Microfluidic fluorescence-activated cell sorting (μFACS) chip with integrated piezoelectric actuators for low-cost mammalian cell enrichment

A low-cost, microfluidic fluorescence-activated cell sorting (μFACS) microchip integrated with two piezoelectric lead–zirconate–titanate actuators was demonstrated for automated, high-performance mammalian cell analysis and enrichment. In this PDMS–glass device, cells were hydrodynamically focused into a single file line in the lateral direction by two sheath flows, and then interrogated with a forward scattering and confocal fluorescent detection system. The selected cells were displaced transversely into a collection channel by two piezoelectric actuators that worked in a pull–push relay manner with a minimal switching time of ~0.8 ms. High detection throughput (~2500 cells/s), high sorting rate (~1250 cells/s), and high sorting efficiency (~98%) were successfully achieved on the μFACS system. Six cell mixture samples containing 22.87% of GFP-expressing HeLa cells were consecutively analyzed and sorted on the chip, revealing a stable sorting efficiency of 97.7 ± 0.93%. In addition, cell mixtures containing 37.65 and 3.36% GFP HeLa cells were effectively enriched up to 83.82 and 78.51%, respectively, on the microchip, and an enrichment factor of 105 for the low-purity (3.36%) sample was successfully obtained. This fully enclosed, disposable microfluidic chip provides an automated platform for low-cost fluorescence-based cell detection and enrichment, and is attractive to applications where cross-contamination between runs and aerosol hazard are the primary concerns.     

Simulation-Based Analysis of Dielectrophoretic Field Flow Fractionation Devices

Simulation of microfluidic devices is very difficult due to the interaction and coupling between multiple energy domains. This article presents an innovative technique to simulate dielectrophoretic forces and laminar flows in microfluidic devices. Lab-on-a-chip systems, or biochips, are one of the fastest growing sectors in the life sciences industry. These systems employ miniaturization of biological separation and assay techniques to enable multiple, complex analyses on a single chip. Separation of micron-sized particles and cells is critical in many biochemical-analysis and high-throughput-screening applications. Field flow fractionation (FFF) using dielectrophoresis (DEP) is fast becoming an established methodology for sorting and manipulating particles and cells.     

基于图像配准的食品包装印刷缺陷检测与实现

传统食品包装印刷缺陷检测系统采集的实时图像和标准图像在空间上存在着较大的差异,在缺陷检测前首先要将实时图像与标准图像配准,再进行图像缺陷检测与识别。针对传统检测方法检测时间长、分拣效率低、漏检率高和对人视觉要求高等缺点,在图像增强处理的基础上,提出了一种适用于食品包装印刷缺陷检测的图像配准算法。该算法利用小波变换改进算法对图像边缘进行检测,有效地解决了噪声所产生的误检问题。实验仿真结果表明,该算法具有较高的稳定性和可靠性,能够精确检测出小于0.1 mm的刀丝和拉条等细微缺陷,实现了食品包装印刷品的无损检测。     

Fluorescence detection in a micro flow cytometer without on-chip fibers

This paper describes a novel concept of integrated on-chip fiber free laser-induced fluorescence detection system. The poly-dimethylsiloxane (PDMS) chip was fabricated using soft lithography and was bonded with a glass substrate of 150 μm thickness that reduced the distance of channel-to-sidewall to less than 180 μm. The cells and particles detection was conducted by an external single fiber close to the glass substrate that transmitted laser light for simultaneous excitation and receipt of the emission light signals. The performance of the proposed device was demonstrated using fluorescence beads, stained white blood cells, and yeast cells. The experimental results showed the simplicity and flexibility of the proposed device configuration which can provide convenient on-chip integration interface for fast, high throughput, and low-cost laser-induced fluorescence detection micro flow cytometer.     

Driving and sorting of the fluorescent droplets on digital microfluidic platform

In this paper, we present a digital microfluidic droplet sorting platform to achieve automated droplet sorting based on fluorescent detection. We design and fabricate a kind of digital microfluidic chip for manipulating nano-liter-sized liquid droplets, and the chip is integrated with a fluorescence-initiated feedback system for real-time sorting control. The driving and sorting characteristics of fluorescent droplets encapsulating fluorescent-labeled particles are studied on this platform. The droplets dispensed from on-chip reservoir electrode are transported to a fluorescence detection site and sorted according to their fluorescence signals. The fluorescent droplets and non-fluorescent droplets are successfully separated and the number of fluorescent particles inside each droplet is quantified by its fluorescent intensity. We realize droplet sorting at 20 Hz and obtain a linear relationship between the fluorescent particle concentrations and the fluorescence signals. This work is easily adapted for sorting out fluorescent-labeled microparticles, cells and bacteria and thus has the potential of quantifying catalytic or regulatory bio-activities.     

