Fluorescent Microbeads Research Articles

A Balance between Turning and Persistent Motion is Critical for Fast and Efficient 3-Dimensional Neutrophil Migration

While much research has been dedicated to identifying the cascade of specific biochemical processes involved in the recruitment of neutrophils, much less is known about the mechanical events driving their directed migration. In particular, it is still unclear how neutrophils generate the necessary traction forces to migrate across three-dimensional (3-D) extravascular spaces and the effects of matrix porosity on turning events and persistence during this process. In this study, we examine the effects of extracellular matrix properties on the mechanics of 3-D neutrophil motility in collagen gels. We embedded neutrophil-like differentiated human promyelocytic leukemia (dHL-60) cells in collagen matrices of different concentrations containing fluorescent micro-beads. To induce directed migration, we introduced the chemokine formyl-Methionyl-Leucyl-Phenylalanine (fMLP) to dHL-60 cells in a custom build device. We then used brightfield microscopy to track chemotaxing cells and both confocal and fluorescent microscopy techniques to image the movement of the embedded micro-beads and fluorescently labeled cells. We find that turning events, mediated by the Arp 2/3 complex, are crucial for fast 3-D neutrophil migration. Additionally, the Arp 2/3 complex and myosin II play an increasingly important role in 3-D migration as matrices become denser. Finally, we find that cellular force patterns vary in response to matrices with various degrees of pore size heterogeneities and cell's intrinsic abilities to engage in path-finding and contractility. The results from our study show that neutrophils migrating in 3-D environments employ distinct mechanical mechanisms that depend on the local structure of their mechanical environments. Our work has implications for understanding how neutrophils interact physically with their surrounding environment what conditions may be necessary for fast 3-D neutrophil migration.

Low‐cost microplastic visualization in feeding experiments using an ultraviolet light‐emitting flashlight

AbstractMicroplastics are pollutants threatening the health of marine, freshwater and terrestrial organisms. To analyze whether an organism is able to ingest microplastics, the organism is usually fed with expensive fluorescent microbeads and placed under a fluorescence microscope for microplastic detection. However, such equipment cannot be afforded by many laboratories. Therefore, we developed a low‐cost method to study the ingestion and egestion of low‐cost, fluorescent microplastic fragments and fibers by aquatic invertebrates. During our feeding experiments, we exposed the freshwater snail Radix balthica to artificial biofilms containing 15% microplastic fragments and fibers, respectively. We then used an ultraviolet (UV) flashlight to irradiate the microplastics. Hence, we could directly observe the microplastics in the food, during ingestion and egestion as well as in the snail feces. Our method, thereby, allowed us to analyze the snails' behavior (during ingestion and egestion), the microplastics' distribution in the snail feces and the microplastics' shape after the snails' digestion. Furthermore, we observed that the snails ceased to egest microplastics after 76 hr following microplastic ingestion. However, chemical digestion of the snails 6 days after microplastic exposure revealed that microplastics were still present in R. balthica, emphasizing the microplastics' persistence in the animals' bodies. This is the first study combining the usage of low‐cost fluorescent microplastics with an inexpensive UV flashlight for microplastic detection during microplastic ingestion and egestion by living organisms. Our new method is not only cost‐effective, but also fast and can be performed by a wide range of researchers without special previous training.

Comparison of viability and phagocytic responses of hemocytes withdrawn from the bivalves Mytilus edulis and Dreissena polymorpha, and exposed to human parasitic protozoa

Bivalve molluscs are now considered indicator species of aquatic contamination by human parasitic protozoa. Nonetheless, the possible effects of these protozoa on the immune system of their paratenic hosts are poorly documented. The aim of this study was to evaluate the effects of two protozoa on hemocyte viability and phagocytosis from two mussels, the zebra mussel (freshwater habitat) and the blue mussel (seawater habitat). For these purposes, viability and phagocytic markers have been analysed on hemocytes from mussels without biological stress (control hemocytes), and on hemocytes exposed to a biological stress (Toxoplasma gondii and Cryptosporidium parvum oocysts). We report, for the first known time, the interactions between protozoa and hemocytes of mussels from different aquatic environments. Zebra mussel hemocytes showed a decrease in phagocytosis of fluorescent microbeads after exposure to both protozoa, while blue mussel hemocytes reacted only to T. gondii oocysts. These decreases in the ingestion of microbeads can be caused by competition between beads and oocysts and can be influenced by the size of the oocysts. New characterisations of their immune capacities, including aggregation, remain to be developed to understand the specificities of both mussels.

