting of the outer retina and distorted inner retina), and 33 into group 1C (prominent inner retina thickening). Overall, postoperative final BCVA and CMT at 1 year improved in all groups. Patients who presented with a better initial BCVA exhibited a more favorable final BCVA. Epiretinal membranes in group 1C demonstrated the greatest decrease in CMT compared with those in groups 1B and 1A, but the final CMT did not differ among the groups. A negative correlation between the density of hyalocytes (P = 0.003) and myofibroblasts (P = 0.047) was noted between the 3 groups. Total cell density and glial cell density of the ERMs were strongly associated with poor final BCVA and BCVA improvement. The present study provides new histopathologic information regarding the formation and progression of idiopathic ERM. Glial cell proliferation plays a predominant role in these processes. Epiretinal membranes with high cellularity and glial cell density may cause damage to the retina structure, resulting in poor postoperative visual outcomes. These findings provide additional evidence supporting early surgical intervention in patients with idiopathic ERM reported with visual disturbance. [ABSTRACT FROM AUTHOR]

[Display omitted] Fluorescent red-emissive carbon dots (RCDs) were synthesized using the solvothermal method with citric acid as a carbon source, N , N -dimethylformamide as a nitrogen source, and formamide as a solvent. The as-synthesized RCDs show red fluorescence in an aqueous solution and have an excellent stability towards photobleaching as well as extremely low cytotoxicity and are successfully used for cell and zebrafish imaging. The results indicate that RCDs have potential applications in both in vitro and in vivo bioimaging. [ABSTRACT FROM AUTHOR]

Despite the progress in assisted reproductive techniques, there is still a lack of rapid and minimally invasive in situ approaches for further enhancements of female fertility. Therefore, we synthesized clinically relevant liposome nanoparticles for ovarian intrafollicular injection to allow in vivo cellular imaging for future drug delivery, using the mare as an animal model. Ovarian follicles of living mares were injected in vivo with fluorescently labeled liposomes. Samples of the follicular wall (mural granulosa, theca interna, and theca externa), granulosa cells, and follicular fluid were harvested 24 h post-injection through the follicle wall biopsy (FWB), flushing, and aspiration techniques, respectively, using a transvaginal ultrasound-guided approach. In parallel, post-mortem dissected, and cultured porcine antral follicles were microinjected with doxorubicin-encapsulated liposomes to assess intracellular delivery potential. All injected mare and pig follicles were macroscopically healthy, and fluorescence imaging revealed successful intrafollicular binding to mural granulosa cells and progressive migration of liposomes to other follicle cell layers (theca interna, and theca externa), regardless of the follicle size. Intracellular delivery of doxorubicin was confirmed in all porcine follicle wall cell types. We conclude that the intrafollicular injection of nanomolecules is a promising approach for real-time monitoring of intrafollicular processes and potential utilization of in vivo cellular drug delivery to assist in follicle disease treatments and fertility improvement. • Equine and swine antral follicles were injected with liposome nanoparticles. • Follicular fluid, granulosa cells and follicle wall biopsy (FWB) were collected. • Fluorescence imaging revealed successful intrafollicular binding of liposomes to mural granulosa cells and progressive migration to other follicle cell layers. • Intrafollicular injection of nanomolecules in combination with the FWB technique is a promising approach for future diagnostic and treatment. [ABSTRACT FROM AUTHOR]

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. Developing methods that can provide insight into correlations between membrane protein colocalization and interactions is challenging. We report here on a correlated stochastic optical reconstruction microscopy/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Using live cell homoFRET imaging of carcinoembryonic antigen-related cellular adhesion molecule 1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, we revealed highly heterogeneous diffraction-limited structures on the surface of HeLa cells. Furthermore, correlated super-resolved stochastic optical reconstruction microscopy imaging showed that these structures comprised a complex mixture and spatial distribution of self-associated carcinoembryonic antigen-related cellular adhesion molecule 1 molecules. In conclusion, this correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function. [ABSTRACT FROM AUTHOR]

Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase. [ABSTRACT FROM AUTHOR]

Native broken-rice starch was used to create starch nanoparticles (StNPs) with particle sizes ranging from 100 nm to 800 nm. The fluorescent isothiocyanate poly- l -lysine StNPs (FITC-PLL-StNPs) were created in two steps. First, the StNPs were electrostatically modified by poly- l -lysine (PLL) molecules rich in amino acids. Second, fluorescein isothiocyanate reacted with some amino groups on PLL molecules (FITC). Fluorescence spectrophotometry was used to determine the degree of substitution (DS) and fluorescent properties of fluorescent starches. The study found that FITC-PLL-StNP-200 has higher fluorescence stability, more phagocytic cells, and a better and clearer fluorescence detecting effect than FITC-PLL-St, FITC-PLL-StNP-100, FITC-PLL-StNP-400, and FITC-PLL-StNP-800. The biological evaluation results showed that FITC-PLL-StNP-200 did not affect the viability of HeLa cells at the lysosome labeling concentration. These findings suggest that FITC-PLL-StNP-200 has strong and stable fluorescence, indicating that FITC-PLL-StNP-200 can be used as a fluorescent probe and lysosome marker in a variety of applications, particularly in biomedicine. [Display omitted] • Fluorecent starch nanoparticles (StNP) were synthesized. • FITC-PLL-StNP-200 has clearer fluorescence detecting effect. • FITC-PLL-StNP-200 has higher phagocytic amount of Hela cells. • FITC-PLL-StNP-200 is safe and possesses excellent biocompatibility. [ABSTRACT FROM AUTHOR]

