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Multiparameter Lab-on-a-Chip flow cytometry of the cell cycle
Abstract
Multiparameter analysis of apoptosis in relation to cell cycle position is helpful in exploring mechanism of action of anticancer drugs that target specific molecular cogs of the cell cycle. This work demonstrates a new rationale for using microfluidic Lab-on-a-Chip flow cytometry (μFCM) with a simple 2D hydrodynamic focusing for the multiparameter analysis of apoptosis and DNA ploidy analysis in human hematopoietic cancer cells. The microfluidic system employs disposable microfluidic cartridges fabricated using injection moulding in optically transparent poly(methylmethacrylate). The dedicated and miniaturized electronic hardware interface enables up to six parameter detections using a combination of spatially separated solid-state 473nm (10mW) and 640nm (20mW) lasers and x-y stage for rapid laser alignment adjustment. We provide evidence that the simple 2D flow focusing on a chip-based device is sufficient to measure cellular DNA content in both fixed and living tumor cells. The feasibility of using the μFCM system for multiparameter analysis of caspase activation and dissipation of mitochondrial inner membrane potential (ΔΨ(m) loss) in relation to DNA content is also demonstrated. The data shows that straightforward microfluidic chip designs are sufficient to acquire high quality biological data when combined with sophisticated electronic interfaces. They can be a viable alternative to conventional FCM for multiparameter detection of programmed cell death.
... One of the most exciting applications of microfluidic, chip-based cytometers is multivariate analysis using sampling volumes as small as 10 μl while matching the multiparameter single-cell data collection of conventional cytometers Wlodkowic and Darzynkiewicz, 2011). In this unit, we outline several innovative protocols to analyze caspase-dependent cell death and also cell cycle (DNA-content) profile using a fully integrated microfluidic flow cytometry system, Fishman-R (Akagi et al., 2012Skommer et al., 2013;Fig. 9.42.1). ...
... 9.42.1). This enabling technology features a user-friendly interface and utilizes disposable polymeric cartridges with a simple 2-D hydrodynamic cell focusing system Akagi et al., 2012;Skommer et al., 2013). Despite the simplicity of chip design and operation, the microfluidic cytometer can perform up to 6-parameter multivariate analysis using very small sampling volumes ( Fig. 9.42.1). ...
... Despite the simplicity of chip design and operation, the microfluidic cytometer can perform up to 6-parameter multivariate analysis using very small sampling volumes ( Fig. 9.42.1). This unit outlines a new rationale for using μFCM, in conjunction with single-step staining protocols for the multi-parameter analysis of apoptosis and DNA ploidy analysis in human hematopoietic cancer cells (Akagi et al., 2012Skommer et al., 2013). Every protocol describes a simple and time-saving assay for quick assessment of caspase-depended cell death. ...
The age of microfluidic flow cytometry (µFCM) is fast becoming a reality. One of the most exciting applications of miniaturized chip-based cytometers is multivariate analysis using sampling volumes as small as 10 µl while matching the multiparameter data collection of conventional flow cytometers. We outline several innovative protocols for analyzing caspase-dependent cell death and cell cycle (DNA-content) profile using a fully integrated microfluidic flow cytometry system, Fishman-R. The first protocol describes the use of a new plasma membrane-permeability marker, DRAQ7, and the fluorogenic caspase substrate PhiPhiLux to track caspase activation during programmed cell death. Also outlined is the use of DRAQ7 fluorochrome in conjunction with the mitochondrial membrane potential-sensitive probe TMRM to track dissipation of inner mitochondrial cross-membrane potential. Another protocol adds the ability to measure dissipation of mitochondrial inner membrane potential (using TMRM probe) in relation to the cell cycle profile (using DRAQ5 probe) in living leukemic cells. Finally, we describe the combined use of fluorogenic caspases substrate PhiPhiLux with DRAQ5 probe to measure caspase activation in relation to the cell cycle profile in living tumor cells. Curr. Protoc. Cytom. 66:9.42.1-9.42.15. © 2013 by John Wiley & Sons, Inc.
... This allows for correlative studies between several different endpoints. As an example, activation of specific enzymes can be precisely correlated to other discrete biomarkers such as immunophenotype, DNA damage, mitochondrial function, expression of diverse proteins or position in the cell cycle Zhao et al., 2012;O'Donnell et al., 2013;Skommer et al., 2013;Wlodkowic et al., 2022). The HCA has historically been used for cell-based assays but there are recent examples of its successful applications in tissue microarrays and in situ screening using genetically tractable small model organisms such as nematode (Caenorhabditis elegans) and zebrafish (Danio rerio) (Vogt et al., 2010;Lantz-McPeak et al., 2015). ...
... The multiplexed assays allow deeply correlative studies between the above parameters where e.g. mitochondrial function, generation of ROS or induction of PCD can be interrelated to other discrete markers such as position within the cell cycle and/or immunophenotype (Darzynkiewicz and Andreeff, 1981;Burchiel et al., 1997;Wlodkowic et al., 2008;Akagi et al., 2013b;O'Donnell et al., 2013;Skommer et al., 2013;Wlodkowic et al., 2022). ...
The rapidly increasing number of new production chemicals coupled with stringent implementation of global chemical management programs necessities a paradigm shift towards boarder uses of low-cost and high-throughput ecotoxicity testing strategies as well as deeper understanding of cellular and sub-cellular mechanisms of ecotoxicity that can be used in effective risk assessment. The latter will require automated acquisition of biological data, new capabilities for big data analysis as well as computational simulations capable of translating new data into in vivo relevance. However, very few efforts have been so far devoted into the development of automated bioanalytical systems in ecotoxicology. This is in stark contrast to standardized and high-throughput chemical screening and prioritization routines found in modern drug discovery pipelines. As a result, the high-throughput and high-content data acquisition in ecotoxicology is still in its infancy with limited examples focused on cell-free and cell-based assays.
In this work we outline recent developments and emerging prospects of high-throughput bioanalytical approaches in ecotoxicology that reach beyond in vitro biotests. We discuss future importance of automated quantitative data acquisition for cell-free, cell-based as well as developments in phytotoxicity and in vivo biotests utilizing small aquatic model organisms. We also discuss recent innovations such as organs-on-a-chip technologies and existing challenges for emerging high-throughput ecotoxicity testing strategies. Lastly, we provide seminal examples of the small number of successful high-throughput implementations that have been employed in prioritization of chemicals and accelerated environmental risk assessment.
... A further advantage of microfabricated devices is that they can be easily integrated with existing laboratory instrumentation. This leads to platforms that enable sophisticated cellular analyses and/or the isolation of target cells for post-processing (Skommer et al. 2013;Wolff et al. 2003). ...
... Page 2 of 13 to laterally focus it down to a size of 10′s μm (Skommer et al. 2013). This concept features in many applications where detection of the 'identification' signal used for the sorting process is easy due to its magnitude (e.g., strong fluorescence signals). ...
Optical-based microfluidic cell sorting has become increasingly attractive for applications in life and environmental sciences due to its ability of sophisticated cell handling in flow. The majority of these microfluidic cell sorting devices employ two-dimensional fluid flow control strategies, which lack the ability to manipulate the position of cells arbitrarily for precise optical detection, therefore resulting in reduced sorting accuracy and purity. Although three-dimensional (3D) hydrodynamic devices have better flow-focusing characteristics, most lack the flexibility to arbitrarily position the sample flow in each direction. Thus, there have been very few studies using 3D hydrodynamic flow focusing for sorting. Herein, we designed a 3D hydrodynamic focusing sorting platform based on independent sheath flow-focusing and pressure-actuated switching. This design offers many advantages in terms of reliable acquisition of weak Raman signals due to the ability to precisely control the speed and position of samples in 3D. With a proof-of-concept demonstration, we show this 3D hydrodynamic focusing-based sorting device has the potential to reach a high degree of accuracy for Raman activated sorting.
... Screening tumor biopsies, immunophenotyping, and DNA content analysis are additional applications where flow cytometry is used in cancer diagnostics. 14,34,39 Although flow cytometry is a reliable tool for immunological and hematological based diagnostics, its potential and utility is limited to large centralized institutions due to its many specialized requirements. A bulky footprint, high equipment cost, large consumption of samples and reagents, and requirement for highly skilled personnel for operation and maintenance significantly diminish the conventional flow cytometer's utility as a POC device. ...