Insights into the feeding behaviors and biomechanics of Varroa destructor mites on honey bee pupae using electropenetrography and histology

Feeding behaviors and biomechanics of female Varroa destructor mites are revealed from AC-DC electropenetrography (EPG) recordings of mites feeding from Apis mellifera honey bee pupae and histology of mite internal ingestion apparatus. EPG signals characteristic of arthropod suction feeding (ingestion) were identified for mites that fed on pupae during overnight recordings. Ingestion by these mites was confirmed afterwards by observing internally fluorescent microbeads previously injected into their hosts. Micrographs of internal ingestion apparatus illustrate the connection between a gnathosomal tube and a pharyngeal lumen, which is surrounded by alternating dilator and constrictor muscles. Inspection of EPG signals showed the muscularized mite pharyngeal pump operates at a mean repetition rate of 4.5 cycles/s to ingest host fluids. Separate feeding events observed for mites numbered between 23 and 33 over approximately 16 h of recording, with each event lasting ~10 s. Feeding events were each separated by ~2 min. Consecutive feeding events separated by either locomotion or prolonged periods of quiescence were grouped into feeding bouts, which ranged in number from one to six. Statistical analyses of EPG data revealed that feeding events were prolonged for mites having lower pharyngeal pump frequencies, and mites having prolonged feeding events went unfed for significantly more time between feeding events. These results suggest that mites may adjust behaviors to meet limitations of their feeding apparatus to acquire similar amounts of food. Data reported here help to provide a more robust view of Varroa mite feeding than those previously reported and are both reminiscent of, as well as distinct from, some other acarines and fluid-feeding insects.

Patterns of antibody responses to nonviral cancer antigens in head and neck squamous cell carcinoma patients differ by human papillomavirus status.

There have been hints that nonviral cancer antigens are differentially expressed in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC). Antibody responses (AR) to cancer antigens may be used to indirectly determine cancer antigen expression in the tumor using a noninvasive and tissue-saving liquid biopsy. Here, we set out to characterize AR to a panel of nonviral cancer antigens in HPV-positive and HPV-negative HNSCC patients. A fluorescent microbead multiplex serology to 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens and 8 oncogenes) and 29 HPV-antigens was performed in 382 HNSCC patients from five independent cohorts (153 HPV-positive and 209 HPV-negative). AR to any of the cancer antigens were found in 272/382 patients (72%). The ten most frequent AR were CT47, cTAGE5a, c-myc, LAGE-1, MAGE-A1, -A3, -A4, NY-ESO-1, SpanX-a1 and p53. AR to MAGE-A3, MAGE-A9 and p53 were found at significantly different prevalences by HPV status. An analysis of AR mean fluorescent intensity values uncovered remarkably different AR clusters by HPV status. To identify optimal antigen selections covering a maximum of patients with ≤10 AR, multiobjective optimization revealed distinct antigen selections by HPV status. We identified that AR to nonviral antigens differ by HPV status indicating differential antigen expression. Multiplex serology may be used to characterize antigen expression using serum or plasma as a tissue-sparing liquid biopsy. Cancer antigen panels should address the distinct antigen repertoire of HPV-positive and HPV-negative HNSCC.

Ingestion of polyethylene microbeads affects the growth and reproduction of medaka, Oryzias latipes

Research using various species of wild and cultured fish has identified negative effects of short-term exposure to microbeads. Although wild animals might be contaminated with microbeads and/or other pharmaceuticals, data regarding the long-term effects remain limited. To clearly elucidate the effects of microbeads, studies of long-term exposure using animal models are necessary. Our aim was to elucidate the effects of microbeads alone on the growth and fecundity of medaka following long-term exposure (12 weeks). In experiment 1, fish groups (except controls) were temporarily exposed to polyethylene microbeads (10–63 μm diameter) a low dose of 0.065 microbeads-mg/L and high dose of 0.65 microbeads-mg/L. In experiment 2, see-through medaka and fluorescent polyethylene microbeads (10–45 μm diameter) were used to estimate the retention time of ingested microbeads in the digestive tract, which was 4–9 days. The low dose of microbeads did not affect growth but did decrease the number of eggs and the hatching rate. The high dose decreased growth, the number of eggs, and hatching rate. Growth differences were recognized for the first time at 7 weeks, and differences in the number of eggs at 12 weeks. Thus, long-term tests using medaka indicated that microbeads per se exhibit growth inhibition and reproductive toxicity. These effects could be associated with nutritional factors resulting from the long retention time of microbeads in the digestive tract. We also determined the dose that affects only fecundity. This suggests that normal growth of medaka in the wild does not mean the environment is free from microbead contamination. We are thus attempting to identify new biological indexes for monitoring the status of microbead contamination using our system.

