Fluorescent Particles Research Articles

Crack identification method for magnetic particle inspection of bearing rings based on improved Yolov5

The fluorescent magnetic particle inspection technique is often used for surface crack detection of bearing rings due to its advantages of simple operation and high sensitivity. With the development of computer vision technology, more and more visual algorithms are used in magnetic particle inspection for defect detection. However, most of these current algorithm models have low detection accuracy and poor efficiency, making it difficult to meet the precision requirements of production testing and affecting the overall pace of production processes. To address this problem, this paper proposes an improved algorithm model based on Yolov5. Firstly, MobileNetV3-small is utilized to construct the backbone feature extraction network, reducing the network’s parameter count and enhancing its detection speed. In addition, Bidirectional Feature Pyramid Network is implemented to facilitate swift and efficient multi-scale feature fusion, while the C3 module in the neck is replaced with C2f to enhance detection precision. Finally, Focal-Loss EIoU is adopted as the loss function to improve the model’s accuracy in positioning the crack borders. Experimental results demonstrate that the precision of this model in detecting surface cracks in bearing rings achieves an impressive 95.1%, while the recall reaches 90.4%. The mAP stands at 0.946. When compared to the original Yolov5s network, this model showcases a reduction in network parameters by 32.1% and a significant increase in frames per second by 40.0%. These improvements effectively fulfill the production process’s demands for crack detection tasks, providing a balance between accuracy and efficiency.

Luminescent hollow spherical nanoparticles with enhanced imaging contrast through hydrogen bonding connected micelles

Herein, we propose an innovative method for synthesizing luminescent hollow spherical nanoparticles. Our method involves the construction of hydrogen bonding connected micelles (HBCMs) utilizing inter-polymer complexes of poly(styrene-alt-hydroxyphenylmaleimide)/poly(4-vinylpyridine) (poly(S-alt-HPMI)/P4VP), which could undergo self-assembly, resulting in the formation of core/shell nanostructures in the selective solvent. The aim was to incorporate two types of fluorescent compounds, namely tris(4–bromophenyl)amine (TPA–Br3) and 1,1,2,2–tetrakis(4–bromophenyl)ethene (TPE–Br4), as the cross-linkers to enhance the shell structures of HBCMs and using dimethylformamide (DMF) was employed to remove the core structures, aiming to pursue the formation of luminescent hollow spheres. Transmission electron microscopy (TEM) images confirmed the formation of self-assembled spherical structures formed by poly(S-alt-HPMI)/P4VP inter-polymer complexes. The morphological features, hydrogen bonding interactions, particle sizes, cross-linking reactions, core removal, and thermal properties were thoroughly investigated by TEM, atomic force microscope (AFM), dynamic light scattering (DLS) instrument, Fourier transform infrared (FTIR) spectroscopy, 1H nuclear magnetic resonance (NMR) spectroscopy, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) analyses. Moreover, the optical properties were also explored by fluorescence microscope and photoluminescence (PL). This evidence indicates that the HBCMs could be directly cross-linked with TPA–Br3 or TPE–Br4 and subsequently transformed into hollow spheres by the addition of DMF. Fluorescent particles featuring hollow spheres could be prepared successfully without traditional dye staining or labeling. This advancement holds promising potential for applications in nano-carriers with improved imaging contrast.

Abstract 3633: Multi-parametric characterization of extracellular vesicles in the aqueous humor of uveal melanoma eyes undergoing brachytherapy