A portable,hand-powered microfluidic device for sorting of biological particles

Manually hand-powered portable microfluidic devices are cheap alternatives for point-of-care diagnostics. Currently, on-field tests are limited by the use of bulky syringe pumps, pressure controller and equipment. In this work, we present a manually operated microfluidic device incorporated with a groove-based channel. We show that the device is capable to effectively sort particles/cells by manual hand powering. First, the grooved-based channel with differently sized polystyrene particles was characterized using syringe pumps to study their distributions under various flow rate conditions. Afterward, the particle mixtures were sorted manually using hand power to verify the capability of this device. Finally, the manually operated device was used to sort platelets from peripheral blood mononuclear cells (PBMCs). The platelets were collected with a purity of ~ 100%. The purity of PBMCs was enhanced from 0.8 to 10.4% after multiple processes which results in an enrichment ratio of 13.8. During the process of manual hand pumping, the flow fluctuation caused by unstable injection will not influence the sorting performance. Due to its simplicity, this manually operated microfluidic chip is suitable for outfield settings.     

无透镜微流控成像流动细胞检测与计数系统

在未来面向个人化的生物医疗诊断中,实时的细胞检测与计数具有重要需求.现有的细胞检测和计数系统例如流式细胞仪和血细胞计数器不适用于小型化流动细胞实时检测和计数.通过将CMOS图像传感器芯片和微流控芯片结合,提出了一种用于流动细胞检测和计数的无透镜微流控成像系统,与用于计数静态细胞的其它无透镜微流控成像系统不同,该系统可以通过基于时域差分的运动检测算法检测和计数微流体通道中连续流动的细胞样本.测试结果表明:该系统可以对微流控通道中流动的人体骨髓基质细胞实现自动检测和计数,并具有-6.53％的低统计错误率.该系统提供了面向未来即时应用的细胞检测和计数解决方案.     

Fabrication of the PDMS microchip for serially diluting sample with buffer

We developed a polymer polydimethylsiloxane (PDMS) based microfluidic device that dilutes biological samples with buffer solutions with serially increasing diluted sample concentrations. The device showed the relatively high accuracy in terms of dilution ratios along with the fact that it was faster and easier to operate. With two simple disposable plastic syringes, the plastic microfluidic chip completes ordinary sample dilution sequences faster and more precisely than the conventional manual pipette process in biological or chemical laboratory. The serially diluting mechanism of the microchip is simply that the number of microchannels with the same flow rate determines the total amount of flow into the wells. The soft lithography fabricated the microchannels of tetragonal section length of 50 m of each side of the microfluidic chip. This paper was supported by the Nano Bioelectronics and Systems Research Center of Seoul National University, which is an Engineering Research Center supported by the Korean Science and Engineering Foundation (KOSEF).     

Detection of Cellular Parameters Using a Microfluidic Chip-Based System

Lab-on-a-chip technology achieves a reduction of sample and reagent volume and automates complex laboratory processes. Here, we present the implementation of cell assays on a microfluidic platform using disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow inside networks of microfluidic channels. Cells are hydrodynamically focused and pass the fluorescence detector in single file. Initial applications are the determination of protein expression and apoptosis parameters. The microfluidic system allows unattended measurement of six samples per chip. Results obtained with the microfluidic chips showed good correlation with data obtained using a standard flow cytometer.     

高速动态图像目标识别算法的设计与实现

为了实现对高速动态图像进行稳定的快速识别,并将识别出的目标图像传给上位机,应用优化的背景差分原理,设计并验证了一种可并行计算的高速动态目标识别算法,利用FPGA作为主控芯片实现了有效图像的高效采集以及实时传输功能;首先该系统采用流水线处理方式实现对数据的实时采集,然后利用乒乓操作来实现目标识别算法,通过对DDR3进行分页操作,将识别后的动态目标图像进行缓存,最后采用USB3.0芯片实现上位机与FPGA进行实时传输数据;实验结果表明,所设计的动态目标高速识别算法可以有效识别出6 mm的BB弹,捕获率高达99%,同时该系统可以实现动态目标数据的实时传输。     

Strategies for implementing hardware-assisted high-throughput cellular image analysis