... Our device's S value of 4.28 indicates sensitivity within acceptable range as defined by Woods (>3 is acceptable). 34 These results demonstrate that our microfluidic flow cytometer is able to resolve dim fluorescence even down to 486 MESF value (dimmest peak) while maintaining satisfactory linearity at higher staining levels. ...
Cost-effective, high-performance diagnostic instruments are vital to providing the society with accessible, affordable, and high-quality healthcare. Here we present an integrated, “microfluidic drifting” based flow cytometry chip as a potential inexpensive, fast, and reliable diagnostic tool. It is capable of analyzing human blood for cell counting and diagnosis of diseases. Our device achieves a throughput of ~3754 events/s. Calibration with Flow-Check calibration beads indicated good congruency with a commercially available benchtop flow cytometer. Moreover, subjection to a stringent 8-peak rainbow calibration particle test demonstrated its ability to perform high-resolution immunological studies with separation resolution of 4.28 between the two dimmest fluorescent populations. Counting accuracy at different polystyrene bead concentrations showed strong correlation (r = 0.9991) with hemocytometer results. Finally, reliable quantification of CD4+ cells in healthy human blood via staining with monoclonal antibodies was demonstrated. These results demonstrate the potential of our microfluidic flow cytometry chip as an inexpensive yet high-performance point-of-care device for mobile medicine.
... Wang et al. present a microfluidic system to capture and monitor C. elegans on a chip and assess the influence of microgravity through fluorescence and motional imaging [ Fig. 4(c)]. [47][48][49][50]57 The result suggests potential muscle atrophy and compromised nematode physiological activity induced by long-term microgravity. During a long-term space trip, it is necessary to monitor the astronauts' physiological conditions, which can be accomplished by daily multi-parametric tests conducted with wearable microfluidic systems such as watches, contact lenses, smart skins, and masks, taking sweat, tears, blood, and exhaled gas as samples for analysis [ Fig. 4(d)]. ...
Numerous revolutionary space missions have been initiated and planned for the following decades, including plans for novel spacecraft, exploration of the deep universe, and long duration manned space trips. Compared with space missions conducted over the past 50 years, current missions have features of spacecraft miniaturization, a faster task cycle, farther destinations, braver goals, and higher levels of precision. Tasks are becoming technically more complex and challenging, but also more accessible via commercial space activities. Remarkably, microfluidics has proven impactful in newly conceived space missions. In this review, we focus on recent advances in space microfluidic technologies and their impact on the state-of-the-art space missions. We discuss how micro-sized fluid and microfluidic instruments behave in space conditions, based on hydrodynamic theories. We draw on analyses outlining the reasons why microfluidic components and operations have become crucial in recent missions by categorically investigating a series of successful space missions integrated with microfluidic technologies. We present a comprehensive technical analysis on the recently developed in-space microfluidic applications such as the lab-on-a-CubeSat, healthcare for manned space missions, evaluation and reconstruction of the environment on celestial bodies, in-space manufacturing of microfluidic devices, and development of fluid-based micro-thrusters. The discussions in this review provide insights on microfluidic technologies that hold considerable promise for the upcoming space missions, and also outline how in-space conditions present a new perspective to the microfluidics field.
... Moreover, LOC facilitates the integration of many active and miniaturised analytical components such as micro-valves, solid state light emitting diodes and electrodes. A highly innovative avenue with an LOC system that has not been thus far explored is the utilisation of microflow cytometry (µFC) [131][132][133]. This technology represents a vastly miniaturised version of conventional FC. ...
Continuous monitoring and early warning of potential water contamination with toxic chemicals is of paramount importance for human health and sustainable food production. During the last few decades there have been noteworthy advances in technologies for the automated sensing of physicochemical parameters of water. These do not translate well into online monitoring of chemical pollutants since most of them are either incapable of real-time detection or unable to detect impacts on biological organisms. As a result, biological early warning systems have been proposed to supplement conventional water quality test strategies. Such systems can continuously evaluate physiological parameters of suitable aquatic species and alert the user to the presence of toxicants. In this regard, single cellular organisms, such as bacteria, cyanobacteria, micro-algae and vertebrate cell lines, offer promising avenues for development of water biosensors. Historically, only a handful of systems utilising single-cell organisms have been deployed as established online water biomonitoring tools. Recent advances in recombinant microorganisms, cell immobilisation techniques, live-cell microarrays and microfluidic Lab-on-a-Chip technologies open new avenues to develop miniaturised systems capable of detecting a broad range of water contaminants. In experimental settings, they have been shown as sensitive and rapid biosensors with capabilities to detect traces of contaminants. In this work, we critically review the recent advances and practical prospects of biological early warning systems based on live-cell biosensors. We demonstrate historical deployment successes, technological innovations, as well as current challenges for the broader deployment of live-cell biosensors in the monitoring of water quality.
... By this manner higher accurate methodologies for fabrication of In Flow channels within the nanoscale were developed and they are currently used in highly accurate and confined studies. Moreover, new potential In Flow studies based on individual Biostructures tracking, analysis and counting based on Bacteria labelling with Ultraluminescent gold Core-shell Nanoparticles [12] and genetically modified Fluorescent Bacteria grafting with silver nanoparticles [13] by MEF Imaging detections would be of high impact as well ( Figure 2). ...
... analytes were the detection antibodies) sensitivity was given in terms of amount of bound fluorophores detectable, with an LOD of ~2.6 × 10 4 fluorescent molecules per microbead. While multi-wavelength flow cytometers require a more complicated optical setup, they also enable multiparameter analysis of complex phenomena, such as the determining the cell cycle position and detecting apoptosis as determined by DNA content, mitochondrial membrane potential, and caspase activation 151 . ...
Flow cytometry is an invaluable tool utilized in modern biomedical research and clinical applications requiring high throughput, high resolution particle analysis for cytometric characterization and/or sorting of cells and particles as well as for analyzing results from immunocytometric assays. In recent years, research has focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or point-of-care diagnostics especially in limited resource facilities or locations requiring on-site analyses. However, designing a device that fulfills the criteria of high throughput analysis, automation and portability, while not sacrificing performance is not a trivial matter. This review intends to present the current state of the field and provide considerations for further improvement by focusing on the key design components of microfluidic flow cytometers. The recent innovations in particle focusing and detection strategies are detailed and compared. This review outlines performance matrix parameters of flow cytometers that are interdependent with each other, suggesting trade offs in selection based on the requirements of the applications. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate and cost-effective flow cytometers.
... These nanostructures combine the biocompatible plasmonic properties of gold nanoparticles with the dielectric properties from the silica shell, a platform for bioconjugation with targeted applications. In a next step, we propose to use this approach in cytometry and application of microfluidic devices 24,25 with detection based on nanoimaging. 26 ...
Gold core-shell nanoparticles were synthesized based on metallic cores, variable silica shell spacers covered with modified fluorescent silica layers. Ultraluminescent properties were obtained based on metal-enhanced fluorescence (MEF). Different silica spacers were synthesized to optimize the MEF enhancement factor (MEFEF). An optimal MEFEF was determined equal to 9.5 for shorter silica spacers (d-SiO2-=10 nm). These nanoparticles were deposed on Escherichia coli bacteria at different concentration levels for Bioimaging generation over their surfaces. The best luminescent nanoparticles were deposed on intermediate and higher bacteria concentrations. In the presence of intermediate bacteria concentrations, the ultraluminescent nanoparticles adsorbed showed an increase of 35% to 45% compared with individual nanoparticles. To modify the surface of individual bacteria, diluted samples of bacteria were used in which a 20% decrease in fluorescence emission was measured. In the presence of higher bacteria concentrations, fewer clear and bright images were obtained. At diluted ultraluminescent nanoparticle concentrations, a decrease in brightness and image detail was observed; and in the absence of nanoparticle deposition, no image was recorded. Accordingly, these ultraluminescent gold core-shell nanoparticles have been shown to be useful as platforms for biodetection and tracking applications. © 2017 Society of Photo-Optical Instrumentation Engineers (SPIE).