Abstract Introduction: Aqueous humor (AH), fluid in the anterior chamber of the eye, is a liquid biopsy source of tumor information in uveal melanoma (UM). Extracellular vesicles (EVs) have recently been characterized in the AH of retinoblastoma eyes, however, have not been investigated in UM. EVs are small membrane-enclosed particles secreted by virtually every cell type with a role in intercellular communications. We hypothesize that tumor-derived EVs are detectable in the AH of UM eyes and that understanding the type of vesicles present may help us better understand their function, particularly for cancer. Small extracellular vesicles (sEVs) have been shown to promote tumorigenesis, treatment resistance, and metastasis, leading to the discovery of clinically-relevant biomarkers for multiple cancer types. As the diversity and role of sEVs in the AH of UM patients has never previously been reported, this project is a first step towards investigating new biomarkers of clinical utility. Our aim is to use single particle analysis to characterize sEV subpopulations in the AH of UM patients by quantifying their size, concentration, and phenotypes based on cell surface markers, specifically tetraspanin co-expression patterns. Methods: sEVs were analyzed from paired pre- and post-brachytherapy AH samples collected from 19 UM patients. AH samples from 5 glaucoma patients served as the control cohort. Unprocessed AH underwent Nanoparticle Tracking Analysis for size quantification and Single Particle-Interferometric Reflectance Imaging Sensor analysis for fluorescent particle count and tetraspanin immunophenotyping; counts were subsequently converted to percentages for analysis. Statistics were performed using Mann-Whitney U, ANOVA, and Tukey’s tests. Results: AH derived from UM eyes (pre-brachytherapy) displayed a broader sEV size distribution than glaucoma eyes, with more diverse sEV subpopulations. Both tumor and nontumor AH samples demonstrated a dominant CD63+ sEV subpopulation. A significantly higher percentage of CD63/81+ sEVs (P= 0.0217) and CD9/63/81+ sEVs (P= 0.0146) were found in post-brachytherapy samples compared to pre-brachytherapy. Conclusions: sEV subpopulations in UM eyes demonstrate increased heterogeneity in comparison to nontumor eyes. Our results are consistent with prior studies reporting that the CD63+ sEV subtype is an AH-specific biomarker enriched across multiple ocular diseases. The increase in CD63/81+ sEVs and CD9/63/81+ sEVs after brachytherapy is presumably a result of radiation-induced release of these vesicles, suggesting a tumor-derived origin of these subpopulations. CD63/81+ sEV subpopulation was previously reported to also be a tumor-derived subpopulation in AH of retinoblastoma eyes. Further study of the cargo carried by these sEV subpopulations may uncover molecular mechanisms pivotal to UM and other ocular cancers. Citation Format: Shreya Sirivolu, Chen-Ching Peng, Paolo Neviani, Jesse L. Berry, Liya Xu. Multi-parametric characterization of extracellular vesicles in the aqueous humor of uveal melanoma eyes undergoing brachytherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3633.

Abstract 483: Unraveling the impact of cancer cell derived membrane and cytosolic proteins using synthetic small extracellular vesicles

Abstract Background: Extracellular vesicles (EVs) are couriers in cell-cell communication. Evidence suggests that EV biology, from cargo packing, biodistribution, to cell uptake is a non-random and well-orchestrated process. Substantial efforts have been devoted to studying the pathological roles and therapeutic potential of EVs. It is speculated that surface properties, such as membrane proteins, mediate the interaction between EVs and recipient cells, while the enclosed “messages” exert downstream effects. This concept is exemplified in cancer metastasis, where cancer cell-derived EVs were shown to establish pre-metastatic niches and guide cancer organotropism. We propose selectively loading cancer cell membrane and cytosolic proteins into synthetic vesicles (SVs) like liposomes. This approach allows for the isolated study of individual factors, in contrast to the complexity of naturally occurring EVs. Methods: Cellular membrane and cytosolic proteins were extracted separately from a cancer cell line (MP41) (Mem-PERTM Plus kit). Empty-SV, Mem-SV (with membrane proteins), Cyto-SV (with cytosolic proteins), and Complete-SV (with both) were synthesized in a 3D-printed microfluidic chip and subsequently dialyzed (1000 KDa) overnight. The volume of protein buffer was controlled in all SV syntheses. EVs were isolated from the conditioned medium by ultrafiltration (Amicon) followed by ultracentrifugation. The size (Nanosight), zeta potential (ZetaView), and morphology (Transmission Electron Microscopy) of SVs and EVs were analyzed. Proteins incorporated into SVs were validated by stain-free gel, western blot, and CytoFLEX. SVs and EVs were stained by sp-DiIC18 fluorescent dye. The number of fluorescent particles was quantified (CytoFLEX) and controlled in cell uptake analysis on hepatocytes (IHH) and fibroblasts (BJ) (Incucyte). Results: All SVs were successfully synthesized with a mean size of ~100 nm. The zeta potential of both Mem-SV (-27.03 mV) and Complete-SV (-24.78 mV) is close to MP41 EVs (-26.01 mV) (P > 0.05) and are more negative than Empty-SV (-11.30 mV) and Cargo-SV (-13.38 mV) (P < 0.05). Total protein incorporation efficiency was estimated to be ~30%. The relative level of specific proteins (e.g. integrin av, TSG101) in Mem-SV and Cyto-SV matched the level in the raw membrane and cytosolic proteins by western blot. Integrin avb5 was detected on the surface of Mem-SV. Membrane and cytosolic protein incorporation increased the uptake efficiency of SVs compared to Empty-SVs, and the efficiency varied with cell type and protein content. Conclusion: In this study, we demonstrate that membrane and cytosolic proteins differentially impact the physical (zeta potential) and functional (cellular uptake) properties of SVs. Investigating these distinct roles could further our understanding of cancer EV biology, as well as advance drug delivery systems. Citation Format: Yunxi Chen, Rubén R. López, Thupten Tsering, Chaymaa Zouggari Ben El Khyat, Vahé Nerguizian, Julia V. Burnier. Unraveling the impact of cancer cell derived membrane and cytosolic proteins using synthetic small extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 483.