Recent advances in imaging technology for biomedicine, including high-speed microscopy, automated microscopy, and imaging flow cytometry are poised to have a large impact on clinical diagnostics, drug discovery, and biological research. Enhanced acquisition speed, resolution, and automation of sample handling are enabling researchers to probe biological phenomena at an increasing rate and achieve intuitive image-based results. However, the rich image sets produced by these tools are massive, possessing potentially millions of frames with tremendous depth and complexity. As a result, the tools introduce immense computational requirements, and, more importantly, the fact that image analysis operates at a much lower speed than image acquisition limits its ability to play a role in critical tasks in biomedicine such as real-time decision making. In this work, we present our strategy for high-throughput image analysis on a graphical processing unit platform. We scrutinized our original algorithm for detecting, tracking, and analyzing cell morphology in high-speed images and identified inefficiencies in image filtering and potential shortcut routines in the morphological analysis stage. Using our "grid method" for image enhancements resulted in an 8.54× reduction in total run time, whereas origin centering allowed using a look up table for coordinate transformation, which reduced the total run time by 55.64×. Optimization of parallelization and implementation of specialized image processing hardware will ultimately enable real-time analysis of high-throughput image streams and bring wider adoption of assays based on new imaging technologies.     

一种嵌入式图像记录设备的设计与实现

主要论述在钞票识别设备中实现整幅钞票图像实时存储的方法，采用嵌入式的存储方法，以降低成本，为了解决嵌入式系统资源有限的矛盾，采用多种高效、低耗的处理方法，能够实现在纸币清分机等高速识别钞票的设备中，实时把钞票图像存储起来，还能够实现钞票图像的实时查询。     

基于FPGA的运动目标远程监视系统设计

运动目标检测是智能安防系统的重要组成部分,为了满足安防系统远距离监视目标以及视频传输实时性等需求,设计了一种基于FPGA平台的运动目标远程监视系统;该系统以Xilinx公司的Artix7系列FPGA芯片为核心,通过OV5640摄像头实现视频图像的采集,将采集到的图像进行灰度化处理,并通过DDR3存储器缓存处理后的图像,采用帧间差分法运动目标检测技术实现对多个运动物体的检测与标记,将检测结果通过以太网的UDP协议传输到上位机实时显示;实验结果表明,在图像分辨率为640*480时,以太网UDP传输速度为133Mbit/s,视频图像帧率为26fps,大于人眼的可视帧率24fps,满足视频传输实时性的要求,同时该系统能够远距离、高效地检测与跟踪多个运动目标,相比于其他系统具有可远程实时检测、小型化、低功耗的特点,可进一步应用到智能安防系统中。     

Continuous-flow ferrohydrodynamic sorting of particles and cells in microfluidic devices

A new sorting scheme based on ferrofluid hydrodynamics (ferrohydrodynamics) was used to separate mixtures of particles and live cells simultaneously. Two species of cells, including Escherichia coli and Saccharomyces cerevisiae, as well as fluorescent polystyrene microparticles were studied for their sorting throughput and efficiency. Ferrofluids are stable magnetic nanoparticles suspensions. Under external magnetic field gradients, magnetic buoyancy forces exerted on particles and cells lead to size-dependent deflections from their laminar flow paths and result in spatial separation. We report the design, modeling, fabrication and characterization of the sorting device. This scheme is simple, low-cost and label-free compared to other existing techniques.     

Development of a biological detection platform utilizing a modular microfluidic stack

The goal of this project is to build a miniaturized, user-friendly cytometry setup (Datta et al. in Microfluidic platform for education and research. COMS, Baton Rouge, 2008; Frische et al. in Development of an miniaturized flow cytometry setup for visual cell inspection and sorting. Baton Rouge, Project Report, 2008) by combining a customized, microfluidic device with visual microscope inspection to detect and extract specific cells from a continuous sample flow. We developed a cytological tool, based on the Coulter particle counter principle, using a microelectrode array patterned on a borosilicate glass chip as electrical detection set-up which is fully embedded into a polymeric multi-layer microfluidic stack. The detection takes place between pairs of coplanar Cr/Au microelectrodes by sensing an impedance change caused by particles continuously carried within a microfluidic channel across the detection area under laminar flow conditions. A wide frequency range available for counting provides information on cell size, membrane capacitance, cytoplasm conductivity and is potentially of interest for in-depth cell diagnostic e.g. to detect damaged or cancerous cells and select them for extraction and further in-depth analysis.     