Fluorescent microbeads for point-of-care testing: a review.

Microbead-based point-of-care testing (POCT) has demonstrated great promise in translating detection modalities from bench-side to bed-side. This is due to the ease of visualization, high surface area-to-volume ratio of beads for efficient target binding, andefficient encoding capability for simultaneous detection of multiple analytes. This review (with 112 references) summarizes the progress made in the field of fluorescent microbead-based POCT. Following an introduction into the field, a first large section sums up techniques and materials for preparing microbeads, typically of dye-labelled particles, various kinds of quantum dots and upconversion materials. Further subsections cover the encapsulation of nanoparticles into microbeads, decoration of nanoparticles on microbeads, and in situ embedding of nanoparticles during microbead synthesis. A next large section summarizes microbead-based fluorometric POCT, with subsections on detection of nucleic acids, proteins, circulating tumor cells and bacteria. A further section covers emerging POCT based on the use of smartphones or flexible microchips. The last section gives conclusions and an outlook on current challenges and possible solutions. Aside from giving an overview on the state of the art, we expect this article to boost the further development of POCT technology. Graphical Abstract Schematic presentation of the fabrication of microbeads, the detection targets of interest including bacteria, circulating tumor cells (CTCs), protein and nucleic acid, and the emerging point-of-care testing (POCT) platform. The colored wheels of the bus represent the fluorescent materials embedded in (red color) or decorated on the surface of microbeads (green color).

A new approach for analyzing an adhesive bacterial protein in the mouse gastrointestinal tract using optical tissue clearing

Several bacterial moonlighting proteins act as adhesion factors, which are important for bacterial colonization of the gastrointestinal (GI) tract. However, little is known about the adherence properties of moonlighting proteins in the GI tract. Here, we describe a new approach for visualizing the localization of moonlighting protein-coated fluorescent microbeads in the whole GI tract by using a tissue optical clearing method, using elongation factor Tu (EF-Tu) as an example. As a bacterial cell surface-localized protein mimic, recombinant EF-Tu from Lactobacillus reuteri was immobilized on microbeads. EF-Tu-coating promoted the interaction of the microbeads with a Caco-2 cell monolayer. Next, the microbeads were orally administered to mice. GI whole tissues were cleared in aqueous fructose solutions of increasing concentrations. At 1 h after administration, the microbeads were diffused from the stomach up to the cecum, and after 3 h, they were diffused throughout the intestinal tract. In the lower digestive tract, EF-Tu-beads were significantly more abundant than non-coated control beads, suggesting that EF-Tu plays an important role in the persistence of the microbeads in the GI tract. The new approach will help in evaluating how moonlighting proteins mediate bacterial colonization.

The hematopoietic organ of Macrobrachium rosenbergii: Structure, organization and immune status

The hematopoietic organ (HO) of the giant freshwater prawn Macrobrachium rosenbergii is a discrete, whitish mass located in the epigastric region of the cephalothorax, posterior to the brain. It is composed of hematopoietic cells arranged in a thick layer of numerous lobules that surround a central hemal sinus from which they are separated by a thin sheath. At the center of the sinus is the muscular cor frontale. The lobules extend radially outward from the sinus in three developmental zones. Basal Zone 1 nearest the sinus contains large hematopoietic stem cells with euchromatic nuclei that stain positive for proliferation cell nuclear antigen (PCNA). Zone 2 contains smaller, actively dividing cells as indicated by positive 5-bromo-20-deoxyuridine (BrdU) staining. Distal Zone 3 contains small, loosely packed cells with heterochromatic nuclei, many cytoplasmic granules and vesicles indicating that they will eventually differentiate into hemocytes and enter circulation. Three main arteries, namely the ophthalmic and the 2 branches of the antennary, connect the heart to the HO. Use of India ink and 0.1 μm fluorescent micro-beads injected into the heart revealed that the cor frontale could immediately remove foreign particles from hemolymph by filtration. Fluorescent beads were also detected in the hematopoietic tissue at 30 min after injection, indicating that it could be penetrated by foreign particles. However, the fluorescent signal completely disappeared from the whole HO after 4 h, indicating its role in removal of foreign particles. In conclusion, the present study demonstrated for the first time the detailed histological structures of the HO of M. rosenbergii and its relationship to hematopoiesis and removal of foreign particles from hemolymph.