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a fatal brain disorder featuring cerebellar neurodegeneration leading to spasticity and ataxia. This disease is caused by mutations in the SACS gene that encodes sacsin, a massive 4579-amino acid protein with multiple modular domains. However, molecular details of the function of sacsin are not clear. Here, using live cell imaging and biochemistry, we demonstrate that sacsin binds to microtubules and regulates microtubule dynamics. Loss of sacsin function in various cell types, including knockdown and KO primary neurons and patient fibroblasts, leads to alterations in lysosomal transport, positioning, function, and reformation following autophagy. Each of these phenotypic changes is consistent with altered microtubule dynamics. We further show the effects of sacsin are mediated at least in part through interactions with JIP3, an adapter for microtubule motors. These data reveal a new function for sacsin that explains its previously reported roles and phenotypes. [ABSTRACT FROM AUTHOR]

Many cell surface stimuli cause calcium release from endoplasmic reticulum (ER) stores to regulate cellular physiology. Upon ER calcium store depletion, the ER-resident protein stromal interaction molecule 1 (STIM1) physically interacts with plasma membrane protein Orai1 to induce calcium release--activated calcium (CRAC) currents that conduct calcium influx from the extracellular milieu. Although the physiological relevance of this process is well established, the mechanism supporting the assembly of these proteins is incompletely understood. Earlier we demonstrated a previously unknown post-translational modification of Orai1 with long-chain fatty acids, known as S-acylation. We found that S-acylation of Orai1 is dynamically regulated in a stimulus-dependent manner and essential for its function as a calcium channel. Here using the acyl resin--assisted capture assay, we show that STIM1 is also rapidly S-acylated at cysteine 437 upon ER calcium store depletion. Using a combination of live cell imaging and electrophysiology approaches with a mutant STIM1 protein, which could not be S-acylated, we determined that the S-acylation of STIM1 is required for the assembly of STIM1 into puncta with Orai1 and full CRAC channel function. Together with the S-acylation of Orai1, our data suggest that stimulus-dependent S-acylation of CRAC channel components Orai1 and STIM1 is a critical mechanism facilitating the CRAC channel assembly and function. [ABSTRACT FROM AUTHOR]

Imaging flow cytometry (IFC), a technique originally designed for cellular imaging, is featured by the parallel acquisition in brightfield (BF), fluorescence (FL), and side scattering channels. Introduced to the field of subvisible particle analysis in biopharmaceuticals roughly ten years ago, it has the potential to yield additional information, e.g., on particle origin. Here, we present an extensive, systematic development of masks for IFC image analysis to optimize the accuracy of size determination of polystyrene beads and pharmaceutically relevant particles (protein, silicone oil) in BF and FL channels. Based on the developed masks, particle sizing and counting by IFC are compared to flow imaging microscopy (FIM). Mask verification based on fluorescent polystyrene particles revealed good agreement between sizes obtained from IFC and FIM. In the evaluation of counting accuracy, IFC reported lower concentrations for polystyrene particle standards than FIM. For the analysis of fluorescently stained silicone oil and protein particles however, IFC FL imaging reported higher particle concentrations in the low micrometer size range. Overall, we identified IFC as suitable tool to generate supportive data for particle characterization purposes or trouble shooting activities, but not as routine quantitative technique, e.g., for subvisible particle analysis during drug product development or quality control. [ABSTRACT FROM AUTHOR]

Mitochondrial viscosity and mitochondrial membrane potential (MMP) are vital parameters for reflecting mitochondrial conditions and they are closely correlative in various biological events. Accordingly, simultaneous detection of mitochondrial viscosity and MMP is imperative for elucidating their interactions, further exploring the mechanism of diseases, and investigating numerous pathological procedures. However, such profound research remains a big challenge due to the lack of high-performing single fluorescent probes capable of simultaneously visualizing of these two parameters. In this study, we developed a new kind of fluorescent carbon dots (CDs) that can successfully realize simultaneous monitoring of mitochondrial viscosity and MMP in living cells. The fluorescence of CDs can be specifically turned on as the viscosity of solvents increases from 0.89 to 945 cP. Meanwhile, the CDs accumulate in mitochondria under normal MMP conditions, switch to exist in both mitochondria and nucleoli under reduced MMP, and present only in nucleoli with the vanishment of MMP. Thus, various MMP situations can be easily differentiated through the changed spatial distributions of CDs. More importantly, based on the developed CDs, an increased mitochondrial viscosity accompanying a decreased MMP was simultaneously tracked in drug-stimulated cells. We envision that the developed CDs has a great potential to be utilized as a promising fluorescent probe for mitochondrion-related biology studies. The carbon dots were elaborately designed for simultaneous determination of mitochondrial viscosity and membrane potential through their fluorescence variation and location migration from mitochondria to nucleoli. [Display omitted] • The fluorescence of Carbon Dots (CDs) was specifically turned on as the viscosity of solvents increased. • Fluctuation of mitochondrial membrane potential was visualized through migration of CDs from mitochondria to nucleoli. • An increased mitochondrial viscosity accompanying a decreased membrane potential was simultaneously monitored in cells. [ABSTRACT FROM AUTHOR]