... Moreover, the size of the bubble can be adjusted to enable hydrodynamic patterning of suspended particles through the main channel. Such a patterning is desirable in many flow cytometry investigations 37 . This phenomenon can be achieved when a large bubble is generated in the main channel, leaving a narrow gap, approximately the same size of the suspended particles, between the bubble and the channel walls. ...
Recently, the bubble-based systems have offered a new paradigm in microfluidics. Gas bubbles are highly flexible, controllable and barely mix with liquids, and thus can be used for the creation of reconfigurable microfluidic systems. In this work, a hydrodynamically actuated bubble-based microfluidic system is introduced. This system enables the precise movement of air bubbles via axillary feeder channels to alter the geometry of the main channel and consequently the flow characteristics of the system. Mixing of neighbouring streams is demonstrated by oscillating the bubble at desired displacements and frequencies. Flow control is achieved by pushing the bubble to partially or fully close the main channel. Patterning of suspended particles is also demonstrated by creating a large bubble along the sidewalls. Rigorous analytical and numerical calculations are presented to describe the operation of the system. The examples presented in this paper highlight the versatility of the developed bubble-based actuator for a variety of applications; thus providing a vision that can be expanded for future highly reconfigurable microfluidics.
... The device utilized integrated optical fibres for laser excitation and detection of fluorescent signals. In another study, Skommer et al. pioneered the use of a μFCM system to measure cellular DNA content in both live and fixed tumour cells, and also to track the drug-induced activation of caspases and the dissipation of mitochondrial inner membrane potential (Δψ m loss) with respect to cell cycle stage of tumour cells (Skommer et al., 2013) (Fig. 3A-B). Despite the essentially simple design, the system incorporated a dedicated hardware interface comprising of a microcontroller-driven syringe pump, spatially separated laser excitation sources, photodiodes, and photomultiplier tubes to achieve an elegant separation. ...
... Our plasmonic lensfree holographic cytometer is also significantly different from previous on-chip cytometry approaches, which are typically miniaturized versions of conventional flow cytometers [35][36][37][38][39] . First, our plasmonic lensfree cytometer does not rely on a microfluidic flow system, and thus it does not demand sophisticated chip fabrication or fluid handling. ...
Computational microscopy tools, in particular lensfree on-chip imaging, provide a large field-of-view along with a long depth-of-field, which makes it feasible to rapidly analyze large volumes of specimen using a compact and light-weight on-chip imaging architecture. To bring molecular specificity to this high-throughput platform, here we demonstrate the use of plasmon-resonant metallic nanoparticles to automatically recognize different cell types based on their plasmon-enhanced lensfree holograms, detected and reconstructed over a large field-of-view of e.g., ~24 mm(2).
... Nevertheless, such solutions usually require precise adjustment of the flow rate ratios and are difficult to handle. On the other hand, recent studies demonstrate that even two-dimensional hydrodynamic focusing can be sufficient to measure cellular DNA content in tumor cells [33,34]. ...
This study demonstrates the suitability of microfluidic structures for high throughput blood cell analysis. The microfluidic chips exploit fully integrated hydrodynamic focusing based on two different concepts: Two-stage cascade focusing and spin focusing (vortex) principle. The sample—A suspension of micro particles or blood cells—is injected into a sheath fluid streaming at a substantially higher flow rate, which assures positioning of the particles in the center of the flow channel. Particle velocities of a few m/s are achieved as required for high throughput blood cell analysis. The stability of hydrodynamic particle positioning was evaluated by measuring the pulse heights distributions of fluorescence signals from calibration beads. Quantitative assessment based on coefficient of variation for the fluorescence intensity distributions resulted in a value of about 3% determined for the micro-device exploiting cascade hydrodynamic focusing. For the spin focusing approach similar values were achieved for sample flow rates being 1.5 times lower. Our results indicate that the performances of both variants of hydrodynamic focusing suit for blood cell differentiation and counting. The potential of the micro flow cytometer is demonstrated by detecting immunologically labeled CD3 positive and CD4 positive T-lymphocytes in blood.
Bio‐inspired cilium‐based mechanosensors offer a high level of responsiveness, making them suitable for a wide range of industrial, environmental, and biomedical applications. Despite great promise, the development of sensors with multifunctionality, scalability, customizability, and sensing linearity presents challenges due to the complex sensing mechanisms and fabrication methods involved. To this end, high‐aspect‐ratio polycaprolactone/graphene cilia structures with high conductivity, and facile fabrication are employed to address these challenges. For these 3D‐printed structures, an “inter‐cilium contact” sensing mechanism that enables the sensor to function akin to an on‐off switch, significantly enhancing sensitivity and reducing ambiguity in detection, is proposed. The cilia structures exhibit high levels of customizability, including thickness, height, spacing, and arrangement, while maintaining mechanical robustness. The simplicity of the sensor design enables highly sensitive detection in diverse applications, encompassing airflow and water flow monitoring, braille detection, and debris recognition. Overall, the unique conductive cilia‐based sensing mechanism that is proposed brings several advantages, advancing the development of multi‐sensing capabilities and flexible electronic skin applications in smart robotics and human prosthetics.
Microfluidic impedance cytometry is emerging as a label-free, low-cost and portable solution for cell analysis. Impedance-based cell or particle characterization is provided by microfluidic and electronic devices. We report the design and characterization of a miniaturized flow cytometer based on a 3-dimensional (3D) hydrodynamic focusing mechanism. A sheath adaptively concentrated the sample laterally and vertically at the bottom of the microchannel, reducing the variance of particle translocation height and increasing the signal-to-noise ratio of the particle impedance pulse. Through simulation and confocal microscopy experiments, it has been verified that an increase in the ratio of sheath to sample decreased the cross-sectional area of the concentrated stream, which can be reduced to 26.50% of the pre-focusing value. The appropriate sheath flow settings increased the impedance pulse amplitude for different particles, and the coefficient of variation reduced by at least 35.85%, contributing to a more accurate representation of the particle impedance characteristic distribution. The system displayed the difference of HepG2 cell impedance before and after drug treatment, which is consistent with the results of flow cytometry, providing a convenient and inexpensive solution for monitoring cell status.
Researches on Sustainable Food Systems
Et ve et ürünleri üretiminin, çiftlikten çatala tüm zincirlerde (üretim, işleme, nakliye ve tüketici aşaması) çevre üzerinde olumsuz etkisi bulunmaktadır. Et endüstrisinde, kesim ve etin ileri ürünlere işlenmesi sırasında kan, kemik, deri, iç organlar, boynuzlar, ayaklar gibi büyük hacimlerde yan ürünler çıkar. Kesim atıkları, insan veya hayvan sağlık risklerine göre Avrupa Birliği ülkelerinde yüksek riskli, orta ve düşük riskli olmak üzere üç kategoride sınıflandırılır. Bu atıkların ve yan ürünlerin ekolojik olarak işlenmesi veya imha edilmesi çevre koruma açısından önemlidir. Et işleme zincirlerinde yan ürünlerin değerlendirmesine yönelik yenilikçi yaklaşımlar geliştirilmektedir. Kesimhane yan ürünleri zengin protein, yağ, vitamin, mineral kaynağı olduğu için yüksek besin değerine sahiptir ve birçok ülkede geleneksel olarak gıda veya gıda bileşenleri olarak tüketilmektedir. Proteinlerin jelleşme, köpürme ve emülsifikasyon gibi teknolojik kullanımları bulunmaktadır. Protein hidrolizatları sindirilebilirlik, lezzet, antihipertansif, antioksidan, antitrombotik ve antimikrobiyal etkiye katkıda bulunabilir. Yağlar ise kozmetik endüstrisinde, hammadde olarak biyodizel üretiminde, plastiklere alternatif olabilecek biyolojik olarak parçalanabilen plastiklerin geliştirilmesinde kullanılabilir. Mineral olarak zengin (fosfor ve kalsiyum gibi) kesim attıkları, evcil hayvan yemleri, gübre olarak kullanılır. Karaciğer, demir ve çinko gibi çeşitli mikro besinlerin kaynağıdır. Ayrıca kan, demir eksikliği olan kişiler için ek olarak kullanılabilecek iyi bir heme demir kaynağıdır ve bir polipeptit ile ilişkilendirilmesi durumunda demir emilimini arttırdığı rapor edilmiştir. Kesim atıklarından tıbbi açıdan önemli olan birçok madde elde edilir ve deri gibi bazı kısımlar yanıklar ve ülserler için pansuman olarak kullanılır. Deri endüstrisinde kesim atıkları kullanılarak birçok ürün üretilmektedir. Bu derlemede kesim atıklarının değerlendirilmesi hakkında bilgi vermek ve et ürünlerin üretimini daha sürdürülebilir hale getirme fırsatlarını tartışmak amaçlanmıştır. Sürdürülebilir et ve et ürünleri ile ilgili yeni uygulamalara özellikle su ve enerji tüketiminin çevresel etkilerinin azaltılması, gelecekteki çalışma önerisi olarak sunulabilir.