Microfabricated fluorescence-activated cell sorter through hydrodynamic flow manipulation

In this paper, we present a novel flow manipulation and signal detection scheme for a microfabricated fluorescence-activated cell sorter (μFACS). Optics setup was built on an inverted fluorescence microscope (IX71, Olympus, USA) and microchips were made of PDMS using soft lithography. Perfectly aligned axial illumination setup of the commercial microscope ensures effective signal detection. Hydrodynamic flow manipulation scheme is adopted for both flow focusing and cell sorting. Fast and robust sorting function is utilized by a nozzle flow from wide actuation channel. The sorting function is verified through real-time high-speed imaging. The captured images are used for adjusting delays between signal detection and sorting activation. The system automatically adjusts itself to any flow conditions, and so no adjustment is made to the flow parameters between runs. We believe that the proposed techniques will help breaking through the obstacles of μFACS system development.     

Cell viability assessment by flow cytometry using yeast as cell model

This paper reports the new combination of cell sorting and counting capabilities on a single device. Most state-of-the-art devices combining these technologies use optical techniques requiring complicated experimental setups and labeled samples. The use of a label-free, electrical device significantly decreases the system complexity and makes it more appropriate for use in point-of-care diagnostics.Living and dead yeast cells are separated by dielectrophoretic forces and counted using coulter counters. The combination of these two methods allows the determination of the percentage of living and dead cells for viability studies of cell samples. The device could further be used for sorting and counting of blood cells in applications such as diagnosis of insufficient cell concentrations, identification of cell deficiencies or bacterial contamination. The use of dielectrophoresis (DEP) as sorting principle allows to separate cells based on their dielectric properties in the place of size-based separation, enabling sorting of large panels of cells and separation of infected and non-infected cells of the same type.     

双目三维定位的视频运动检测控制系统设计

针对TI公司的视频处理芯片TMS320DM642设计一款视频检测运动控制卡,采用CPLD图像采集,运用视频解码芯片SAA7111和视频编码芯片SAA7105实现采集图像的编解码过程,从而实现对32路舵机的并行控制。此款控制卡可以进行颜色识别,实现双目三维定位,可应用在移动机器人领域。主控程序采用DSP/BIOS嵌入式实时操作系统,实现控制卡对目标形心的实时标定,以及发送对32路舵机并行控制的相关命令,从而实现对目标物体的实时视频追踪与抓取。     

Pre-programmable polymer transformers as on-chip microfluidic vacuum generators

This paper develops novel polymer transformers using thermally actuated shape memory polymer (SMP) materials. This paper applies SMPs with thermally induced shape memory effect to the proposed novel polymer transformers as on-chip microfluidic vacuum generators. In this type of SMPs, the morphology of the materials changes when the temperature of materials reaches its glass transition temperature (T _g). The structure of the polymer transformer can be pre-programmed to define its functions, which the structure is reset to the temporary shape, using shape memory effects. When subjected to heat, the polymer transformer returns to its pre-memory morphology. The morphological change can produce a vacuum generation function in microfluidic channels. Vacuum pressure is generated to suck liquids into the microfluidic chip from fluidic inlets and drive liquids in the microchannel due to the morphological change of the polymer transformer. This study adopts a new smart polymer with high shape memory effects to achieve fluid movement using an on-chip vacuum generation source. Experimental measurements show that the polymer transformer, which uses SMP with a T _g of 40°C, can deform 310 μm (recover to the permanent shape from the temporary shape) within 40 s at 65°C. The polymer transformer with an effective cavity volume of 155 μl achieved negative pressures of −0.98 psi. The maximum negative up to −1.8 psi can be achieved with an effective cavity volume of 268 μl. A maximum flow rate of 24 μl/min was produced in the microfluidic chip with a 180 mm long channel using this technique. The response times of the polymer transformers presented here are within 36 s for driving liquids to the end of the detection chamber. The proposed design has the advantages of compact size, ease of fabrication and integration, ease of actuation, and on-demand negative pressure generation. Thus, this design is suitable for disposable biochips that need two liquid samples control. The polymer transformer presented in this study is applicable to numerous disposable microfluidic biochips.     

Automated Lab-on-a-Chip Analysis of DNA Fragments

The design of a fully automated system for the analysis of DNA fragments in 96-well plates is described. Microfluidic technology is used to integrate sample loading, electrophoretic analysis, and fragment detection onto a miniature lab-on-a-chip device. The microfluidic chip operates in an instrument platform that automates sample access, data collection, and data reporting. Each microfluidic chip provides sizing and concentration values for more than 1000 DNA samples.