Ultra-High Resolution 3D Imaging of Whole Cells.

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

Mass cytometry: blessed with the curse of dimensionality.

Immunologists are being compelled to develop new high-dimensional perspectives of cellular heterogeneity and to determine which applications best exploit the power of mass cytometry and associated multiplex approaches.

Breaking the diffraction barrier: super-resolution imaging of cells.

Anyone who has used a light microscope has wished that its resolution could be a little better. Now, after centuries of gradual improvements, fluorescence microscopy has made a quantum leap in its resolving power due, in large part, to advancements over the past several years in a new area of research called super-resolution fluorescence microscopy. In this Primer, we explain the principles of various super-resolution approaches, such as STED, (S)SIM, and STORM/(F)PALM. Then, we describe recent applications of super-resolution microscopy in cells, which demonstrate how these approaches are beginning to provide new insights into cell biology, microbiology, and neurobiology.

Repeatability and reproducibility of microscopic examination of adhesive tape strip cytology slides for the quantification of Malassezia spp. in canine skin.

BACKGROUND: The optimal microscopic magnification and number of optical fields of adhesive tape strip cytological slides that should be examined when searching for Malassezia yeasts on canine skin are unknown. OBJECTIVES: To determine the optimal magnification and the minimum number of optical fields that should be examined to maximise intraobserver repeatability and interobserver reproducibility. MATERIALS AND METHODS: Seven experienced examiners counted, twice, the number of yeasts in 10, 20, 30, 40 and 50 optical fields of 40 slides at ×400 and ×1000 magnification. RESULTS: The number of yeasts per unit surface area was significantly higher at ×1000 compared to ×400 magnification. Repeatability and reproducibility for counting the yeasts was very poor. CONCLUSIONS AND CLINICAL RELEVANCE: Adhesive tape strip cytological slides should be examined microscopically for Malassezia spp. at ×1000 magnification. The repeatability of this examination for counting the yeasts is poor.

Contexte - Le grossissement microscopique optimal et le nombre de champs optiques des lames cytologiques de bandes adhésives à examiner lors de la recherche de levures Malassezia sur la peau de chien sont inconnus. Objectifs - Déterminer le grossissement optimal et le nombre minimal de champs à examiner pour maximiser la répétabilité intra-observateur et la reproductibilité inter-observateur. Matériels et méthodes - Sept examinateurs expérimentés ont compté, deux fois, le nombre de levures dans 10, 20, 30, 40 et 50 champs de 40 lames aux grossissements ×400 et ×1 000. Résultats - Le nombre de levures par unité de surface était significativement plus élevé au grossissement ×1 000 par rapport au grossissement ×400. La répétabilité et la reproductibilité du comptage des levures étaient très médiocres. Conclusions et pertinence clinique - Les lames cytologiques de bandes adhésives doivent être examinées au microscope pour Malassezia spp. à un grossissement ×1 000. La répétabilité de cet examen de comptage des levures est faible.

Introducción- se desconoce el aumento microscópico óptimo y el número de campos ópticos de los portaobjetos citológicos en tiras de cinta adhesiva que deben examinarse al buscar levaduras Malassezia en la piel canina. Objetivos- determinar el aumento óptimo y el número mínimo de campos ópticos que deben examinarse para maximizar la repetibilidad intraobservador y la reproducibilidad interobservador. Materiales y métodos- siete examinadores experimentados contaron dos veces el número de levaduras en campos ópticos de 10, 20, 30, 40 y 50 de 40 portaobjetos con aumentos de x ×400 y ×1000. Resultados- el número de levaduras por unidad de superficie fue significativamente mayor con un aumento de ×1000 en comparación con un aumento de ×400. La repetibilidad y reproducibilidad para contar las levaduras fue muy pobre. Conclusiones y relevancia clínica - Los portaobjetos citológicos en tiras de cinta adhesiva deben examinarse microscópicamente para detectar Malassezia spp. con un aumento de ×1.000. La repetibilidad de este examen para contar las levaduras es pobre.