Surface expression of cell populations are often sought as diagnostic and prognostic biomarkers in hematology-oncology and infectious diseases, making flow cytometry an invaluable technique for both clinical and basic research applications. On the other hand, the reliance of flow cytometry on manual input parameters and user protocols for operation introduces variation between analyses while potentially leading to errors in measurements. In this work, we introduce an integrated flow cytometry microchip that automatically adapts to the sample it interrogates. Our device measures the antigen expression in a sample by automatically analyzing the response of immunomagnetically labeled cells to an external magnetic field through integrated electrical sensors and by continuously modulating the time the cells are subjected to the field for optimal sensitivity and dynamic range. Furthermore, the lack of optical illumination and fluorescence detectors enables automated analysis to be carried on a fully integrated platform that is particularly well suited for translation into point-of-care testing and mobile screening. We applied our automated cytometry chip on both pure and mixed cell populations and validated its operation by benchmarking against a conventional flow cytometer. By transforming the utility-proven flow cytometry, a technique that has long been dependent on an operator in centralized laboratories, into a standardized disposable test for bedside or home testing, the automated flow cytometry microchip introduced here has the potential to enable self-screening for telemedicine and wellness.
Environmental toxicology focuses on identifying and predicting impact of potentially toxic anthropogenic chemicals on biosphere at various levels of biological organization. Presently there is a significant drive to gain deeper understanding of cellular and sub‐cellular mechanisms of ecotoxicity. Most notable is increased focus on elucidation of cellular‐response networks, interactomes, and greater implementation of cell‐based biotests using high‐throughput procedures, while at the same time decreasing the reliance on standard animal models used in ecotoxicity testing. This is aimed at discovery and interpretation of molecular pathways of ecotoxicity at large scale. In this regard, the applications of cytometry are perhaps one of the most fundamental prospective analytical tools for the next generation and high‐throughput ecotoxicology research. The diversity of this modern technology spans flow, laser‐scanning, imaging, and more recently, Raman as well as mass cytometry. The cornerstone advantages of cytometry include the possibility of multi‐parameter measurements, gating and rapid analysis. Cytometry overcomes, thus, limitations of traditional bulk techniques such as spectrophotometry or gel‐based techniques that average the results from pooled cell populations or small model organisms. Novel technologies such as cell imaging in flow, laser scanning cytometry, as well as mass cytometry provide innovative and tremendously powerful capabilities to analyze cells, tissues as well as to perform in situ analysis of small model organisms. In this review, we outline cytometry as a tremendously diverse field that is still vastly underutilized and often largely unknown in environmental sciences. The main motivation of this work is to highlight the potential and wide‐reaching applications of cytometry in ecotoxicology, guide environmental scientists in the technological aspects as well as popularize its broader adoption in environmental risk assessment.
Modern microflow cytometers are sophisticated instruments capable of measuring multiple physical characteristics of a single cell simultaneously as the cells flow in suspension through a measuring device. Cytometers are the tool of choice for the high-speed acquisition and analysis of large single cell populations. However, traditional devices lack the ability to provide intracellular spatial information. In the past few decades, various flow cytometer systems with the ability to combine cell/particle detection and the acquisition of two or three-dimensional spatial information have been proposed. However, these devices suffer a number of drawbacks Accordingly, the problem of developing more sophisticated microflow cytometers based on electrical impedance, optical detection and image analysis methods has received significant attention in the literature. This review describes some of the major advances made in the microfluidic cytometry field over the past ten years. The review focuses specifically on recent proposals for enhanced microfluidic focusing techniques and detection/analysis methods, respectively. Overall, the review provides a useful insight into the microflow cytometer technology field for both new and existing users.
We are developing an integrated flow cytometry chip, which comprises a pre-treatment part and a sheath flow-forming part with a twisted microchannel structure, for continuous monitoring of bacteria in drinking water. Due to its unique elastic property, polydimethylsiloxane (PDMS) was used to fabricate the twisted microchannel that forms the sheath flow, which is a three-dimensional focusing flow. Cells from a human lung cancer cell line were stained in the pre-treatment part and the fluorescence of the stained cells was successfully detected at the sheath flow-forming part. With no dead volume between the two parts, the transfer time was nearly zero, and only 30 µl of sample fluid and approximately 6 µl of reagent were required.
We report a facile and non-invasive way to disintegrate a microdroplet into a string of further miniaturized ones under the influence of an external electrohydrodynamic field inside a microchannel. The deformation and breakup of the droplet was engendered by the Maxwell's stress originating from the accumulation of induced and free charges at the oil-water interface. While at smaller field intensities, e.g. less than 1 MV/m, the droplet deformed into a plug, at relatively higher field intensities, e.g. ∼1.16 MV/m, a pair of droplets having opposite surface charge was formed. The charged droplets showed an interesting periodic bridging and breakup during their translation motion across the channel. For even higher field intensities, e.g. more than 1.2 MV/m, the entire droplet underwent dielectrophoresis towards one of the electrodes before experiencing a strong attractive force from the other electrode to deform into a shape of a Taylor cone. With progress in time, mimicking the electrospraying phenomenon, the cone-tip periodically ejected a string of miniaturized water droplets to form a microemulsion inside the channel. The frequency and size of the droplet ejection could be tuned by varying the applied field intensity. A water droplet of ∼214 μm diameter could continuously eject droplets of size ∼10 μm or even smaller to form a microemulsion inside the channel. This article is protected by copyright. All rights reserved
Numerical simulations supplemented by experiments together uncovered that strategic integration of discrete electric fields in a non-invasive manner could substantially miniaturize the droplets into smaller parts in a pressure driven oil-water flow inside microchannels. The Maxwell's stress generated from the electric field at the oil-water interface could deform, stretch, neck, pin, and disintegrate a droplet into many miniaturized daughter droplets, which eventually ushered a one-step method to form water-in-oil microemulsion employing microchannels. The interplay between electrostatic, inertial, capillary, and viscous forces led to various pathways of droplet breaking, namely, fission, cascade, or Rayleigh modes. While a localized electric filed in the fission mode could split a droplet into a number of daughter droplets of smaller size, the cascade or the Rayleigh mode led to the formation of an array of miniaturized droplets when multiple electrodes generating different field intensities were ingeniously assembled around the microchannel. The droplets size and frequency could be tuned by varying the field intensity, channel diameter, electrode locations, interfacial tension, and flow ratio. The proposed methodology shows a simple methodology to transform a microdroplet into an array of miniaturized ones inside a straight microchannel for enhanced mass, energy, and momentum transfer, and higher throughput. This article is protected by copyright. All rights reserved.
The flow morphology of two immiscible fluids in a microfluidic device finds numerous applications such as emulsification, synthesis of nanomaterials, lab-on-a-chip devices, cell cytometry and biological analysis. It offers many advantages over the conventional macroscopic devices when comes to handling of small volume of fluids because of its availability of higher surface to volume ratio, easier process control and reduction in operating cost. In cell cytometry studies, biomarkers such as shape, size and deformability of cells and also chemical properties of cells play a pivotal role in order to sort healthy cells from the contaminated cells. A microfluidic device to categorize cells by biophysical markers can be low cost, suitable to maintain, and characterized by good throughput and shorter assay times. In the recent years many theoretical studies have been done to successfully deform a flow structure inside a microchannel by using external force fields.