A hydrodynamic-based dual-function microfluidic chip for high throughput discriminating tumor cells

A hydrodynamic-based microfluidic chip consisted of two function units that could not only separate tumor cells (TCs) from whole blood but also remove residual blood cells was designed. The separation of TCs was achieved by a straight contraction-expansion array (CEA) microchannel on the front end of the chip. The addition of contractive structure brought a micro-vortex like Dean vortex that promoted cell focusing in the channel, while when cells entered the dilated region, the wall-induced lift force generated by the channel wall gave cells a push away from the wall. As the wall-induced lift force is proportional to the third power of the cell diameter, TCs with larger diameter will have a larger lateral migration under the wall-induced lift force, realizing the separation of TCs from blood sample. Fluorescent particles with diameters of 19.3 μm and 4.5 μm were used to simulate TCs and red blood cells, respectively, to verify the separation capacity of the proposed CEA microchannel for particles with different diameter. And a separation efficiency 98.7% for 19.3 μm particles and a removal rate 96.2% for 4.5 μm particles was observed at sample flow rate of 10 μL min−1 and sheath flow rate of 190 μL min−1. In addition, a separation efficiency about 96.1% for MCF-7 cells (stained with DiI) and removal rates of 96.2% for red blood cells (RBCs) and 98.7% for white blood cells (WBCs) were also obtained under the same condition. However, on account of the large number of blood cells in the blood, there will be a large number of blood cells remained in the isolated TCs, so a purification unit based on hydrodynamic filtration (HDF) was added after the separation microchannel. The purification channel is a size-dictated cell filter that can remove residual blood cells but retain TCs, thus achieving the purification of TCs. Combined the CEA microchannel and the purifier, the microchip facilitates sorting of MCF-7 cells from whole blood with a separation rate about 95.3% and a removal rate over 99.99% for blood cells at a sample flow rate of 10 μL min−1, sheath flow rate of 190 μL min−1 and washing flow rate of 63 μL min−1.

The 1 μm wear particles entrainment in situ observation via fluorescent staining silica particles by silane coupling with Rhodamine B

Observing wear debris during friction is crucial for comprehending the wear behavior of lubrication systems. Despite various techniques attempted for observation, a persistent challenge is the oversight of wear debris with a diameter less than 1 μm, mainly due to limitations in measurement systems. Consequently, we still lack a comprehensive understanding of whether these small particles can infiltrate contact points and serve as abrasives. In this study, we conducted in-situ friction tests to investigate the entrainment of imitation wear particles at the contact point under boundary lubrication conditions. These imitation wear particles were glass beads with diameters of approximately 0.8 μm, 1.0 μm, and 3.0 μm, respectively. To address optical limitations, we stained these particles using silane coupling to attach Rhodamine B to the glass beads. We examined the effect of particle diameter on entrainment numbers under varying outside oil film thicknesses. The results showed that the entrainment number was highest when the outside oil film thickness matched the particle diameter. This clearly indicated that the outside oil film thickness significantly influenced the entrainment of particles.

