Microchip imaging cytometer:making healthcare available,accessible,and affordable

The Microchip Imaging Cytometer(MIC)is a class of integrated point-of-care detection systems based on the combination of optical microscopy and flow cytometry.MIC devices have the attributes of portability,cost-effectiveness,and adaptability while providing quantitative measurements to meet the needs of laboratory testing in a variety of healthcare settings.Based on the use of microfluidic chips,MIC requires less sample and can complete sample preparation automatically.Therefore,they can provide quantitative testing results simply using a finger prick specimen.The decreased reagent consumption and reduced form factor also help improve the accessibility and affordability of healthcare services in remote and resource-limited settings.In this article,we review recent developments of the Microchip Imaging Cytometer from the following aspects:clinical applications,microfluidic chip integration,imaging optics,and image acquisition.Following,we provide an outlook of the field and remark on promising technologies that may enable significant progress in the near future.

Algorithm to identify circulating tumor cell clusters using in vivo flow cytometer

Recent studies in oncology have addressed the importance of detecting circulating tumor cell clusters because circulating tumor cell clusters might survive and metastasize more easily than single circulating tumor cells.Signals with larger peak widths detected by in vivo flow cytometer(IVFC)have been used to identify cell clusters in previous studies.However,the accuracy of this criterion might be greatly degraded by variance in blood°ow and the rolling behaviors of circulating tumor cells.Here,we propose a criterion and algorithm to distinguish cell clusters from single cells.In this work,we first used area-based and volume-based models for single°uorescent cells.Simulating each model,we analyzed the corresponding morphology of IVFC signals from cell clusters.According to the Rayleigh criterion,the valley between two adjacent peak signals from two distinguishable cells should be lower than 73.5%of the peak values.A novel signal processing algorithm for IVFC was developed based on this criterion.The results showed that cell clusters can be reliably identied using our proposed algorithm.Intravital imaging was also performed to further support our algorithm.With enhanced accuracy,IVFC is a powerful tool to study circulating cell clusters.

流式细胞仪平台支撑本科细胞生物学实验课教学改革的实践

在流式细胞术检测细胞凋亡实验中,借助流式细胞仪平台,从教学模式、实验内容、仪器准备、教学素材等方面对细胞生物学实验课进行改革,取得了较好的教学效果。这种仪器平台与教学团队共同探索教学改革的模式,充分发挥大型仪器设备在人才培养方面的潜能,可为其他高校仪器平台建设提供参考。

流式细胞仪样品预处理系统的设计与实现

根据流式细胞仪对样品“均一化”的需求,研制了一套基于微流控细胞分选技术的流式细胞仪样品预处理系统,装置可以实现对粒径小于100µm细胞或微粒的驱动和分选.系统主要包含微流控分选芯片和样品驱动模块两部分,通过聚焦不同位置,实现了对不同粒径细胞/颗粒的有效分离.系统无需对细胞进行标记处理,经分选的细胞便于后续流式细胞仪检测.经验证,系统能够有效去除牡蛎血淋巴细胞样品中的大粒子杂质,提高细胞样品的稳定性和均一性,增加流式细胞仪检测结果的准确性.

依托大型科研仪器开展创新型本科实验教学的设计与研究

大型科研仪器是先进科学技术的体现,是培养创新型人才的重要基础条件和硬件保障,是开展高水平实验教学的宝贵资源。为促进创新型研究人才的培养,拓展生物医学本科实验教学内容,该研究设计了一套依托流式细胞仪开展的本科实验教学的具体方案,针对经典的“细胞增殖/细胞周期”问题,分别设计两个不同原理的流式细胞术进行对比实验。实践表明,依托流式细胞仪开展对比实验教学,能加深本科生对大型科研仪器的了解,培养学生扎实的动手能力,激发学生的创新性思维和科研热情。