In this present study, we show a simple strategy by which a water droplet suspended in a continuous oil media can be deformed into various shapes due to an externally applied and linearly increasing electric field. Here we show a noninvasive pathway to distort the structural integrity of a water droplet to deform into various shapes depending upon the physical properties of the water and also forcefully decelerate it inside the electric field by introducing a locally applied and linearly increasing electric field.
Selective isolation of cell subpopulations with defined biological characteristics is crucial for many biological studies and clinical applications. In this work, we present the development of an acoustofluidic fluorescence activated cell sorting (FACS) device that simultaneously performs on-demand, high-throughput, high-resolution cell detection and sorting, integrated onto a single chip. Our acoustofluidic FACS device uses the "microfluidic drifting" technique to precisely focus cells/particles three dimensionally and achieves a flow of single-file particles/cells as they pass through a laser interrogation region. We then utilize short bursts (150 μs) of standing surface acoustic waves (SSAW) triggered by an electronic feedback system to sort fluorescently labeled particles/cells with desired biological properties. We have demonstrated continuous isolation of fluorescently labeled HeLa cells from unlabeled cells at a throughput of ∼1200 events/s with a purity reaching 92.3 ± 3.39%. Furthermore, 99.18% postsort cell viability indicates that our acoustofluidic sorting technique maintains a high integrity of cells. Therefore, our integrated acoustofluidic FACS device is demonstrated to achieve two-way cell sorting with high purity, biocompatibility, and biosafety. We believe that our device has significant potential for use as a low-cost, high-performance, portable, and user-friendly FACS instrument.
A simple and highly sensitive technique for laser-induced fluorescence detection on multiple channels in a plastic microchip was developed, and its effectiveness was demonstrated by laser-beam ray-trace simulations and experiments. In the microchip, with refractive index nC, A channels and B channels are arrayed alternately and respectively filled with materials with refractive indexes nA for electrophoresis analysis and nB for laser-beam control. It was shown that a laser beam entering from the side of the channel array traveled straight and irradiated all A channels simultaneously and effectively because the refractive actions by the A and B channels were counterbalanced according to the condition nA < nC < nB. This technique is thus called "side-entry laser-beam zigzag irradiation". As a demonstration of the technique, when nC = 1.53, nA = 1.41, nB = 1.66, and the cross sections of both eight A channels and seven B channels were the same isosceles trapezoids with 97° base angle, laser-beam irradiation efficiency on the eight A channels by the simulations was 89% on average and coefficient of variation was 4.4%. These results are far superior to those achieved by other conventional methods such as laser-beam expansion and scanning. Furthermore, fluorescence intensity on the eight A channels determined by the experiments agreed well with that determined by the simulations. Therefore, highly sensitive and uniform fluorescence detection on eight A channels was achieved. It is also possible to fabricate the microchips at low cost by plastic-injection molding and to make a simple and compact detection system, thereby promoting actual use of the proposed side-entry laser-beam zigzag irradiation in various fields.
We report the capillary and frictional force mediated transitions of morphologies of an oil-water flow inside a microchannel using experiments and computational fluid dynamic simulations. A number of steady and time-periodic flow patterns were reported with the variations in the interfacial tension, exchange of inlets, flow ratio, and viscosity ratio of the phases. Transitions from slug to plug to droplet to stratified flow patterns were obtained by tuning the interfacial tension. Progressive reduction in the interfacial tension transformed big slugs into smaller plugs, plugs into droplets, and droplets into a stratified flow pattern. Interestingly, the simulations uncovered a non-monotonic and nonlinear reduction in pressure drop with the decrease in interfacial tension. The change in the pressure drop was correlated to the variation in the slug, plug, or droplet frequency of water at the outlet. The variations in the pressure drop were also associated with the transition from dripping to jetting of water droplet ejection near the channel inlet. Apart from the interfacial tension, the viscosity stratification across the phases was also found to play an important role in converting the slug flow patterns into smaller plugs or droplets. The study also reports the parametric space in which the droplet flow patterns could be obtained inside a microchannel tuning the flow and viscosity ratios of the phases alongside the interfacial tension. The reported transitions of flow patterns and the pressure drop characteristics can be of significance in improving the efficiency of future microfluidic devices.
We describe the development of an integrated, lensed Arrayed Waveguide Grating (AWG) microspectrometer for localized multiplexing fluorescence measurements. The device, which has a footprint of only 1mm wide and 1cm long, is capable of spectroscopic measurements on chip. Multiple fluorescence signals were measured simultaneously based upon simple intensity readouts from a CCD camera. We also demonstrate the integration of the AWG spectrometer with a microfluidics platform using a lensing function to confine the beam shape for focused illumination. This capability enhances signal collection, gives better spatial resolution, and provides a route for the analysis of small volume samples (e.g. cells) in flow. To show these capabilities we developed a novel “bead-AWG” platform with which we demonstrate localized multiplexed fluorescence detection either simultaneously or successively. Such an integrated system provides the basis for a portable system capable of optical detection of multi-wavelength fluorescence from a single, defined location.
Strategic application of external electrostatic field on a pressure-driven two-phase flow inside a microchannel can transform the stratified or slug flow patterns into droplets. The localized electrohydrodynamic stress at the interface of the immiscible liquids can engender a liquid-dielectrophoretic deformation, which disrupts the balance of the viscous, capillary, and inertial forces of a pressure-driven flow to engender such flow morphologies. Interestingly, the size, shape, and frequency of the droplets can be tuned by varying the field intensity, location of the electric field, surface properties of the channel or fluids, viscosity ratio of the fluids, and the flow-ratio of the phases. Higher field intensity with lower interfacial tension is found to facilitate the oil droplet formation with a higher throughput inside the hydrophilic microchannels. The method is successful in breaking down the regular pressure-driven flow patterns even when the fluid inlets are exchanged in the microchannel. The simulations identify the conditions to develop interesting flow morphologies such as, (i) an array of miniaturized spherical or hemispherical or elongated oil drops in continuous water phase, (ii) ‘oil-in-water’ microemulsion with varying size and shape of oil droplets. The results reported can be of significance in improving the efficiency of multiphase microreactors where the flow patterns composed of droplets are preferred because of the availability of higher interfacial area for reactions or heat and mass exchange.This article is protected by copyright. All rights reserved
The development of microfluidic chip-based cytometers has become an important area due to their advantages of compact size and low cost. Herein, we demonstrate a sheathless microfluidic cytometer which integrates a standing surface acoustic wave (SSAW)-based microdevice capable of 3D particle/cell focusing with a laser-induced fluorescence (LIF) detection system. Using SSAW, our microfluidic cytometer was able to continuously focus microparticles/cells at the pressure node inside a microchannel. Flow cytometry was successfully demonstrated using this system with a coefficient of variation (CV) of less than 10% at a throughput of ~1000 events s(-1) when calibration beads were used. We also demonstrated that fluorescently labeled human promyelocytic leukemia cells (HL-60) could be effectively focused and detected with our SSAW-based system. This SSAW-based microfluidic cytometer did not require any sheath flows or complex structures, and it allowed for simple operation over a wide range of sample flow rates. Moreover, with the gentle, bio-compatible nature of low-power surface acoustic waves, this technique is expected to be able to preserve the integrity of cells and other bioparticles.
In this article, we demonstrate single-layered, "microfluidic drifting" based three-dimensional (3D) hydrodynamic focusing devices with particle/cell focal positioning approaching submicron precision along both lateral and vertical directions. By systematically optimizing channel geometries and sample/sheath flow rates, a series of "microfluidic drifting" based 3D hydrodynamic focusing devices with different curvature angles are designed and fabricated. Their performances are then evaluated using confocal microscopy, fast camera imaging, and side-view imaging techniques. Using a device with a curvature angle of 180°, we have achieved a standard deviation of ±0.45 μm in particle focal position and a coefficient of variation (CV) of 2.37% in flow cytometric measurements. To the best of our knowledge, this is the best CV that has been achieved using a microfluidic flow cytometry device. Moreover, the device showed the capability to distinguish 8 peaks when subjected to a stringent 8-peak rainbow calibration test, signifying the ability to perform sensitive, accurate tests similar to commercial flow cytometers. We have further tested and validated our device by detection of HEK-293 cells. With its advantages in simple fabrication (i.e., single-layered device), precise 3D hydrodynamic focusing (i.e., submicrometer precision along both lateral and vertical directions), and high detection resolution (i.e., low CV), our method could serve as an important basis for high-performance, mass-producible microfluidic flow cytometry.