99mTc-AgNPs-ICG: nanoparticle for hybrid image

Introduction: Currently nanotechnology has radically changed the diagnosis of many human pathologies. The aim of this work is to obtain silver nanoparticles for hybrid imaging (99mTc-AgNPs-ICG) having potential clinical imaging applications. Materials and method: We mixed 2 ml of ascorbic acid (1.7x10-4 M), 5 mCi of 99mTcO4- , 2 ml of citric acid (8.0x10-4 M) and 0.5 ml of silver nitrate (2.5x10-3 M). Solution pH was 5, and it was shaken for 20 minutes at 37º C. Afterwards, 2 µL of Indocyanine Green (1.3x10-3 M) was added (99mTc-AgNPs-ICG). Physiochemical properties of the solution were characterized by UV (λ1 = 420 nm, λ2 = 254 nm) and gamma detector. Fluorescence image, particle size and IR spectrum were evaluated. Results: Silver nanoparticles were obtained in aqueous solution a pH of 5. Their pH, color and spectrum were stable for seven days. Furthermore, the principal peak characterized by HPLC, UV and Gamma detector had similar retention times. Its UV spectrum showed an absorption band of 420 nm, which corresponds to the plasmon absorption band of these nanoparticles. The particle size was 46 nm ± 1.5 nm. The IR spectrum showed absorption bands in 3193, 2624, 1596 y 1212 cm-1. Conclusions: We describe for the first time in literature the synthesis of hybrid (radioactive and fluorescent) silver nanoparticles. Their physiochemical properties were characterized, being stable and their labelling was reproducible having potential biomedical applications. Received for review: November 2023. Accepted for publication: February 2024. Correspondence: Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. Mataojo 2055, P.O. 11400, Montevideo, Uruguay. Tel: (+5982) 5250901/108; fax: (+5982) 5250895. Contact e-mail: pabloc7@gmail.com This article was approved by the Editorial Committee.

Characterising phagocytes and measuring phagocytosis from live Galleria mellonella larvae

ABSTRACT Over the last 20 years, the larva of the greater waxmoth, Galleria mellonella, has rapidly increased in popularity as an in vivo mammalian replacement model organism for the study of human pathogens. Experimental readouts of response to infection are most often limited to observing the melanization cascade and quantifying larval death and, whilst transcriptomic and proteomic approaches, and methods to determine microbial load are also used, a more comprehensive toolkit of profiling infection over time could transform the applicability of this model. As an invertebrate, Galleria harbour an innate immune system comprised of both humoral components and a repertoire of innate immune cells – termed haemocytes. Although information on subtypes of haemocytes exists, there are conflicting reports on their exact number and function. Flow cytometry has previously been used to assay Galleria haemocytes, but protocols include both centrifugation and fixation – physical methods which have the potential to affect haemocyte morphology prior to analysis. Here, we present a method for live haemocyte analysis by flow cytometry, revealing that Galleria haemocytes constitute only a single resolvable population, based on relative size or internal complexity. Using fluorescent zymosan particles, we extend our method to show that up to 80% of the Galleria haemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicates in vitro data, showing that cell wall β-1,3-glucan masking by Candida albicans subverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics in Galleria.

A novel strategy for partial purification of alkane hydroxylase from P. chrysogenum SNP5 through reconstituting its native membrane into liposome

Integral proteins or enzymes are still challenging to purify into their native state because of their need for an amphipathic environment and cofactors. Alkane hydroxylase (AlkB) is a membrane-bound enzyme that catalyzes the hydroxylation of a range of alkanes that have a broad spectrum of applications. In the current study, a novel approach has been explored for partial purification of alkane hydroxylase (AlkB) in its native state through restructuring the lipid bilayer of Penicilliumchrysogenum SNP5 into a liposome to extend the native and protective environment to AlkB enzyme. Three different methods i.e., reverse-phase evaporation method (RPEM), detergent-based method (DBM), and ethanol injection method (EIM) have been used for reconstituting its native membrane into liposome. On characterizing liposomes through fluorescence imaging, AFM, and particle size analysis, the reverse-phase evaporation method gave the best results based on the size distribution (i.e., 100–300 nm), the morphology of liposomes, and maximum AlkB specific activity (i.e., 140.68 U/mg). The maximum reconstitution efficiency of 29.48% was observed in RPEM followed by 17.3% in DBM and 12.3% in EIM. On the characterization of the purified AlkB, the molecular weight was measured of 44.6 KDa and the thermostability of liposomes synthesized with the RPEM method was obtained maximum at 55 °C. This approach may open a new strategy for the purification of integral enzymes/proteins in their native state in the field of protein purification and its applications in diversified industries.

FRET-Sensing of Multivalent Protein Binding at the Interface of Biomimetic Microparticles Functionalized with Fluorescent Glycolipids.