Contexto - A ampliação microscópica ideal e o número de campos ópticos das lâminas citológicas de fita adesiva que devem ser examinados nas pesquisas de leveduras do gênero Malassezia em cães são desconhecidos. Objetivos - Determinar a magnificação ideal e o número mínimo de campos ópticos que devem ser examinados para maximizar a repetibilidade intraobservador e a reproducibilidade interobservador. Materiais e métodos - Sete examinadores experientes contaram duas vezes o número de leveduras em 10, 20, 30, 40 e 50 campos ópticos de 40 lâminas nas magnificações de x400 e x1000. Resultados - O número de leveduras por unidade de área de superfície foi significativamente maior em x1000 em comparação com a ampliação de x400. A repetibilidade e a reprodutibilidade para a contagem de leveduras foi muito pobre. Conclusões e relevância clínica - Lâminas de citologia por fica adesiva devem ser examinadas microscopicamente para Malassezia spp a uma magnificação de x1.000. A repetibilidade deste exame para contagem de leveduras foi pobre.

An impedimetric assay for the identification of abnormal mitochondrial dynamics in living cells.

Mitochondrial dynamics (fission and fusion) plays an important role in cell functions. Disruption in mitochondrial dynamics has been associated with diseases such as neurobiological disorders and cardiovascular diseases. Analysis of mitochondrial fission/fusion has been mostly achieved through direct visualization of the fission/fusion events in live-cell imaging of fluorescently labeled mitochondria. In this study, we demonstrated a label-free, non-invasive Electrical Impedance Spectroscopy (EIS) approach to analyze mitochondrial dynamics in a genetically modified human neuroblastoma SH-SY5Y cell line with no huntingtin protein expression. Huntingtin protein has been shown to regulate mitochondria dynamics. We performed EIS studies on normal SH-SY5Y cells and two independent clones of huntingtin-null cells. The impedance data was used to determine the suspension conductivity and further cytoplasmic conductivity and relate to the abnormal mitochondrial dynamics. For instance, the cytoplasm conductivity value was increased by 11% from huntingtin-null cells to normal cells. Results of this study demonstrated that EIS is sensitive to characterize the abnormal mitochondrial dynamics that can be difficult to quantify by the conventional microscopic method.

Microfluidic chambers using fluid walls for cell biology.

Many proofs of concept have demonstrated the potential of microfluidics in cell biology. However, the technology remains inaccessible to many biologists, as it often requires complex manufacturing facilities (such as soft lithography) and uses materials foreign to cell biology (such as polydimethylsiloxane). Here, we present a method for creating microfluidic environments by simply reshaping fluids on a substrate. For applications in cell biology, we use cell media on a virgin Petri dish overlaid with an immiscible fluorocarbon. A hydrophobic/fluorophilic stylus then reshapes the media into any pattern by creating liquid walls of fluorocarbon. Microfluidic arrangements suitable for cell culture are made in minutes using materials familiar to biologists. The versatility of the method is demonstrated by creating analogs of a common platform in cell biology, the microtiter plate. Using this vehicle, we demonstrate many manipulations required for cell culture and downstream analysis, including feeding, replating, cloning, cryopreservation, lysis plus RT-PCR, transfection plus genome editing, and fixation plus immunolabeling (when fluid walls are reconfigured during use). We also show that mammalian cells grow and respond to stimuli normally, and worm eggs develop into adults. This simple approach provides biologists with an entrée into microfluidics.

New Concept and Apparatus for Cytocentrifugation and Cell Processing for Microscopy Analysis.

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.

Towards a perceptive understanding of size in cellular biology.

Cells are minute-typically too small to be seen by the human eye. Even so, the cellular world encompasses a range of scales, from roughly a tenth of a nanometer (10-10 m) to a millimeter (10-3 m) or larger, spanning seven orders of magnitude or more. Because they are so far from our experience, it is difficult for us to envision such scales. To help our imagination grasp such dimensions, I propose the adoption of a 'perceptive scale' that can facilitate a more direct experience of cellular sizes. From this, as I argue below, will stem a new perception also of biological shape, cellular space and dynamic processes.

Exosome aggregation mediated stop-flow paper-based portable device for rapid exosome quantification.