The structural changes of airway smooth muscle (ASM) that characterize airway remodeling (AR) are crucial to the pathogenesis of asthma. During AR, ASM cells dedifferentiate from a quiescent to a proliferative, migratory, and secretory phenotype. Calcium (Ca2+) is a ubiquitous second messenger that regulates many cellular processes, including proliferation, migration, contraction, and metabolism. Furthermore, mitochondria have emerged as major Ca2+ signaling organelles that buffer Ca2+ through uptake by the mitochondrial Ca2+ uniporter and extrude it through the Na+/Ca2+ exchanger (NCLX/Slc8b1). Here, we show using mitochondrial Ca2+-sensitive dyes that NCLX only partially contributes to mitochondrial Ca2+ extrusion in ASM cells. Yet, NCLX is necessary for ASM cell proliferation and migration. Through cellular imaging, RNA-Seq, and biochemical assays, we demonstrate that NCLX regulates these processes by preventing mitochondrial Ca2+ overload and supporting store-operated Ca2+ entry, activation of Ca2+/calmodulin-dependent kinase II, and transcriptional and metabolic reprogramming. Using small animal respiratory mechanic measurements and immunohistochemistry, we show that smooth muscle-specific NCLX KO mice are protected against AR, fibrosis, and hyperresponsiveness in an experimental model of asthma. Our findings support NCLX as a potential therapeutic target in the treatment of asthma. [ABSTRACT FROM AUTHOR]

The morphology of bone marrow cells is crucial for the diagnostics of blood disorders. However, the traditional approach of cell sorting and counting under a microscope is time-consuming and error-prone. To assist experts in diagnosis, there is considerable interest at present in using neural networks to develop automatic recognition algorithms. However, the results obtained from these approaches indicate that the achieved accuracy is not entirely convincing, and there remains a risk of misdiagnosis. In fact, a reliable collaborative recognition algorithm not only needs to have high recognition accuracy but also must be able to confidently reject low-confidence classification results and leave them for human experts to handle. Therefore, this paper proposes a method that combines high accuracy with a rejection recognition option for bone marrow cell recognition. This method incorporates Crossformer (Cross) and Class Centroid Learning (CCL), which integrate multi-scale information from cell images, enhance image feature differentiation, and effectively reject low-confidence predictions. In the experiment, the recognition accuracy of Cross-CCL reaches 94.41%, surpassing the performance of the current mainstream and advanced methods for bone marrow cell recognition. Simultaneously, CCL serves as a dependable collaborative algorithm by effectively rejecting 21.07% of cell images that represent low-confidence prediction results. Moreover, the accuracy of identification for the remaining portions exceeds 98%. This rejection mechanism not only enhances the efficiency of expert resource allocation but also ensures a higher level of confidence in the identified cell images. • Enhancing recognition accuracy by leveraging the characteristics of bone marrow cells. • Transformer architecture models outperform convolutional neural networks in bone marrow cell recognition tasks. • Excluding low-confidence results to enhance the reliability of predictions. • In high-risk medical domains, human-machine collaboration is essential. [ABSTRACT FROM AUTHOR]

White blood cells (WBC) are vital elements of the immune system, and their number and differential count are crucial for diagnosing blood-related disorders. While existing research has primarily focused on classifying easily distinguishable major WBC types, our study delves into a model encompassing up to 19 WBC classes, some of which exhibit irregular shapes and are challenging to differentiate manually. Convolutional Neural Networks (CNNs) have shown remarkable progress in accurately classifying these intricate WBC classes. However, the accuracy of these models depends mainly on the availability of enough appropriate datasets, which can be challenging to obtain for rare WBC classes. To address this, we introduce a generative model, the diffusion-based Wasserstein Generative Adversarial Network with Gradient Penalty (WGAN-GP). This model innovatively combines the Denoising Diffusion Probabilistic Model (DDPM) forward diffusion process with the WGAN-GP, leveraging DDPM's noisy vectors as inputs for WGAN-GP's generator. This fusion accelerates the generative process and significantly enhances the output's fidelity, particularly for complex WBC images. Our model demonstrated its effectiveness on a dataset comprising 4,503 images across 19 WBC classes from Soonchunhyang University Bucheon Hospital, Korea, showing significant improvement in generating high-quality images for rare WBC classes and addressing data imbalance. We further combined pre-trained CNNs with Support Vector Machines (SVM) for classification, where our augmentation strategy led to the ResNet50-SVM model achieving an average accuracy of 95% in classifying the 19 WBC classes. This study not only addresses the data imbalance but also sets a new benchmark in WBC image analysis, demonstrating our model's efficacy in generating high-quality data for rare classes. [ABSTRACT FROM AUTHOR]