Abstract B130: Induced lymphangiogenesis enhances antigen-specific T-cell response in anticancer vaccination

Abstract Tumor-associated lymphatic vessels play multiple and complex roles in the regulation of antitumor immunity. In both mouse and human melanomas, lymphatic markers correlate with increased tumor immune infiltrates and enhanced responsiveness to immunotherapy. Despite the immune-promoting functions of lymphatics in cancer immunotherapy, manipulation of lymphatic vessels directly within the tumor site appears controversial as a therapeutic strategy, due to the correlation of tumor lymphatic growth (lymphangiogenesis) with cancer cell dissemination and metastasis. Here, we sought to manipulate and exploit lymphatic vessels remotely from the tumor by developing and characterizing a lymphangiogenic cancer vaccine. Lymphangiogenic whole-cell vaccines were formulated using lethally irradiated tumor cells overexpressing the pro-lymphangiogenic growth factor VEGF-C in combination with topically retained immune adjuvants (VEGFC vax). In the B16F10 and B16F10-OVA mouse melanoma models, VEGFC vax induced extensive lymphatic growth at the cutaneous injection site, as assessed by both flow cytometry and histologic analysis. Induced lymphangiogenesis increased the rate of fluorescent microbead transport from the skin to the draining lymph nodes, suggesting an enhanced antigen trafficking. In addition, VEGF-C overexpression drove the recruitment in the vaccine injection site of naïve T-cells (responsive to lymphatic-derived CCL21), which could be primed in situ. Compared to a control VEGF-C-free vaccine formulation, VEGFC vax induced a broader T-cell response targeting multiple mouse melanoma antigens (including tumor neoantigens), as assessed by IFNγ ELISPOT following antigen-specific stimulation. In a prophylactic setting, VEGFC vax provided complete protection against B16F10 melanoma challenge, outperforming both the control vaccine and a GM-CSF-secreting whole-cell vaccine (GVAX). Overall this study provides a proof of concept for the use of VEGF-C as an immunostimulatory agent in cancer vaccines, based on its ability to activate and expand lymphatic vessels and consequently promote local immune cell trafficking, antigen transport and T-cell activation. In addition, we here developed and characterized a whole-cell VEGF-C vaccine formulation with potential for clinical translation. Citation Format: Maria Stella Sasso, Sylvie Hauert, Priscilla Briquez, Yue Wang, Jun Ishihara, Jeffrey A. Hubbell, Melody A. Swartz. Induced lymphangiogenesis enhances antigen-specific T-cell response in anticancer vaccination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B130.

Uptake and accumulation of microplastics in an edible plant

Microplastic (MP, 100 nm−5 mm) may present an attributable risk to ecosystem and human health, and its pollution has become a global environmental concern. Despite a wealth of information on the accumulation of MPs in aquatic species, there is no information on the uptake and accumulation of MPs by higher plants. Terrestrial edible plants are directly exposed to MPs when agricultural soil was applied with organic manure, sewage sludge as fertilizer or plastic mulching. In this paper, the uptake of two sizes of polystyrene (PS) microbeads (0.2 and 1.0 μm) and then their distribution and migration in an edible plant lettuce were firstly investigated based on laboratory experiments. We used fluorescent markers to track PS microbeads in plant tissues and found fluorescence to be a sensitive and reliable detection method. Sections from untreated control lettuce showed no autofluorescence. When roots were treated with fluorescently labeled PS microbeads, the microbeads could be identified by its fluorescence. Our main study investigated the uptake of 0.2 μm beads, as few luminescence signals were observed in lettuce roots for 1.0 μm beads in our experiment. We observed that 0.2 μm fluorescent microbeads were extracellularly trapped in the root cap mucilage (which is a highly hydrated polysaccharide) and a “dark green tip” (which was typical of lettuce roots exposed to label PS beads) was usually visible to the naked eye. Confocal images revealed that the PS luminescence signals were mainly located in the vascular system and on the cell walls of the cortex tissue of the roots, indicated that the beads passed through the intercellular space via the apoplastic transport system. Once inside the central cylinder, the 0.2 μm PS beads were transferred from the roots to the stems and leaves via the vascular system following the transpiration stream. We also observed that the PS beads adhered to one another and self-assembled systematically into “grape-like” and “(chain) string-like” clusters in the intercellular space of the root and stem vascular tissue of lettuce plant. In contrast to the root and stem, PS beads were dispersed in the leaf tissue. Here, for the first time we provide evidence of the adherence, uptake, accumulation, and translocation of submicrometer MPs within an edible plant. Our findings highlight the previously underappreciated human exposure pathway to MPs through the consumption of contaminated crops and emphasize the need for new management strategies to control the release of MPs waste products into the terrestrial environment. Ultimately, the potential impacts of low range sized MPs on food safety of crop plants and human health need to be urgently considered.