Exosome quantification is important for estimation of informative messengers (e.g., proteins, lipids, RNA, etc.) involving physiological and pathological effects. This work aimed to develop a simple and rapid distance-based paper portable device using exosome-capture vesicles (polydiacetylene conjugated with antiCD81) for exosome quantification in cell cultures. This novel concept relied on distinct aggregation of exosomes and exosome-capture vesicles leading to different solvent migration. Distances of the migration were used as signal readouts, which could be detected by naked eye. PDA-antiCD81 as exosome-capture vesicles were optimized (e.g., size, reaction ratio, and concentration) and the paper designs were investigated (e.g., diameter of sample reservoir and lamination layer) to enhance the solvent stop-flow effects. Finally, exosome screening on three cell culture samples (COLO1, MDA-MB-231, and HuR-KO1 subclone) was demonstrated. The method could linearly measure exosome concentrations in correlation with solvent migration distances in the range of 106 -1010 particles/mL (R2  > 0.98) from the cell culture samples. The exosome concentration measurements by the developed device were independently assessed by nanoparticle tracking analysis. Results demonstrated no statistically significant difference (p > 0.05) by t-test. This low-cost and rapid device allows a portable platform for exosome quantification without the requirement of expensive equipment and expertise of operation. The developed device could potentially be useful for quantification of other biomarker-related extracellular vesicles.

Label-free characterization of different kinds of cells using optoelectrokinetic-based microfluidics.

We report a novel, to the best of our knowledge, method to rapidly characterize different kinds of cells and drug-treated cancer cells using a label-free biomarker of self-rotation in an optoelectrokinetics (OEK)-based microfluidic platform. OEK incorporates optics and electrokinetics into microfluidics, thereby offering a contact-free, label-free, and rapid approach to the cellular manipulation community. Self-rotational behaviors of four different kinds of cells were experimentally investigated by the frequency-sweeping of an AC bias potential in an optically induced nonuniform and irrotational electric field. The results revealed that these kinds of cells displayed a Gaussian distribution versus the AC frequency as well as different self-rotational speeds under the same conditions. Furthermore, the peak self-rotational speed varied from one kind of cell to another, with that of cancer cells higher than that of normal cells. In addition, MCF-7 cells treated by various concentrations of drug showed remarkably different self-rotational speeds. This finding suggests a high potential of developing a new label-free biomarker to rapidly distinguish different kinds of cancer cells and quantitatively monitor the response of cancer patients to various treatments.

Three-tissue microphysiological system for studying inflammatory responses in gut-liver Axis.

The interaction between the gut and the liver, often known as the gut-liver axis, play crucial roles in modulating the body's responses to the xenobiotics as well as progression of diseases. Dysfunction of the axis can cause metabolic disorders as well as obesity, diabetes, and fatty liver disease. During the progression of such diseases, inflammatory responses involving the immune system also play an important part. In this study, we developed a three-tissue microphysiological system (MPS) that can accommodate three different cell types in separated compartments connected via fluidic channels in a microfluidic device. Using computational fluid dynamics, geometry of fluidic channels and flow conditions were optimized for seeding and culturing different cell types in the three-tissue MPS. Caco-2 (gut), RAW264.7 (immune), and HepG2 (liver) cells were seeded and cultured in the chip. Stimulation of the gut cells in the MPS with lipopolysaccharide (LPS) resulted in induction of inflammatory response and production of nitric oxide (NO) in all connected chambers. The anti-inflammatory effect of luteolin was demonstrated. Our study demonstrates that the three-tissue MPS can recapitulate the inflammatory responses involving the gut, liver and immune cells.

High-throughput cell migration assay under combinatorial chemical environments by a novel 24-well-plate based device.

The quantitative studies of cell proliferation and migration under different chemical environments are important for both scientists and clinicians searching for new therapeutics. In this study, we developed a new device to pattern several types of cells in 24-well-plate and demonstrated its' application in cancer cell proliferation and migration assay. The new device combined 3D-printed-silica-part for multi cell types loading with PDMS-through-hole-layer-part for cell micro-patterning which was matched with commercial 24-well-plate. This 24-well-plate based device is flexible and feasible in many applications and can be used in one piece or multi pieces. Besides the application for two types of cells proliferation and migration assay in one chemical condition, as a demonstration, the migration behaviors of four types of cells under 24 types of EGF + bFGF combinatorial conditions were studied. We believed this device could be widely used in clinical searching for new anti-cancer therapeutics and other related studies.