In recent times, harmonic nanoparticles (HNPs) have gained considerable interest in many fields like nanophotonics, cell imaging, nanomedicine, etc. Unlike fluorescent particles and quantum dots, HNPs are photo-stable, non-bleaching, bio-compatible, and wavelength-flexible. Here, we explore second harmonic generation (SHG) from sub-micron size BaTiO 3 (BTO) particles. BTO is a well-known perovskite material extensively used for its nonlinear optical properties. Our investigation of out-of-focus diffraction patterns of second harmonic light has helped us know about particle assembly's nature. We have observed interference fringes at the overlapped regions of the out-of-plane SHG patterns that emerged from two, three, and even multiple adjacent particles. Our study may help in the 3D tracking of nonlinear particles in a living cell and deep tissue imaging. • Second harmonic (SH) from a single isolated Sn-doped BaTiO 3 (BTO) sub-micron-sized particle. • Identification of the nature of particle assembly from out-of-focus diffraction of SH light. • Interference among two or multiple neighboring BTO particles. • Speckle formation of nonlinear light with many particles. [ABSTRACT FROM AUTHOR]

• In this paper, we report a 1,8-naphthalimide/ triphenylamine-based fluorescent probe (TPA-NI-NO 2) for the specific detection of 4‑methoxy-thiophenol (MTP) with good selectivity among the thiophenols. • The probe can "turn on" detect MTP with the limit of detection (LOD) of 80 nM and no obvious response to relevant analytes and homologues. • The sensing mechanism was explored by HPLC experiment and the PET process was determined by density functional theory (DFT) calculations. • The Gibbs free energy (DG) analysis for the reaction between the probe and different thiophenols were obtained by the theoretical calculations to study the specificity of the probe. • The probe was successfully applied to monitor the MTP in Hela cells. Thiophenols as a series of industrial products are widely used and high toxicity to the organisms and the environment. The fluorescent probe technique offers important insights into thiophenols convenient sensing. Herein, we report a 1,8-naphthalimide/ triphenylamine-based fluorescent probe (TPA-NI-NO 2) for the specific detection of 4‑methoxy-thiophenol (MTP). The probe can "turn on" specific detect MTP with the limit of detection (LOD) of 80 nM among various relevant analytes and homologues (p -Cl-C 6 H 4 SH, p -NO 2 - C 6 H 4 SH, C 6 H 5 SH and o -CH 3 - C 6 H 5 SH). The sensing mechanism was explored by the HPLC experiment and the PET process was determined by density functional theory (DFT) calculations. In addition, the specificity of the probe was also studied by the Gibbs free energy (ΔG) analysis of the reaction between the probe and different thiophenols. The reaction between probe and MTP gives the minimum of the ΔG value (∼ -9.7280 kcal/mol) and that of other thiophenols-mediated is -2.5283 to -5.8911 kcal/mol, which proves the thermodynamic chemical equilibrium can optimize the design of reaction-based fluorescent probes with excellent specificity. At last, TPA-NI-NO 2 was successfully applied to Hela cells imaging. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Luminescent dipyrrinato complexes of Re and Ir with different types of co-ligands are discussed. • Re and Ir complexes are known as triplet sensitizers and they can generate significant amount of singlet oxygen. • Their synthetic methodologies, structures and optical properties are discussed in detail. • Their potential as cellular imaging agents or as photosensitizers for anticancer applications is discussed. Luminescent dipyrrinato complexes of d-block metals are well explored in literature for diverse applications in materials as well as in biology. They offer exceptional fine tuning of their electrochemical and optical properties with the variety of substitutions on the dipyrrin core. The development of mixed ligand type dipyrrinato complexes with heavy metals like: Ru, Os, Pd, Pt, Re, Ir are of great interest in biomedical applications due to significant ROS (Reactive Oxygen Species) generation, anti-microbial and anti-cancer applications. Such dipyrrinato complexes display bright triplet emission in the visible to red region due to heavy atom effect. This review is focused on the synthesis of luminescent dipyrrinato complexes of Re(I) and Ir(III) with different types of co-ligands around the metal center. The overviewed complexes are subcategorized further on the basis of metal and substitutions on the dipyrrin C-5 position or around the metal center. Re and Ir complexes of subporphyrin, N- confused porphyrin, sapphyrin, rubyrin and N- fused porphyrins have also been included. Their absorption and emission properties and their ability to work as triplet photosensitizers are discussed. Deliberations are made to understand their potential as cellular imaging agents and/or photosensitizers for ROS generation and phototherapy to treat cancer. [ABSTRACT FROM AUTHOR]