Cell adhesion is a central process in cellular communication and regulation. Adhesion sites are triggered by specific ligand-receptor interactions inducing the clustering of both partners at the contact point. Investigating cell adhesion using microscopy techniques requires targeted fluorescent particles with a signal sensitive to the clustering of receptors and ligands at the interface. Herein, we report on simple cell or bacterial mimics, based on liquid microparticles made of lipiodol functionalized with custom-designed fluorescent lipids. These lipids are targeted toward lectins or biotin membrane receptors, and the resulting particles can be specifically identified and internalized by cells, as demonstrated by their phagocytosis in primary murine bone marrow-derived macrophages. We also evidence the possibility to sense the binding of a multivalent lectin, concanavalin A, in solution by monitoring the energy transfer between two matching fluorescent lipids on the surface of the particles. We anticipate that these liquid particle-based sensors, which are able to report via Förster resonance energy transfer (FRET) on the movement of ligands on their interface upon protein binding, will provide a useful tool to study receptor binding and cooperation during adhesion processes such as phagocytosis.

Roles of marine biota in the formation of atmospheric bioaerosols, cloud condensation nuclei, and ice-nucleating particles over the North Pacific Ocean, Bering Sea, and Arctic Ocean

Abstract. We investigated the association of marine biological indicators (polysaccharides, protein-like gel particles, and chl a) with the formation of fluorescent aerosol particles, cloud condensation nuclei (CCNs), and ice-nucleating particles (INPs) over the North Pacific Ocean, Bering Sea, and Arctic Ocean during September–November 2019. The abundance of bioindicators was high in the North Pacific Ocean and the Bering Sea (e.g., up to 1.3 mg m−3 of chl a), suggesting high biological activity due to a phytoplankton bloom. In the North Pacific Ocean, particles were characterized by high mass fractions of organics and sulfate with a predominance of terrestrial air masses. Conversely, in the Bering Sea and the Arctic Ocean, particles were characterized by high mass fractions of sea salt and sulfate with a predominance of maritime air masses. The averaged range/value of the CCN concentration at 0.4 % supersaturation were 99–151, 43–139, and 36 cm−3 over the North Pacific Ocean with terrestrial influences, over the Bering Sea with marine biogenic influences, and over the Arctic Ocean with marine influences, respectively, and the corresponding range/value of the hygroscopicity parameter κ were 0.17–0.59, 0.42–0.68, and 0.66, respectively. The averaged INP concentration (NINP) measured at temperatures of −18 and −24 ∘C with marine sources in the North Pacific and Bering Sea was 0.01–0.09 and 0.1–2.5 L−1, respectively, and that over the Arctic Ocean was 0.001–0.016 and 0.012–0.27 L−1, respectively. When marine sources were dominant, fluorescent bioaerosols in the fine mode were strongly correlated with all bioindicator types (R: 0.81–0.88) when the effect of wind-induced uplift from the sea surface to the atmosphere was considered. Correlations between NINP measured at −18 and −24 ∘C and all bioindicator types (R: 0.58–0.95 and 0.79–0.93, respectively) were positive, even when the extreme outlier point was omitted, as were those between NINP and fluorescent bioaerosols (R: 0.50 and 0.60, respectively), suggesting that marine bioindicators contributed substantially as sources of bioaerosols and to cloud formation.

Vortex-Enhanced Microfluidic Chip for Efficient Mixing and Particle Capturing Combining Acoustics with Inertia.

Vortex-based microfluidics has received significant attention for its unique characteristics of high efficiency, flexible control, and label-free properties for the past decades. Herein, we present a vortex-based acousto-inertial chip that allows both fluid and particle manipulation within a significantly wider flow range and lower excitation voltage. Composed of contraction-expansion array structures and vibrating microstructures combined with bubbles and sharp edges, such a configuration results in more vigorous vortical fluid motions. The overall improvement in device performance comes from the synergistic effect of acoustics and inertia, as well as the positive feedback loop formed by vibrating bubbles and sharp edges. We characterize flow patterns in the microchannels by fluorescence particle tracer experiments and uncover single- and double-vortex modes over a range of sample flow rates and excitation voltages. On this basis, the ability of rapid and efficient sample homogenization up to a flow rate of 200 μL/min under an excitation voltage of 15 Vpp is verified by a two-fluid fluorescence mixing experiment. Moreover, the recirculation motion of particles in microvortices is investigated by using a high-speed imaging system. We also quantitatively measure the particle velocity variation on the trajectory and illustrate the capturing mechanism, which results from the interaction of the microvortices, particle dynamics, and composite microstructure perturbations. Further utilizing the shear forces derived by microvortices, our acousto-inertial chip is demonstrated to lysis red blood cells (RBCs) in a continuous, reagent-free manner. The high controllability and multifunction of this technology allow for the development of multistep miniaturized "lab-on-chip" analytical systems, which could significantly broaden the application of microvortex technology in biological, chemical, and clinical applications.