Magnetic Patterning of Vorticella convallaria in a Microfluidic Device.

We describe an inexpensive magnetic cell patterning method as a tool for protozoologists. The ciliate Vorticella convallaria is useful for various biofluidics applications. Here, we show that V. convallaria will ingest metal beads and that permanent magnets can be used to pattern cells in Petri dishes or a microfluidic device. Patterning is reversibly achieved by placing magnets at the point of desired cell attachment. Analogous magnetic manipulation could be performed using other phagocytic cells.

Cells Under Stress: An Inertial-Shear Microfluidic Determination of Cell Behavior.

The deformability of a cell is the direct result of a complex interplay between the different constituent elements at the subcellular level, coupling a wide range of mechanical responses at different length scales. Changes to the structure of these components can also alter cell phenotype, which points to the critical importance of cell mechanoresponse for diagnostic applications. The response to mechanical stress depends strongly on the forces experienced by the cell. Here, we use cell deformability in both shear-dominant and inertia-dominant microfluidic flow regimes to probe different aspects of the cell structure. In the inertial regime, we follow cellular response from (visco-)elastic through plastic deformation to cell structural failure and show a significant drop in cell viability for shear stresses >11.8 kN/m2. Comparatively, a shear-dominant regime requires lower applied stresses to achieve higher cell strains. From this regime, deformation traces as a function of time contain a rich source of information including maximal strain, elastic modulus, and cell relaxation times and thus provide a number of markers for distinguishing cell types and potential disease progression. These results emphasize the benefit of multiple parameter determination for improving detection and will ultimately lead to improved accuracy for diagnosis. We present results for leukemia cells (HL60) as a model circulatory cell as well as for a colorectal cancer cell line, SW480, derived from primary adenocarcinoma (Dukes stage B). SW480 were also treated with the actin-disrupting drug latrunculin A to test the sensitivity of flow regimes to the cytoskeleton. We show that the shear regime is more sensitive to cytoskeletal changes and that large strains in the inertial regime cannot resolve changes to the actin cytoskeleton.

Safety and Acceptability of Esophageal Cytosponge Cell Collection Device in a Pooled Analysis of Data From Individual Patients.

BACKGROUND & AIMS: Diagnosis and surveillance of Barrett's esophagus (BE) and eosinophilic esophagitis (EoE) have become emerging public health issues. Cytosponge is a novel, minimally invasive esophageal cell collection device. We aimed to assess the data on safety and acceptability of this device. METHODS: We performed a patient-level review of 5 prospective trials assessing Cytosponge performance in patients with reflux disease, BE and EoE in primary and secondary care. Acceptability of Cytosponge and subsequent endoscopy were recorded with visual analogue scale (VAS), wherein 0 and 10 denoted lowest and highest acceptability. Median VAS scores were compared using a Mann-Whitney test. The number of attempts, failures in swallowing the device and occurrence of adverse events were analyzed. Risk factors for failure in swallowing were analyzed using a multivariate regression model. RESULTS: In total, 2672 Cytosponge procedures were performed, in 2418 individuals from 2008 through 2017. There were 2 adverse events related to the device: a minor pharyngeal bleed and a case of detachment (<1:2000). The median acceptability score for the Cytosponge was 6.0 (interquartile range [IQR], 5.0-8.0), which was higher than the score for endoscopy without sedation (median 5.0; IQR, 3.0-7.0; P < .001) and lower than the score for endoscopy with sedation (median 8.0; IQR, 5.0-9.0; P < .001). Nearly all patients (91.1%) successfully swallowed the Cytosponge, most on the first attempt (90.1%). Failure to swallow the device was more likely to occur in secondary care (odds ratio, 5.13; 95% CI, 1.48-17.79; P < .01). CONCLUSIONS: The Cytosponge test is a safe procedure with good acceptability ratings in a variety of health care settings.

Flow force augmented 3D suspended polymeric microfluidic (SPMF3 ) platform.