The first difunctional probe H-1 for detecting ONOO− and Zn2+ at the same time showed high selectivity, good anti-interference capability, lower detect limit and short response time to them. [Display omitted] • · A new difuctional probe H-1 for ONOO− and Zn2+ detection at the same time was obtained firstly. • ·H-1 showed good selectivity and strong anti-interference ability for detecting ONOO− and Zn2+. • ·H-1 could fast response to ONOO− and Zn2+ in 15 s. • ·H-1 was used to monitor the ONOO− and Zn2+ level in living cells. Peroxynitrite (ONOO−) is one of the most significant reactive oxygen species (ROS) in living cells. Zn2+ in living cells plays an essential part in different physiological processes. The abnormal concentration of ONOO− and Zn2+ in living cells are related to many kinds of diseases, such as anemia, epilepsy, diarrhea, Alzheimer's disease, and so on. The relationship of ONOO− and Zn2+ in living cells when the relative disease occurs remains unknown. So we develop the first probe H-1 for detecting ONOO− and Zn2+ at the same time. The probe H-1 shows high selectivity, good anti-interference capability, low detection limit and short response time to ONOO− and Zn2+. When the probe was applied to detect ONOO− and Zn2+ in HeLa cells, we could observe the fluorescence changing in the green and blue channels separately without interference in real time. It has the potential to employ the relation of ONOO− and Zn2+ in some disease mechanism research. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel biotin-tagged fluorescent probe Nap-biotin-glu for in situ imaging of GGT on cell surface was developed. • The construction of the Nap-biotin-glu probe required only three steps with a simple and convenient method. • The probe shows obvious fluorescence change with high sensitivity and selectivity toward GGT. • The probe was applied to distinguish tumor tissues from normal tissues. γ-Glutamyl transpeptidase (GGT), a cell-surface enzyme, is strongly implicated in mammalian malignancy growth and migration processes including human hepatocarcinogens. However, simply and conveniently detect of GGT on the cell membrane remains highly challenging. In this study, a biotin-tagged fluorescent probe Nap-biotin-glu was developed using glutamic acid, naphthalimide, and biotin as the reaction site, fluorescent reporter, and membrane-targeting group, which required only three steps. Colocalization fluorescence imaging and immunofluorescence analysis indicated that probe Nap-biotin-glu was successfully realized in situ visualizing of GGT on the cell membrane.Owing to the significant over-expressed GGT level in tumor, the probe was successfully applied to distinguish cancer tissues from adjacent normal tissues. [ABSTRACT FROM AUTHOR]

[Display omitted] • Ratiometric peptide-based fluorescent probe DKA with large Stokes shift (230 nm) was developed. • DKA demonstrated absorbance ratiometric and fluorescent "off-on" sensing for Hg2+ with low LOD of 13.4 nM. • DKA -Hg2+ complex was applied to sequential detect S2− with good selectivity and excellent sensitivity.. • DKA performed well in sequential detecting the distribution of Hg2+ and S2− in living cells. • DKA was used to visually and semi-quantitatively recognize Hg2+ and S2− by smartphone-assisted detection platform. In this work, a new ratiometric fluorescent probe DKA was synthesized based on the double sides of lysine backbone conjugated with alanine and dansyl groups. DKA exhibited fluorescence ratiometric response for Hg2+ with high sensitivity (13.4 nM), specific selectivity (only Hg2+), strong anti-interference ability (no interference), fast recognition (within 60 s) and wide pH range (5–10). The stoichiometry of binding of DKA and Hg2+ was determined to be 1:1 via Job's plot, ESI-HRMS and 1HNMR titration analysis. Subsequently, the in situ formation of DKA -Hg2+ complex was used for highly selective detection of S2− as a novel fluorescence "on–off" probe, and the lowest detection limit for S2− was 12.9 nM. In addition, DKA possessed excellent cells permeation and low toxicity, and fluorescence imaging of Hg2+ and S2− was performed in living Hacat cells. Most importantly, the digital imaging using a smartphone color recognition APP indicated that DKA could semi-quantitatively and visually detected Hg2+ and S2− without expensive equipment. [ABSTRACT FROM AUTHOR]

[Display omitted] • A first dual-response probe for norepinephrine and viscosity is synthesized. • The crosstalk between the two channels is negligible. • Reducing viscosity accelerates the release of norepinephrine in depression cells. Depression is a serious mental disease that causes grievous harm to human health and quality of life. The vesicular exocytosis of noradrenaline (NE), rather than its intrinsic intracellular concentration, is more associated with depression. Based on the reports on exocytosis of NE, it is reasonable to assume that the viscosity of cells has an important effect on the release of NE. Herein, a dual-response fluorescent probe (RHO-DCO-NE) for detecting NE and viscosity was designed and synthesized. The probe can simultaneously detect NE concentration and viscosity level with negligible crosstalk between the two channels. We utilized the probe to study the effect of viscosity changes on the NE release of PC12 and the corticosterone-induced PC12 cells. The experiment data revealed that the decrease in viscosity level can accelerate the release of NE of depression cell models. The finding provides new insight into the study of the pathological mechanisms of depression. [ABSTRACT FROM AUTHOR]