Cell focusing into a narrow stream is an essential step prior to counting and sorting cells in microfluidic devices for flow cytometry and cell sorting applications. Hydrodynamic focusing techniques, however, rely on the need for large volumes of sheath liquid and complex mechanical setup for flow control, preventing miniaturization of the systems. Although microfluidic methods based on active or passive particle control offer sheathless and efficient focusing, they often accompany fabrication complexities or bulky external setups, and operate in a certain range of flow rates. We present here a microfluidic device to focus cells into a narrow stream. The device employs hydrophoresis to guide cells by locally patterned slanted grooves, and channel expansion to improve focusing efficiency and produce a narrow stream of cells. This device principle allows easy improvement of focusing efficiency by adding more expansion steps. Adjusting channel expansion also ensures successful cell focusing without defocusing by inertial effects even at high Reynolds numbers. Using this device, we successfully produced a narrow stream of cells having size variation of >11% in a coefficient of variation (CV), achieving a narrow cell stream with a focusing variation below CV of 3.0%. © 2013 International Society for Advancement of Cytometry.
Limitations imposed by conventional analytical technologies for cell biology, such as flow cytometry or microplate imaging, are often prohibitive for the kinetic analysis of single-cell responses to therapeutic compounds. In this paper, we describe the application of a microfluidic array to the real-time screening of anticancer drugs against arrays of single cells. The microfluidic platform comprises an array of micromechanical traps, designed to passively corral individual nonadherent cells. This platform, fabricated in the biologically compatible elastomer poly(dimethylsiloxane), PDMS, enables hydrodynamic trapping of cells in low shear stress zones, enabling time-lapse studies of nonadherent hematopoietic cells. Results indicate that these live-cell, microfluidic microarrays can be readily applied to kinetic analysis of investigational anticancer agents in hematopoietic cancer cells, providing new opportunities for automated microarray cytometry and higher-throughput screening. We also demonstrate the ability to quantify on-chip the anticancer drug induced apoptosis. Specifically, we show that with small numbers of trapped cells (approximately 300) under careful serial observation we can achieve results with only slightly greater statistical spread than can be obtained with single-pass flow cytometer measurements of 15,000-30,000 cells.
This review describes recent developments in microfabricated flow cytometers and related microfluidic devices that can detect, analyze, and sort cells or particles. The high-speed analytical capabilities of flow cytometry depend on the cooperative use of microfluidics, optics and electronics. Along with the improvement of other components, replacement of conventional glass capillary-based fluidics with microfluidic sample handling systems operating in microfabricated structures enables volume- and power-efficient, inexpensive and flexible analysis of particulate samples. In this review, we present various efforts that take advantage of novel microscale flow phenomena and microfabrication techniques to build microfluidic cell analysis systems.
This article provides an overview of recent research achievements in miniaturized flow cytometry. The review focuses on chip-based microfluidic flow cytometers, classified by cell transport method, detection technology, and biomedical application. By harnessing numerous ideas and cutting-edge microfabrication technologies, microfluidic flow cytometry benefits from ever-increasing functionalities and the performance levels achieved make it an attractive biomedical research and clinical tool. In this article, we briefly describe an update of recent developments that combine novel microfluidic characteristics and flow cytometry on chips that meet biomedical needs.
The deep red fluorescing agent (DRAQ5) is a synthetic anthraquinone with a high affinity for DNA and a high capacity to rapidly enter living cells or stain fixed cells. DRAQ5 is optimally excited by red-light emitting sources and yields a deep red emission spectrum which extends into the low infra-red. DRAQ5 shows excitation at sub-optimal wavelengths including the 488 nm line and the multi-line UV wavelengths emitted by argon-ion lasers. Single beam (488 nm) flow cytometry has been used to demonstrate the utility of DRAQ5-nuclear DNA fluorescence as a discriminating parameter for human leucocytes and lymphoma cells, in combination with fluorochrome-labelled antibodies for the detection of surface antigens and subpopulation recognition. DRAQ5 fluorescence was found to reflect cellular DNA content as evidenced by cell cycle distribution profiles for asynchronous and cell cycle-perturbed populations. Importantly, DRAQ5 can be used in combination with FITC and RPE-labelled antibodies, without the need for fluorescence compensation.
A cell undergoing apoptosis demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the inducer of apoptosis, cell type and the "time window" at which the process of apoptosis is observed. Because the gross majority of apoptotic hallmarks can be revealed by flow and image cytometry, the cytometric methods become a technology of choice in diverse studies of cellular demise. Variety of cytometric methods designed to identify apoptotic cells, detect particular events of apoptosis and probe mechanisms associated with this mode of cell death have been developed during the past two decades. In the present review, we outline commonly used methods that are based on the assessment of mitochondrial transmembrane potential, activation of caspases, DNA fragmentation, and plasma membrane alterations. We also present novel developments in the field such as the use of cyanine SYTO and TO-PRO family of probes. Strategies of selecting the optimal multiparameter approaches, as well as potential difficulties in the experimental procedures, are thoroughly summarized.
Despite over 2 million papers published on cancer so far, malignancy still remains a puzzlingly complex disease with overall low survival rates. Expanding our knowledge of the molecular mechanisms of malignancy and of resistance to therapy is crucial in guiding the successful design of anti-cancer drugs and new point-of-care diagnostics. The up-and-coming microfluidic Lab-on-a-Chip (LOC) technology and micro-total analysis systems (μTAS) are arguably the most promising platforms to address the inherent complexity of cellular systems with massive experimental parallelization and 4D analysis on a single cell level. This review discusses the emerging applications of microfluidic technologies and their advantages for cancer biology and experimental oncology. We also summarize the recent advances in miniaturized systems to study cancer cell microenvironment, cancer cytomics, and real-time (4D) pharmacological screening. Microfabricated systems, such as cell microarrays, together with on-chip label-less cytometry, and micro-sorting technologies, are all highlighted with the view of describing their potential applications in pharmacological screening, drug discovery, and clinical oncology. It is envisaged that microfluidic solutions may well represent the platform of choice for next generation in vitro cancer models.
Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance-based characterization. Such systems are being developed to provide simple, low-cost, label-free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro-organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high-content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance-based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip-based cytometry can make a large impact.
Over a decade has passed since publication of the last review on "Cytometry in cell necrobiology." During these years we have witnessed many substantial developments in the field of cell necrobiology such as remarkable advancements in cytometric technologies and improvements in analytical biochemistry. The latest innovative platforms such as laser scanning cytometry, multispectral imaging cytometry, spectroscopic cytometry, and microfluidic Lab-on-a-Chip solutions rapidly emerge as highly advantageous tools in cell necrobiology studies. Furthermore, we have recently gained substantial knowledge on alternative cell demise modes such as caspase-independent apoptosis-like programmed cell death (PCD), autophagy, necrosis-like PCD, or mitotic catastrophe, all with profound connotations to pathogenesis and treatment. Although detection of classical, caspase-dependent apoptosis is still the major ground for the advancement of cytometric techniques, there is an increasing demand for novel analytical tools to rapidly quantify noncanonical modes of cell death. This review highlights the key developments warranting a renaissance and evolution of cytometric techniques in the field of cell necrobiology.