Tracking the micro- and nanoplastics in the terrestrial-freshwater food webs. Bivalves as sentinel species

Micro- (MPs) and nanoplastics (NPs) are currently ubiquitous in the ecosystems, and freshwater biota is still insufficiently studied to understand the global fate, transport paths, and consequences of their presence. Thus, in this study, we investigated the role of bivalves and a trophic transfer of MPs and NPs in an experimental food chain. The food chain consisted of terrestrial non-selective detritivore Dendrobaena (Eisenia) sp., freshwater benthic filter feeder Unio tumidus, and freshwater benthic detritivore-collectors Asellus aquaticus or Gammarus sp. Animals were exposed to different fluorescently labeled micro- and nanoplastics (PMMA 20 μm, nanoPS 15–18 nm, and 100 nm, PS 1 μm and 20 μm, PE from cosmetics) as well as to the faeces of animals exposed to plastics to assess their influence on the environmental transportation, availability to biota, and bioaccumulation of supplied particles. Damaged and intact fluorescent particles were observed in the faeces of terrestrial detritivores and in the droppings of aquatic filter feeders, respectively. They were also present in the guts of bivalves and of crustaceans which were fed with bivalve droppings. Bivalves (Unio tumidus, and additionally Unio pictorum, and Sphaerium corneum) produced droppings containing micro- and nanoparticles filtered from suspension and deposited them onto the tank bottom, making them available for broader feeding guilds of animals (e.g. collectors, like crustaceans). Finally, the natural ageing of PS and its morphological changes, leakage of the fluorescent labelling, and agglomeration of particles were demonstrated. That supports our hypothesis of the crucial role of the characterization of physical and chemical materials in adequately understanding the mechanisms of their interaction with biota. Microscopical methods (confocal, fluorescent, scanning electron) and Raman and FT-IR spectroscopy were used to track the particles' passage in a food web and monitor structural changes of the MPs' and NPs' surface.

VISUALIZATION OF MIXING UPPER AND LOWER STRATIFIED SUSPENSIONS USING RED AND BLUE FLUORESCENT PARTICLES

We conducted visualization experiments to investigate the macroscopic mixing of upper and lower stratified suspensions. We used spectroscopy to distinguish the upper and lower suspensions in the images recorded by a color camera, in which the upper and lower suspended particles were replaced with blue and red fluorescent particles, respectively. We also proposed a method to determine the mass of adsorbed particles and the volume of fluorescent solution required to produce fluorescent particles by using the two calibration curves and the Freundlich equation. Visualization with fluorescent particles proved to be effective for macroscopic mixing by comparing mono and color images. Finally, we found good agreement in the trends between the experimental and numerical results.

Inertial co-focusing of heterogeneous particles in hybrid microfluidic channels with constantly variable cross-sections.

Heterogeneous particles co-focusing to a single stream is a vital prerequisite for cell counting and enumeration, playing an essential role in flow cytometry and single-cell analysis. Microfluidics-based inertial focusing holds great research prospects due to its simplicity of devices, ease of operation, high throughput, and freedom from external fields. Combining microfluidic channels with two or more different geometries has become a powerful tool for high-efficiency particle focusing. Here, we explored hybrid microfluidic channels for heterogeneous particle co-focusing. Four different annular channels with obstacles distributed on the inner wall were constructed and simulated, obtaining constantly variable secondary flows. Then we used four different fluorescent particles with the size of 10 μm, 12 μm 15 μm, and 20 μm as well as their mixture to perform the inertial focusing experiments of multi-sized particles. Theoretical simulation and experimental results demonstrated a focusing efficiency of >99%. Finally, we further utilized human white blood cells to estimate the co-focusing performance of our hybrid microfluidic channel, resulting in a high focusing efficiency of >92% and a high throughput of ≈8000 cell s-1. The hybrid microfluidic channels, capable of high-precision heterogeneous particle co-focusing, could pave a broad avenue for microfluidic flow cytometry and single-cell analysis.