[Display omitted] • We have developed a novel quercetin (a SARS-CoV-2 inhibitor) coupled benzyl ethers (QBE) chemosensor for selective detection of Hg2+. • To date, there are very few reports where quercetin itself have been used in sensing field (table 1) but the use of quercetin derivative in sensing field is almost nil. • To the best of our knowledge our chemosensor QBE is the first chemosensor based on quercetin derivative for the selective detection of Hg2+. • The binding phenomenon of QBE with Hg2+ has been proved by UV–vis, fluorescence, DFT and cyclic voltammograms study. • Lower LOD (Limit of Detection) at 8.47 µM and high binding constant value as 2 × 104 M−1 showed the strong binding affinity of Hg2+ towards QBE. • The intracellular activity of QBE with Hg2+ binding has been examined by using living plant tissue by using green gram seeds. Quercetin is an important antioxidant with high bioactivity and it has been used as SARS-CoV-2 inhibitor significantly. Quercetin, one of the most abundant flavonoids in nature, has been in the spot of numerous experimental and theoretical studies in the past decade due to its great biological and medicinal importance. But there have been limited instances of employing quercetin and its derivatives as a fluorescent framework for specific detection of various cations and anions in the chemosensing field. Therefore, we have developed a novel chemosensor based on q uercetin coupled b enzyl e thers (QBE) for selective detection of Hg2+ with "naked-eye" colorimetric and "turn-on" fluorometric response. Initially QBE itself exhibited very weak fluorescence with low quantum yield (Φ = 0.009) due to operating photoinduced electron transfer (PET) and inhibition of excited state intramolecular proton transfer (ESIPT) as well as intramolecular charge transfer (ICT) within the molecule. But in presence of Hg2+, QBE showed a sharp increase in fluorescence intensity by 18-fold at wavelength 444 nm with high quantum yield (Φ = 0.159) for the chelation-enhanced fluorescence (CHEF) with coordination of Hg2+, which hampers PET within the molecule. The strong binding affinity of QBE towards Hg2+ has been proved by lower detection limit at 8.47 µM and high binding constant value as 2 × 104 M−1. The binding mechanism has been verified by DFT study, Cyclic voltammograms and Jobs plot analysis. For the practical application, the binding selectivity of QBE with Hg2+ has been capitalized in physiological medium to detect intracellular Hg2+ levels in living plant tissue by using green gram seeds. Thus, employing QBE as a fluorescent chemosensor for the specific identification of Hg2+ will pave the way for a novel approach to simplifying the creation of various chemosensors based on quercetin backbone for the precise detection of various biologically significant analytes. [ABSTRACT FROM AUTHOR]

A phenolato-thiazole based small molecule is synthesized and structurally characterized for the naked-eye detection of carbonate anion (LOD, 0.18 µM) in physiological conditions. The new colorimetric probe shows selective reversible chemosensory activity through the formation of a stable adduct (L∙CO 3 2). The cell uptake of the probe is mainly in the cytoplasm. [Display omitted] • A novel "turn-on" chemosensor fluorescent probe (L) for aqueous CO 3 2– anion is reported with crystal structure. • The 1:1 adduct L∙CO 3 2– (Binding constant, K = 1.6 x 10–7 M−1) in the quinonoid form is established by DFT/TDDFT. • The goodness of sensor activity concerns the selectivity, reversibility, and regeneration. • Selectivity with LOD (0.18 µM) in the pH range of 4 to 8. • MTT assay and cell uptakes and degradation of the probe through image study. A new phenolate-thiazole derivative (L) has been synthesized and structurally characterized. The chemo-sensing activity of L is detected by the naked eye for the aqueous carbonate anion in the pH range of 4 to 8. The selective 'turn-on' fluorescence occurs through the formation of a stable intermediate L∙CO 3 2–(1) following the PET mechanism. The limit of detection (LOD) is found 0.18 µM based on the absorbance-based assay. The quinonoid form of bromophenol unit binds strongly with CO 3 2– through thiazole nitrogen and hydrazinic nitrogen. Further, the selective holding of CO 3 2– anion over other planar tetranuclear anions (e.g., SO 3 2–, NO 3 –) happens with several intra and intermolecular hydrogen bonds as envisaged by the DFT/TDFT study. The formation mechanism of L∙CO 3 2– is proposed based on experimental and theoretical studies. The biological experiments (MTT and cell imaging) reveal the non-cytotoxicity nature of L and the biocompatible uptake of L mostly in the cytoplasm at physiological pH. [ABSTRACT FROM AUTHOR]

• We propose deep learning models for classification of cancer cells belonging to different organs on holographic images. • We present self-supervised pretraining and hyperparameter tuning for efficient training on biomedical holographic datasets. • Perform extensive experiments with self-supervised frameworks and compared self-supervised learning with supervised learning and proved the effectiveness of the former for holographic images classification. • Proposed work can be an effective tool for the automatic classification of cancer cells for medical diagnosis using self-supervised learning. Image-based stain-free elliptical cancer cell classification is challenging, due to the inter-class morphological similarity. In this paper, we address the classification of different types of cancer cell lines (lung, breast, bladder, and skin) by utilizing self-supervised learning, and compare it with supervised learning based on convolutional neural network. Digital holography in a microscopic configuration was used to obtain stain-free quantitative phase images representing the intracellular content and morphology of cells. The performance of self-supervised learning in natural images shows promising results, and consistently closes the gap between self-supervised and supervised learning. The ability of self-supervised learning to effectively utilize unlabeled data for training is instrumental in the biomedical domain, where labeled data is scarce. Our goal is to study different self-supervised frameworks of biomedical holographic data, and determine how they can be utilized to advance liquid biopsy for the detection of cancer cells. After extensive experimentation, we conclude that self-supervised learning improves the classification performance on cancer cell datasets, and outperforms supervised learning when training data is limited, which is mostly the case in biomedical imaging. [ABSTRACT FROM AUTHOR]