The gastric sieve of penaeid shrimp species is a sub-micrometer nutrient filter.

Unlike that of vertebrates, the penaeid shrimp stomach is of ectodermic origin and is thus covered by a cuticle that is sloughed upon molting. It is composed of two chambers, here called the anterior and posterior stomach chambers, ASC and PSC, respectively. The PSC contains a filtration structure variously called a pyloric filter, filter press, gastric filter or gastric sieve (GS), and the last of these will be used here. The GS resembles an elongated, inverted-V, dome-like, chitinous structure with a midline ridge that is integral to the ventral base of the PSC. The dome surface is covered with a carpet-like layer of minute, comb-like setae bearing laterally branching setulae. This carpet serves as a selective filter that excludes large partially digested food particles but allows smaller particles and soluble materials to enter hepatopancreatic ducts that conduct them into the shrimp hepatopancreas (HP), where further digestion and absorption of nutrients takes place. Although the GS function is well known, its exclusion limit for particulate material has not been clearly defined. Using histological and ultra-structure analysis, we show that the GS sieve pore diameter is approximately 0.2-0.7 µm in size, indicating a size exclusion limit of substantially less than 1 µm. Using fluorescent microbeads, we show that particles of 1 µm diameter could not pass through the GS but that particles of 0.1 µm diameter did pass through to accumulate in longitudinal grooves and move on to the HP, where some were internalized by tubule epithelial cells. We found no significant difference in these sizes between the species Penaeus monodon and Penaeus vannamei or between juveniles and adults in P. vannamei This information will be of value for the design of particulate feed ingredients such as nutrients, therapeutic drugs and toxin-absorbing materials that may selectively target the stomach, intestine or HP of cultivated shrimp.

Three-dimensional localization microscopy using deep learning.

Single molecule localization microscopy (SMLM) is one of the fastest evolving and most broadly used super-resolving imaging techniques in the biosciences. While image recordings could take up to hours only ten years ago, scientists are now reaching for real-time imaging in order to follow the dynamics of biology. To this end, it is crucial to have data processing strategies available that are capable of handling the vast amounts of data produced by the microscope. In this article, we report on the use of a deep convolutional neural network (CNN) for localizing particles in three dimensions on the basis of single images. In test experiments conducted on fluorescent microbeads, we show that the precision obtained with a CNN can be comparable to that of maximum likelihood estimation (MLE), which is the accepted gold standard. Regarding speed, the CNN performs with about 22k localizations per second more than three orders of magnitude faster than the MLE algorithm of ThunderSTORM. If only five parameters are estimated (3D position, signal and background), our CNN implementation is currently slower than the fastest, recently published GPU-based MLE algorithm. However, in this comparison the CNN catches up with every additional parameter, with only a few percent extra time required per additional dimension. Thus it may become feasible to estimate further variables such as molecule orientation, aberration functions or color. We experimentally demonstrate that jointly estimating Zernike mode magnitudes for aberration modeling can significantly improve the accuracy of the estimates.