Ultrabright fluorescent particles via physical encapsulation of fluorescent dyes in mesoporous silica: a mini-review.

Harnessing the power of mesoporous silica to encapsulate organic fluorescent dyes has led to the creation of an extraordinary class of nanocomposite photonic materials. These materials stand out for their ability to produce the brightest fluorescent particles known today, surpassing even the luminosity of quantum dots of similar spectrum and size. The synthesis of these materials offers precise control over the shape and size of the particles, ranging from the nano to the multi-micron scale. Just physical encapsulation of the dyes opens new possibilities for mixing different dyes within individual particles, paving the way for nearly limitless multiplexing capabilities. Moreover, this approach lays the groundwork for the development of highly sensitive sensors capable of detecting subtle changes in temperature and acidity at the nanoscale, among other parameters. This mini-review highlights the mechanism of synthesis, explains the nature of ultrabrightness, and describes the recent advancements and future prospects in the field of ultrabright fluorescent mesoporous silica particles, showcasing their potential for various applications.

Making Rift Valley Fever Viral Particles Fluorescent.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that represents a significant threat to both human and veterinary public health. Since its discovery in the Great Rift Valley of Kenya in the 1930s, the virus has spread across Africa and beyond, now posing a risk of introduction into Southern Europe and Asia. Despite recent progresses, early RVFV-host cell interactions remain largely uncharacterized. In this method chapter, we delineate the procedure for labeling RVFV particles with fluorescent organic dyes. This approach makes it feasible to visualize single viral particles in both fixed and living cells and study RVFV entry into host cells. We provide additional examples with two viruses closely related to RVFV, namely, Toscana virus and Uukuniemi virus. Furthermore, we illustrate how to utilize fluorescent viral particles to examine and quantify each step of the cell entry program of RVFV, which includes state-of-the-art fluorescence-based detection techniques such as fluorescence microscopy, flow cytometry, and fluorimetry.

Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering.

Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

Infectious Virus Tracking by Fluorescent Live Cell Imaging in Primary Cells.

To successfully infect a cell, HIV-1 has to overcome several host barriers while exploiting cellular cofactors. HIV-1 infection is highly inefficient with the great majority of viral particles not being able to successfully integrate into the target cell genome. Nonproductive HIV-1 particles are degraded or accumulated in cellular compartments. Thus, it becomes hard to distinguish between viral behaviors that lead to effectively infecting the cell from the ones that do not by using traditional methods. Here, we describe the infectious virus tracking method that detects and quantifies individual fluorescent viral particles over time and links viral particle behavior to its infectivity. This method employs live-cell imaging at ultra-low MOIs to detect the outcome of infection for every HIV-1 particle.

Comparison of the Residual Amount of an Ophthalmic Viscosurgical Device Among Different Types of Intraocular Lens Implants in vitro.

Although ophthalmic viscosurgical devices are quite important for safe cataract surgery, currently, postoperative residual ophthalmic viscosurgical devices can cause various complications. Previously, we developed a method to visualize residual ophthalmic viscosurgical devices after irrigation/aspiration in vitro and found that the amount of residual ophthalmic viscosurgical device on a single-piece intraocular lens was greater than that on a three-piece intraocular lens. In the present study, we compared the amounts of residual ophthalmic viscosurgical device among various foldable intraocular lenses to investigate the factors that determine the quantity of residual ophthalmic viscosurgical device. Simulated cataract surgery was performed in pig eyes using an ophthalmic viscosurgical device labeled with fluorescent silica particles. After the simulated surgery procedure, the fluorescent silica attached to the intraocular lens was observed and quantified by inductively coupled plasma-atomic emission spectrometry after intraocular lens removal. The amount of residual ophthalmic viscosurgical device was compared among five representative single-piece intraocular lenses and one three-piece intraocular lens. The distribution and amount of the residual ophthalmic viscosurgical device differed for each intraocular lens. The amount of silicon in the lens capsule differed among the intraocular lens types. The postoperative residual tendency of ophthalmic viscosurgical devices differed among various single-piece intraocular lenses. The behavior of the intraocular lenses within the capsule affected the residual tendency. The removal of ophthalmic viscosurgical device in the lens capsule should be tailored for each intraocular lens to improve efficiency.