[Display omitted] • The review explores the versatile capabilities of fluorocolorimetric probes developed in Dr S Velmathi's lab at NIT Trichy for analyte detection, imaging, and real-time applications. • They exhibit distinctive visual signals when interacting with specific targets, marking a significant advancement in molecular interaction analysis. • The probes cover a broad spectrum of analytes, including metal ions, anions, biomolecules, ROS, RSS, toxic agents, and nanoparticles. • The review emphasizes the profound impact of fluorocolorimetric probes on developing analytical potential. • These probes contribute to innovative imaging techniques and expand the possibilities of real-time applications. Fluorescent and colorimetric probes are gaining interest in analyte detection, imaging, and real-time applications. However, it faces several challenges in achieving high sensitivity and selectivity, maintaining a strong signal-to-noise ratio, detecting analytes at extremely low concentrations, ensuring stability under different conditions and addressing issues related to cost, accessibility and instrumentation. Additionally, ensuring biological compatibility and enabling real-time monitoring are also challenges that need to overcome. We have investigated the sensing application of probe molecules towards various analytes like metal ions, anions, biomolecules, reactive oxygen species (ROS), reactive sulphur species (RSS), toxic agents and nanoparticles performed by our group together with critically comparing with the existing reports. This featured article provides the impact of fluorescent and colorimetric probes in advancing analytical techniques, imaging and real-time applications that transcend traditional boundaries. [ABSTRACT FROM AUTHOR]

Butyrylcholinesterase (BChE) is associated with many neurodegenerative diseases, especially Alzheimer's disease (AD). Developing specific imaging tools for BChE is now highly pursued because BChE has substantially similar biochemical properties to acetylcholinesterase (AChE) but distinct roles in AD. Inspired by the BChE-catalyzed hydrolysis of specific substrates (benzoylcholine, tetracaine, and cocaine) containing benzoyl group in physiological condition, a bioinspired probe PM1 with high specificity towards BChE was designed. The docking models indicated that PM1 could easily access the active center of BChE rather than AChE, showing the significance of benzoyl group in specific recognition of BChE. The control probes PM2–5 with smaller recognition groups displayed lower specificity to BChE than PM1 , showing the high efficiency of the bioinspired design strategy. The living cells experiments showed that PM1 signals were weakened by BChE inhibitor but not influenced by AChE inhibitor, which was in contrast to the performance of probe PM2–5 , showing the specific recognition of PM1 for BChE in biological system. Moreover, PM1 has been used to study the positive correlation between BChE level and cell apoptosis for the first time, and the further study showed that Aβ deposition could induce the increase in BChE level and accompany the occurrence of cell apoptosis. Notably, PM1 was successfully traced the enhancement of BChE activity in the brain slice of AD mouse model. Such a rational approach may provide a solid basis for designing specific probes with different properties to study BChE in biosystems. [Display omitted] • Inspired by the BChE-catalyzed hydrolysis of specific substrates, a bioinspired probe towards BChE was designed. • Screening probes with different recognition groups and the docking models showed rationality of our probe. • Probe PM1 has been used to study the positive correlation between BChE level and cell apoptosis for the first time. • The study showed that Aβ could induce the increase in BChE level and accompany the occurrence of cell apoptosis. • PM1 was successfully traced the enhancement of BChE activity in the brain slice of AD mouse model. [ABSTRACT FROM AUTHOR]

[Display omitted] • Hydrazone based dual mode fluorescence sensor for Zn2+ ("turn-on") and Hg2+ ("turn off"). • Probe C1 selectively sense Zn2+and Hg2+ ions with their lowest detection limits 0.042 μM and 0.081 μM respectively. • The designed probe C1 was studied latent fingerprint analysis in various surfaces. • Live cell imaging studies was performed for probe C1 in presence and absence of Zn2+, Hg2+ ions. A facile Schiff base probe C1 has been developed and evaluated using 1H, 13C NMR, and mass spectroscopy. Fluorescence investigations were conducted to determine probe-sensing behavior. Probe C1 demonstrated sensitive, and selective recognition of Zn2+ and Hg2+ ions by using two distinct processes (i.e., chelation enhanced fluorescence effect and fluorescence quenching) over other cations in ACN-H 2 O (8:2, v/v). The Zn2+ and Hg2+ detection limits of 0.042 and 0.081 μM are achieved with the probe C1. C1 has a binding constant of 3.33 × 103 M−1 for Zn2+ and an emission quenching constant of 1.22 × 104 M−1 for Hg2+. A Jobs plot was used to determine the binding stoichiometry of C1 and Zn2+/Hg2+ ions ratio is 1:1. By using Zn2+/Hg2+ and EDTA additions alternately, the probe's reusability was investigated. Moreover, the probe was found to be highly cytocompatibility using an MTT assay in a mouse fibroblast cell line (NIH3T3). Further, the probe was found to visualize Zn2+ and Hg2+ ions inside the cells, indicating the potential application of the probe as an intracellular probe to detect Zn2+ and Hg2+ ions. The probe shows very good solid-state emission behavior, this motivates us to develop latent fingerprint detection and anticounterfeiting applications. [ABSTRACT FROM AUTHOR]