Single microbead-based fluorescence “turn on” detection of biothiols by flow cytometry

Biological thiols (biothiols), such as glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), play a vital role in the process of reversible redox reactions in physiological systems. In this work, flow cytometry-based fluorescent sensor is for the first time developed for the detection of biothiols in a fluorescence “turn on” manner. The probe which we name “Polystyrene/Quantum Dots/Gold Nanoparticles” or (PS/QDs/Au) is constructed by immobilizing QDs onto the surface of PS microbeads to obtain fluorescent microbeads. The probe (PS/QDs/Au) is constructed by immobilizing QDs onto the surface of PS microbeads to obtain fluorescent microbeads, followed by gold NPs absorption through electrostatic interaction to quench their fluorescence. In the presence of biothiols, the fluorescence of our probe can be restored in less than 5 min, and the detection limits for GSH, Cys and Hcy are 0.5 μM, 0.1 μM and 0.3 μM, respectively. Most importantly, the fluorescence signal of each of our probe microbeads can be collected individually by flow cytometry, realizing single microbead-based biothiols detection for the first time. Moreover, the probe is successfully applied to imaging of intracellular biothiols in A549 cells, demonstrating its potential in biological application.

Abstract 17205: Contractility in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes is Highly Dependent on Stochastic Variability in Myofilament Development

Introduction: Modeling heart diseases using cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) enables direct study of cellular and molecular mechanisms. However, the variable phenotypes of individual cells has limited the reproducibility of contractile measurements. We hypothesized that variability in myofibrillar development in hiPSC-CMs influences the contractile kinetics and could be controlled for by visualizing and quantifying myofilaments in vitro . Methods and Results: Control hiPSCs were differentiated into cardiomyocytes (hiPSC-CMs) and purified. hiPSC-CMs were replated at day ~25 onto 8.7 kPa polyacrylamide gels containing fluorescent microbeads and micropatterned with fibronectin in 7:1 aspect ratio rectangles (1750 um 2 ). Single iPSC-CMs grew into the rectangular shapes and exhibited spontaneous contractions by day 2 of replating. hiPSC-CMs were stained with live-cell actin dye and imaged on day 5-7 for fluorescent bead displacements and actin videos depicting myofilaments. Traction force analysis of bead displacements was performed to calculate whole-cell force vs. time curves at 33 msec temporal resolution. Myofilament area was measured by thresholding a binary mask on the actin live cell images. Myofilament number varied markedly among individual iPSC-CMs. iPSC-CMs with few myofilaments (myofilament area / cell area <30%) developed low force (<400 uN per cell) and were not included in subsequent analyses. Among cells with myofilament area >30%, the mean traction force was 1368±357 uN and the myofilament area demonstrated a linear correlation with maximum force (R 2 =0.19, runs test p=0.8) with slope of 0.8±0.2 uN/um 2 (p=0.002 vs zero slope). Maximum contraction velocity strongly correlated with maximum force (R 2 =0.31, runs test p=0.4) with slope of 3.9±0.9 sec -1 (p<0.0001 vs zero slope), as did maximum relaxation velocity (R 2 =0.27, runs test p=0.6, slope 4.1+1.0 sec -1 , p=0.0003 vs zero slope). Similar results were observed in two control cell lines and with two different purification methods. Conclusion: Variability in myofilament development is a critical determinant of contractile force and kinetics in individual iPSC-CMs that should be accounted for in iPSC-CM contractile experiments.

Three-dimensional stimulation and imaging-based functional optical microscopy of biological cells.

A new type of functional optical microscope system called three-dimensional (3D) stimulation and imaging-based functional optical microscopy (SIFOM) is proposed, to the best of our knowledge. SIFOM can precisely stimulate user-defined targeted biological cells and can simultaneously record the volumetric fluorescence distribution in a single acquisition. Precise and simultaneous stimulation of fluorescent-labeled biological cells is achieved by multiple 3D spots generated by digital holograms displayed on a phase-mode spatial light modulator. Single-shot 3D acquisition of the fluorescence distribution is accomplished by common-path off-axis incoherent digital holographic microscopy in which a diffraction grating with a focusing lens is displayed on another phase-mode spatial light modulator. The effectiveness of the proposed functional microscope system was verified in experiments using fluorescent microbeads and human lung cancer cells located at various defocused positions. The system can be used for manipulating the states of cells in optogenetics.