Rapid and accurate differentiation of cell types within a heterogeneous solution is a challenging but important task for various applications in biological research and medicine. Flow cytometry is the gold standard in cell analysis and is regularly used for blood analysis (i.e., complete blood counts). Flow cytometry, however, lacks sufficient throughput to analyze rare cells in blood or other dilute solutions in a reasonable time period because it is an inherently serial process. In this study, we exploit inertial effects for label- and sheath-free parallel flow cytometry with extreme throughput. We demonstrate a microfluidic device that consists of 256 high-aspect (W = 16 microm, H = 37 microm) parallel channels yielding a sample rate up to 1 million cells s(-1), only limited by the field-of-view of our high-speed optical interrogation method. The particles or cells flowing through the channels are focused to one uniform z-position (SD = +/-1.81 microm) with uniform downstream velocity (U(ave) = 0.208 +/- 0.004 m s(-1)) to reduce the probability of overlap and out-of-focus blur and provide similar cell signature images for accurate detection and analysis. To demonstrate a proof-of-concept application of our system operating at these throughputs, we conducted automated RBC and leukocyte counts on diluted whole blood and achieved high counting sensitivity and specificity (86-97%) compared to visual inspection of raw images. As no additional external forces are required to create ordered streams of cells, this approach has the potential for future applications in cost-effective hematology or rare-cell analysis platforms with extreme throughput capabilities when integrated with suitable large field-of view imaging or interrogation methods.
We report a contraction-expansion array (CEA) microchannel that allows three-dimensional hydrodynamic focusing with a single sheath flow in a single-layer device. The CEA microchannel exploits centrifugal forces acting on fluids travelling along the contraction and expansion regions of the microchannel. Around an entrance of the contraction region, the centrifugal forces induce a secondary flow field where two counter-rotating vortices enable to envelop a sample flow with a sheath flow in three dimensions. We herein describe an underlying principle and a design of the CEA microchannel and demonstrate complete sheathing of a sample fluid (water and human red blood cells) in three dimensions. The focusing characteristics of the CEA microchannel are investigated in terms of the number of the rectangular structures, flow rate, and flow ratio between sample and sheath flows. This microfluidic channel for three-dimensional hydrodynamic focusing is easy to fabricate in a single-layer fabrication process and simple to operate with a single sheath flow.
We demonstrate an integrated platform that merges a microfluidic chip with lensless imaging to target CD4(+) T-lymphocyte counts for HIV point-of-care testing at resource-limited settings. The chips were designed and fabricated simply with a laser cutter without using expensive cleanroom equipment. To capture CD4(+) T-lymphocytes from blood, anti-CD4 antibody was immobilized on only one side of the microfluidic chip. These captured cells were detected through an optically clear chip using a charge coupled device (CCD) sensor by lensless shadow imaging techniques. Gray scale image of the captured cells in a 24 mm x 4 mm x 50 microm microfluidic chip was obtained by the lensless imaging platform. The automatic cell counting software enumerated the captured cells in 3s. Captured cells were also imaged with a fluorescence microscope and manually counted to characterize functionality of the integrated platform. The integrated platform achieved 70.2+/-6.5% capture efficiency, 88.8+/-5.4% capture specificity for CD4(+) T-lymphocytes, 96+/-1.6% CCD efficiency, and 83.5+/-2.4% overall platform performance (n=9 devices) compared to the gold standard, i.e. flow cytometry count. The integrated system gives a CD4 count from blood within 10 min. The integrated platform points a promising direction for point-of-care testing (POCT) to rapidly capture, image and count subpopulations of cells from blood samples in an automated matter.
The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV), and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981), and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2, or p53 phosphorylation. However, pronounced ATM, Chk2, and p53 phosphorylation and perturbed G(2) progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2, and p53, dramatic changes marked by a high degree of H2AX, ATM, Chk2, and p53 phosphorylation, all occurring predominantly in S phase cells, and a block in cell cycle progression, were seen after 18 h exposure. These changes suggest that the DRAQ5-induced DNA lesions may become converted into double-strand DNA breaks during replication. Exposure to DCV also led to an increase in the level of activated ATM and Chk2 as well as of phosphorylated p53 and accumulation of cells in G(2)M and S phase. Exposure to SYTO 17 had no significant effect on any of the measured parameters. The data indicate that supravital use of Ho 42, DRAQ5, and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis.
The microfluidic platform is an important tool for diagnosis and biomedical studies because it enables us to handle precious cells and infectious materials safely. We have developed an on-chip microfluidic sorter with fluorescence spectrum detection and multiway separation. The fluorescence spectrum of specimens (495-685 nm) in the microchannels was obtained every 2 ms using a 1 x 16 arrayed photomultiplier tube. The specimen was identified by its spectrum and collected into the corresponding channel based on our previously reported thermoreversible gelation polymer technique (Y. Shirasaki, J. Tanaka, H. Makazu, K. Tashiro, S. Shoji, S. Tsukita and T. Funatsu, Anal. Chem., 2006, 78, 695-701). Four kinds of fluorescence microspheres and three kinds of Escherichia coli cells, expressing different fluorescent proteins, were successfully separated with accuracy and purity better than 90% at a throughput of about one particle per second.
Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and "stain-no wash" protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry. Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors.
Caspase activation is a critical early step in the onset of apoptosis. Cell-permeable fluorogenic caspase substrates have proven valuable in detecting caspase activation by flow cytometry. Nevertheless, detection of early low-level caspase activation has been difficult using conventional area or peak fluorescence analysis by flow cytometry, despite the apparent presence of these cells as observed by microscopy. We describe a method utilizing maximum fluorescence pixel analysis by laser scanning cytometry (LSC) to detect early apoptotic cells.
The PhiPhiLux-G(1)D(2) caspase 3/7 substrate was used in combination with DNA dye exclusion and annexin V binding to identify several stages of apoptosis in EL4 murine thymoma cells by both traditional flow and LSC. LSC analysis of maximum pixel brightness in individual cells demonstrated an intermediate caspase-low subpopulation not detectable by flow or LSC integral analysis. LSC analysis of caspase activity was then carried out using the larger UMR-106 rat osteosarcoma cell line to determine if this apparent early caspase activity could be correlated with localized, punctate caspase activity observed by microscopy.
The caspase-low subpopulation found in apoptotic EL4 cells was also observable in UMR-106 cells. Relocation to cells with low fluorescence due to caspase activity and subsequent examination by microscopy demonstrated that these latter cells indeed show punctate, highly localized caspase activation foci that might represent an early stage in caspase activation.
Cells with low-level, localized caspase expression can be detected using maximum pixel analysis by LSC. This methodology allows an early step of apoptotic activation to be resolved for further analysis.
This review summarizes our observations on the mechanism of induction of apoptosis in vitro in leukaemic cell lines and in vivo in patients with leukaemia undergoing chemotherapy, in relation to the cell cycle. Multiparameter flow cytometric methods allowed us to identify apoptotic cells and position them with respect to their cell cycle phase. Several antitumor agents of different classes have been characterized in terms of the cell cycle phase specificity of induction of apoptosis. Three types of apoptosis could be distinguished in relation to the initial damage to the cell vis-a-vis cell cycle position: (1) homo-phase apoptosis where the cells underwent apoptosis during the same phase in which they were initially affected; (2) homo-cycle apoptosis, where the cells underwent apoptosis during the same cell cycle in which they were initially affected, i.e., prior to or during the first mitosis, and (3) post-mitotic apoptosis, where cells underwent apoptosis during the cell cycle(s) subsequent to that in which the cell was initially affected, most likely at the G1 or G2 checkpoints of these cycle(s). Four ranges of drug concentration can be distinguished in vitro for most drugs, where either: (1) no immediate effects; (2) cytostasis or post-mitotic apoptosis; (3) homo-cycle or homo-phase apoptosis; or (4) necrosis are observed. Analysis of cell death of blast cells from peripheral blood or bone marrow of over 250 leukaemia patients (AML, ALL, CML in blast crisis) treated with various drugs during routine chemotherapy reveals that in the case of DNA topoisomerase inhibitors (e.g., mitoxantrone, VP-16) apoptosis is often rapid (peaks at 1-2 days after drug administration) and has features of homo-phase apoptosis. In contrast, cell death observed after administration of paclitaxel (taxol) or cytarabine (cytosine arabinoside) occurs later and has features of post-mitotic apoptosis: the cells divide but die in G1 of the subsequent cycle(s).