Bacterial meningitis is an acute infection which requires rapid diagnosis and treatment due to the high mortality and serious consequences of the disease. The purpose of this study was to design a homemade multiplex PCR and a novel fluorescence biosensor on chip (FBC) to detect three important agents of meningitis including Streptococcus pneumoniae (S. pneumoniae) , Neisseria meningitidis (N. meningitidis), and Haemophilus influenzae (H. influenzae). The homemade multiplex PCR can diagnose three bacterial species simultaneously. Fabrication of FBC was carried out based on the deposition of lead nanoparticles on a quartz slide using the thermal evaporation method. Then, the SH-Cap Probe/Target ssDNA /FAM-Rep probe was loaded on lead film. The evaluation of the fluorescence reaction when the probes bind to the target ssDNA was assessed by a Cytation 5 Cell Imaging Multimode Reader Bio-Tek. The limit of detections (LOD) in homemade PCR and FBC to identify S. pneumoniae were 119 × 102 CFU/mL (0.27 ng/μL) and 380 CFU/mL (9 pg/μL), respectively. The LODs of homemade PCR and FBC for detection of N. meningitidis were 4.49 CFU/mL (1.1 pg/μL) and 13 × 103 CFU/mL (30 pg/μL), respectively. Our results confirmed the LODs of homemade PCR and FBC in detection of H. influenzae were 15.1 CFU/mL (30 fg/μL) and 41 × 102 CFU/mL (90 pg/ μL), respectively. Both techniques had appropriate sensitivity and specificity in detection of S. pneumoniae , N. meningitidis and H. influenzae. [Display omitted] • A novel biosensor was developed based on fluorescence probes to three important bacteria that cause meningitis. • Structure of the FBC biosensor was characterized by the use of cell citation 5. • Performance of the biosensors towards detection of bacteria was verified. • By an interesting procedure number of the bacteria was determined based on response of the biosensors. • The biosensors showed an admirable performance for detection and determination of the bacteria. [ABSTRACT FROM AUTHOR]

Light is indispensable to every aspect of our life. It is also a very important component of photodynamic therapy to cure cancer. Photodynamic therapy is a modern and effective technique among all these prevailing anticancer therapies because of its uniqueness in selectivity. Organometallic complexes are seen to be better candidates for photodynamic therapy. Cyclometalated complexes have been developed for targeting cellular organelles with remarkable photoactivity. Different types of metal complexes can display PDT activity. Considering its photophysical characteristics, the iridium complex is the most effective alternative to use as a PDT agent. Iridium-based cyclometalated complexes have shown decent results as a PDT-active anti-cancer agent. Reactive oxygen species (ROS) and singlet oxygen with high singlet oxygen quantum yield are both extremely capable of being produced by complexes. High cytotoxicity has been exhibited by various Iridium-based cyclometalated complexes in the presence of light and negligible cytotoxicity in dark conditions. These complexes are considerably fluorescent with the capability of cellular imaging and have displayed very significant potency against HeLa, A549, MCF-7, and other various cell lines. Complexes can arrest the different paths of the cell cycle through different mechanisms and help to force cellular apoptosis. Hence, a galore of organelle-targeted cyclometalated iridium complexes has been showcased in this review article. • Summarizing Current Research: This review papers provide an overview of existing literature on a particular topic. It summarizes the current research and advancements in the field of photodynamic therapy using cyclometalated iridium complexes. • Identifying Trends and Gaps: By analyzing various studies, the review can identify trends in research, such as the most effective types of complexes or promising targeting strategies. It highlights gaps in knowledge or areas where further research is needed. • Consolidating Information: Research in the field of photodynamic therapy using cyclometalated iridium complexes may be scattered across various journals and publications. This review paper consolidates this information into one place, making it easier for researchers and clinicians to access and understand. • Informing Future Research Directions: This review paper can suggest future research directions or areas that warrant further investigation. This can help guide researchers in their studies and experiments. • Educational Resource: This review paper serves as a valuable educational resource for students, researchers, and practitioners. It provides an in-depth understanding of this specific topic and can be used as a reference in academic settings. • Impact on Clinical Practice: Ultimately, the goal of research in this area is to develop more effective treatments for various diseases, such as cancer. This review paper highlights the promising cyclometalated iridium complexes for photodynamic therapy that could have a direct impact on clinical practice by informing the development of new therapeutic strategies. [ABSTRACT FROM AUTHOR]