Multi-Parameter Laser Imaging Reveals Complex Microscale Biofilm Matrix in a Thick (4,000 μm) Aerobic Methanol Oxidizing Community

Although methanol has frequently been used as an inexpensive supplementary carbon source to support treatment processes, knowledge of the resultant microbial biofilms, their 3D architecture, microenvironments, exopolymer chemistry and populations remains limited. We supplied methanol as a supplementary carbon source to biofilms developing in rotating annular reactors. Analysis of circulation waters (1.0 l d−1) indicated that dissolved organic carbon was reduced by 25%, NO3-nitrogen by 95%, and total phosphorus by 70%. Analyses of populations using culture based techniques and fluorescence in situ hybridization indicated enrichment of nitrifiers, denitrifiers, and methylotrophic bacteria relative to reference biofilms not receiving methanol. The biofilms that developed were up to 4,000 μm thick. Staining with fluor conjugated lectins in combination with nucleic acid stains, revealed the presence of discrete bacterial cells inside complex globular polymeric structures. These structures were in turn surrounded by an interstitial polymer containing a variety of bacterial cell types. The globular structures bound FITC-conjugated lectins, from Canavalia ensiformis and Ulex europeaus. The FITC-lectin of Phaseolus vulgaris bound the surface of the globular structures and more generally within the matrix. Chemical analyses of the polymer paralleled the results of lectin analyses indicating that the dominant neutral sugars were glucose, galactose, mannose, rhamnose, with fucose and ribose as minor constituents. Amino sugars were not detected. Dual channel imaging with pH sensitive probes indicated that pH gradients from pH 4 to 7 occurred across the globular microcolonies. Critically for the maintenance of aerobic conditions throughout the thick biofilm it was extensively penetrated by a fine fissure network revealed by the location of fluorescent latex microbeads as detected by confocal laser scanning microscopy. Microelectrode studies confirmed the absence of any detectable Eh gradients within the biofilm. However, mobility of various size-fractionated fluorescent probes indicated that the basal region was only penetrated by the lowest molecular weight probes with a hydrated radius of 2.2 nm or less. These observations indicate the selection of a unique, thick (>4,000 μm) microbial community in which a self-organized architecture promotes the maintenance of optimal conditions and metabolism throughout the biofilm community.

Label-Free Monitoring of Microorganisms and Their Responses to Antibiotics Based on Self-Powered Microbead Sensors.

Rapid detection of bacteria and their susceptibility to specific antibiotics plays a vital role in microbial infection treatments. Antimicrobial susceptibility testing (AST) is a common measure to select effective drugs. However, the conventional practices, such as broth dilution, E-test, and disk diffusion, in clinical applications require a long turnaround time (∼3 days), thereby compromising treatments and increasing mortality. This study presents self-powered sensors for on-site microorganism monitoring and rapid AST based on functionalized microbeads. The microbead sensors are driven by Brownian motion, rendering external power unnecessary. Fluorescent microbeads ( dp = 2 μm) were coated with vancomycin to capture bacteria. The growth and responses of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus under antibiotic treatment were evaluated. The method showed stable selective binding despite the presence of some interferential substances, such as proteins and cells. Diffusivity change was strongly related to bacterial concentration. Accordingly, the diffusivity values of microbeads bound with motile and nonmotile bacteria exhibited specific patterns because of extra motility from microbes and increased particle diameter. Only a drop of microbead-bacteria suspension (∼5 μL) was needed in a microchip for each measurement. The microchip provided a steady environment for measurement over a few hours. By distinguishing the slope of the last four data points in the temporal diffusivity curve, bacterial susceptibility or resistance to specific antibiotics could be determined within a time frame of 2 h. The study provides insights into saving more lives by using a fast and robust AST technique in future clinical practice.

Development of micro‐inclined well array for trapping single cells

AbstractWe investigated the shape of a well to trap single cells for the purpose of improving the cell trapping rate after cell seeding and liquid exchange in a cell microarray. The wells having vertical and inclined tapered side‐wall were made using three‐dimensional UV photolithography. Changes in cell trapping rate by the shape of the wells were evaluated by trapping tests with fluorescent microbeads and living cells. As a result of trapping test with living cells, bead residual rate of the microarray with inclined tapered wells is up to 2.9 times higher than that with vertical wells.

Transmission matrix approaches for nonlinear fluorescence excitation through multiple scattering media.

Several matrix approaches were developed to control light propagation through multiple scattering media under illumination of ultrashort pulses of light. These matrices can be recorded with either spectral or temporal resolution. Thanks to wavefront shaping, temporal and spatial refocusing has been demonstrated. In this Letter, we study how these different methods can be exploited to enhance a two-photon excitation fluorescence process. We first compare the different techniques on micrometer-size isolated fluorescent beads. We then demonstrate point-scanning imaging of these fluorescent microbeads located after a thick scattering medium at a depth where conventional imaging would be impossible because of scattering effects.