Detection and study of bioelements using microfluidic systems has been of great interest in the biodiagnostics field. Microcantilevers are the most used systems in biodetection due to their implementation simplicity which have been used for a wide variety of applications ranging from cellular to molecular diagnosis. However, increasing further the sensitivity of the microcantilever systems have a great effect on the cantilever based sensing for chemical and bio applications. In order to improve further the performance of microcantilevers, a flow force augmented 3D suspended microchannel is proposed using which microparticles can be conveyed through a microchannel inside the microcantilever to the detection area. This innovative microchannel design addresses the low sensitivity issue by increasing its sensitivity up to 5 times than the earlier reported similar microsystems. Moreover, fabricating this microsystem out of Polydimethylsiloxane (PDMS) would eliminate external exciter dependency in many detection applications such as biodiagnostics. In this study, the designed microsystem has been analyzed theoretically, simulated and tested. Moreover, the microsystem has been fabricated and tested under different conditions, the results of which have been compared with simulation results. Finally, its innovative fabrication process and issues are reported and discussed.

Continuous flow microfluidic cell inactivation with the use of insulating micropillars for multiple electroporation zones.

Electroporation is a powerful tool for inactivating cells and transfecting biological cells and has applications in biology, genetic engineering, medicine, environment, and many others. We report a new continuous flow device embedded with insulating micropillars to achieve better performance of cell inactivation. The use of micropillars creates multiple electroporation zones with enhanced local electric field strengths. Using a model solution of Saccharomyces cerevisiae, we examined the inactivation performance of the device under various applied electric voltages and flow rates. Results from the numerical simulations and experiments showed that even with an induced transmembrane potential of 0.58 V, close to 63% of cell inactivation was achieved at a flow rate of 2.5 mL/h. This was higher than the 24% cell inactivation observed for a reference device without micropillars that was subjected to the same conditions.

Automatic detecting and counting magnetic beads-labeled target cells from a suspension in a microfluidic chip.

A novel microfluidic method of continually detecting and counting beads-labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre-labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T-lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell-magnetic beads complex for beads-labeled cell detection is simple, automatic, and particularly suitable for beads-based immunoassay without using fluorescence labeling.

Micropillar-based microfluidic device to regulate neurite networks of uniform-sized neurospheres.

The inability of neurons to undergo mitosis renders damage to the central or peripheral nervous system. Neural stem cell therapy could provide a path for treating the neurodegenerative diseases. However, reliable and simple tools for the developing and testing neural stem cell therapy are still required. Here, we show the development of a micropillar-based microfluidic device to trap the uniform-sized neurospheres. The neurospheres trapped within micropillar arrays were largely differentiated into neuronal cells, and their neurite networks were observed in the microfluidic device. Compared to conventional cultures on glass slides, the neurite networks generated with this method have a higher reproducibility. Furthermore, we demonstrated the effect of thapsigargin on the neurite networks in the microfluidic device, demonstrating that neural networks exposed to thapsigargin were largely diminished and disconnected from each other. Therefore, this micropillar-based microfluidic device could be a potential tool for screening of neurotoxins.

Ejection of cell laden RPMI-1640 culture medium by Electrohydrodynamic method.

Sample deposition based on micro-droplet ejection has broad application prospects in the field of biomedicine. Ejection of RPMI-1640 medium (with and without cells) is investigated experimentally using a home-build electrohydrodynamic (EHD) ejection system, consisting of a liquid supplier and a nozzle, a high voltage source, a droplet collector, and a high speed photography module. High electric voltage is applied between the nozzle and the droplet collector. The liquid surface is electrically charged and the ejection takes place when electric force overcomes the surface tension. The ejection process is studied by using high speed photography and image processing. At low voltage, a stable ejection state is established with ejection frequency ranging from a few to a few tens of Hertz. At high voltage, another stable ejection state is reached with ejection frequency as high as 1300 Hz. At the transition voltage range, the ejection exhibits a periodic behaviour. During each cycle, the meniscus rapidly oscillates with gradually increased amplitude, and with several non-uniform droplets ejected at the final stage of the cycle. Human peripheral blood mononuclear cells, after ejection, shows survival rates higher than 79%, manifesting EHD ejection as a promising technique for cell printing.