The use of spatially sculpted laser interference pattern or optical lattice to sort suspended particles within a microfluidic network is discussed. Some particles deflect from their original trajectories, whereas others pass almost straight through the interference lattice. The ability to separate microscopic biological matter by size is demonstrated by separating out protein microcapsules from the stream. A user can assemble structures, perform optical fractionation, and control the functionality of light-driven pumps, valves and mixers inside microfluidic channels using spatially sculpted light .
Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named gammaH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of gammaH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.
The compounds aimed to directly bind and inhibit Bcl-2 and related anti-apoptotic proteins have entered the clinical or pre-clinical stage of evaluation and represent a promising new strategy to combat cancer. Having reported a pro-apoptotic function of a small molecule inhibitor of Bcl-2, HA14-1, in follicular lymphoma cell lines, we provide herein further insights into the action of this compound in our model. Employing both pharmacological inhibitor studies and multiparametric flow cytometry assays, we demonstrated that following HA14-1 treatment caspase activation occurs solely as a consequence of mitochondrial breach. Moreover, applying bivariate analysis of the DNA content and fluorochrome-labeled inhibitor of caspases (FLICA) binding, we investigated for the first time the cell cycle specificity of HA14-1-evoked apoptosis in FL cells upon different exposure scenarios. Overall, the study provides both mechanistic and clinically relevant information about the action of HA14-1.
We describe the design, fabrication, and operation of two types of flow cytometers based on microfluidic devices made of a single cast of poly(dimethylsiloxane). The stream of particles or cells injected into the devices is hydrodynamically focused in both transverse and lateral directions, has a uniform velocity, and has adjustable diameter and shape. The cytometry system built around the first microfluidic device has fluorescence detection accuracy comparable with that of a commercial flow cytometer and can analyze as many as 17 000 particles/s. This high-throughput microfluidic device could be used in inexpensive stand-alone cytometers or as a part of integrated microanalysis systems. In the second device, a stream of particles is focused to a flow layer of a submicrometer thickness that allows imaging the particles with a high numerical aperture microscope objective. To take long-exposure, low-light fluorescence images of live cells, the device is placed on a moving stage, which accurately balances the translational motion of particles in the flow. The achieved resolution is comparable to that of still micrographs. This high-resolution device could be used for analysis of morphology and fluorescence distribution in cells in continuous flow.
This study utilizes MEMS technology to realize a novel low-cost microfluidics-based biochip system for flow-type cell handling. Powered by vacuum pump, the microfluidic driving system enables cells to move in order one by one in the biochip by an effect of sheath flow prefocus. Then, cells are guided to a fluorescent inspection region where two detection tasks such as cell image identification and cell counting are conducted. Currently, the glass-based biochip has been manufactured and all the related devices have been well set up in our laboratory. With this proposed prototype system, typical results about cell separation of yeast cell and PC-3 cell are available and their separated images are also presented, respectively.
Follicular lymphoma (FL) remains a fatal disease of increasing worldwide incidence. Since patients with FL eventually develop resistance to conventional anticancer agents, and due to BCL-2 overexpression present with profoundly compromised execution of mitochondrial pathway of apoptosis, targeting alternative pathways of cell demise may appear therapeutically beneficial. Herein we report for the first time the effects of an ER-Golgi transport inhibitor, Brefeldin A (BFA), alone and in combination with a small molecule Bcl-2 inhibitor HA14-1 or agonistic anti-Fas mAb, in the recently established human FL cell lines. All cell lines tested were sensitive to BFA-induced cytotoxicity and apoptosis. Moreover BFA-induced cell death was associated with profound ER stress, mitochondrial breach and subsequent caspase cascade activation, including caspase 2 activation. Interestingly, BFA-induced ER stress did not result in appearance of autophagic morphology in FL cells. Of importance, small molecule Bcl-2 antagonist, HA14-1 and agonistic anti-Fas mAb significantly enhanced BFA-mediated cytotoxicity and apoptosis, revealing novel and previously unexplored means to enhance ER stress-mediated cell killing in follicular lymphoma cells.
Over the past decade the importance of signaling from reporter molecules inside live cells and tissues has been clearly established. Biochemical events related to inflammation, tumor metastasis and proliferation, and viral infectivity and replication are examples of processes being further defined as more molecular tools for live cell measurements become available. Moreover, in addition to quantitating parameters related to physiologic processes, real-time imaging of molecular interactions that compose basic cellular activities are providing insights into understanding disease mechanisms as well as extending clinical efficacy of therapeutic regimens. In this review the use of highly cell-permeable fluorogenic substrates that report protease activities inside live cells is described; applications to defining the molecular events of two cellular processes, i.e., apoptosis and cell-mediated cytotoxicity, are then illustrated.
Functional assays allowing phenotypic characterization of different cell death parameters at a single-cell level are important tools for preclinical anticancer drug screening. Currently, the selection of cytometric assays is limited by the availability of fluorescent probes with overlapping spectral characteristics. Following on our earlier reports on green and orange fluorescent SYTO probes, we provide herein further insights into applicability of novel red-excitable SYTO stains (SYTO 17, 59-64) for multiparameter analysis of cell fate. In particular, SYTO 62 appears to be a spectrally favorable candidate. Using a correlative comparison between SYTO 16, Annexin V, YO-PRO 1, and fluorescently labeled inhibitors of caspases (FLICA), we demonstrate the specificity of SYTO 62 in detection of apoptotic cell death. Used in conjunction with FLICA or Annexin V, SYTO 62 stain proved amenable for multivariate kinetic analysis of apoptotic events. Considering simplicity of staining protocols, low cost, and avoidance of spectral compensation problems, we expect that red-excitable SYTO dyes will find a wide range of cytometric applications.
Electrical impedance-based particle detection or Coulter counting, offers a lab-on-chip compatible method for flow cytometry. Developments in this area will produce devices with greater portability, lower cost, and lower power requirements than fluorescence-based flow cytometry. Because conventional Coulter apertures are prone to clogging, hydrodynamic focusing improves the device by creating fluid-walled channels with variable width to increase sensitivity without the associated risk of blocking the channel. We describe a device that focuses the sample in three dimensions, creating a narrow sample stream on the floor of the channel for close interaction with sensing electrodes. The key to this design is a stepped outlet channel fabricated in a single layer with soft lithography. In contrast to previous impedance-based designs, the new design requires minimal alignment with the substrate. Three-dimensional focusing maximizes the sensitivity of the device to cell-size particles within much larger channels. Impedance-based particle sensing experiments within this device show an increase in percentage conductivity change by a factor of 2.5 over devices that only focus the sample in the horizontal direction.
Micro-fluidic single-cell array cytometry for the analysis of tumor apoptosis Cytometry in cell necrobiol-ogy revisited: recent advances and new vistas
- D Wlodkowic
- S Faley
- M Zagnoni
- J P Wikswo
- J M Cooper
Current Opinion in Chemical Biology 14 (5), 556–567. Wlodkowic, D., Faley, S., Zagnoni, M., Wikswo, J.P., Cooper, J.M., 2009a. Micro-fluidic single-cell array cytometry for the analysis of tumor apoptosis. Analytical Chemistry 81 (13), 5517–5523. Wlodkowic, D., Skommer, J., Darzynkiewicz, Z., 2010. Cytometry in cell necrobiol-ogy revisited: recent advances and new vistas. Cytometry A 77 (7), 591–606. Wlodkowic, D., Skommer, J., Faley, S., Darzynkiewicz, Z., Cooper, J.M., 2009b
Grant sponsors: On-chip biotechnologies Co. Ltd
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- Ja
- Kt
- Yf
- Dw
Grant sponsors: On-chip biotechnologies Co. Ltd. (JS, JA, KT, YF, DW);
Three-dimensional hydrodynamic focusing in a microfluidic Coulter counter High-throughput and high-resolution flow cytometry in molded microfluidic devices
- R Scott
- P Sethu
- C K Harnett
Scott, R., Sethu, P., Harnett, C.K., 2008. Three-dimensional hydrodynamic focusing in a microfluidic Coulter counter. Review of Scientific Instruments 79 (4), 046104. Simonnet, C., Groisman, A., 2006. High-throughput and high-resolution flow cytometry in molded microfluidic devices. Analytical Chemistry 78 (16), 5653–5663.