STUDYING THE ROLE OF MACROPHAGES IN CIRCULATING PROSTATE CANCER CELLS BY IN VIVO FLOW CYTOMETRY

Metastasis is a very complicated multi-step process and accounts for the low survival rate of the cancerous patients.To metastasize,t he malignant cells must detach from the primary tumor and migrate to secondary sites in the body through either blood or lymph circulation.Macrophages appear to be directly involved in tumor progression and metastasis.However,the role of macrophages in affecting cancer metast asis has not been fully elucidated.Here,we have utilized an emerging technique,namely in vivo flow cytometry(IVFC)to study the depletion kinetics of circulating prostate cancer cells in mice and determine how depletion of macrophages by the liposome encapsulated clodronate affects the depletion kinetics.Our results show diferent depletion kinetics of PC-3 cells between the macrophagedeficient group and the control group.The number of circulating tumor cells(CTCs)in the macrophage-deficient group decreases in a slower manner compared to the control mice group.The differences in depletion kinetics indicate that the absence of macrophages facilitates the stay of prostate cancer cells in circulation.In addition,our imaging data suggest that macrophages might be able to arrest,phagocytose and digest PC-3 cells.Therefore,phagocy tosis may mainly contribute to the de-pletion kinetic diferences.The developed methods elaborated here would be useful to study the relationship between macr ophages and tumor metastasis in small animal cancer models.

Capillary Micro-flow Through a Fiber Bundle(Part 2)

A numerical model was proposed to simulate the capillary micro-flow through a fiber bundle. The capillary pressure was predicted by the Young-Laplace equation and the corresponding optimal values of permeability were found by a trial-and-error method. The empirical Kozeny constants which are dependent on fiber volume fraction were recormnended for the prediction of permeability.

Fabrication of micro-flow channels on graphite composite bipolar plates using microplaning

A new method of manufacturing micro-flow channels on graphite composite bipolar plate(GCBPP) microplaning using specially designed multi-tooth tool is proposed. In this method, several or even dozens of parallel micro-flow channels ranging from 100 μm to 500 μm in width can be produced simultaneously. But, edge chippings easily occur on the rib surface of GCBPP during microplaning due to brittleness of graphite composites. Experimental results show that edge chippings result in the increase of contact resistance between bipolar plate and carbon paper at low compaction force. While the edge chippings scarcely exert influence on the contact resistance at high compaction force. Contrary to conventional view, the edge chippings can significantly improve performance of microfuel cell and big edge chippings outperform small edge chippings. In addition, the influence of technical parameters on edge chippings was investigated in order to obtain big, but not oversized edge chippings.

Research on Micro-Flow Self-Sensing Actuators Based on Piezoelectric Ceramic Stack `

The paper is concerned with the micro-flow self-sensing actuators,the work of which is based on the secondary piezoelectric effect. The piezoelectric ceramic stack can yield micro-displacement due to its first inverse piezoelectric effect. Therefore,we apply this micro-displacement to cell micro-flow injection. Moreover, due to the charge of the secondary direct piezoelectric effect,the piezoelectric ceramic stack is able to detect the force and displacement in the injection by itself. The experiments of first inverse piezoelectric effect and secondary direct piezoelectric effect are conducted. The experiment results show that,subjected to 0- 60 V input,the piezoelectric ceramic stack can generate 13. 45 μm displacement,and control accuracy can achieve 2 nm. It can completely meet the needs of cell micro-flow injection. Also,the experiments demonstrate that the micro-displacement due to the first inverse piezoelectric effect can be well self-sensed by the electric charge due to the secondary direct piezoelectric effect.

Formation mechanism of micro-flows in aqueous poly(ethylene oxide) droplets on a substrate at different temperatures

The drying of aqueous poly（ethylene oxide） （PEO） droplet on a substrate at different temperatures was studied. It was found that the contact line receded when the substrate was at a temperature above 60 ℃. Different nucleation behavior and surface profiles of PEO films were found in different droplets drying processes. The rheological properties of aqueous PEO solutions were studied to understand the mechanism of contact line recession and micro-flow in drying aqueous PEO droplets. It was found that at low temperature, the contact line was static because of great viscous stress; while at high temperature, it receded because of great Marangoni force and the decrease of viscous stress. It was indicated that Marangoni convection was inhibited by the outward capillary flow and viscous stress at low temperature, whereas it became dominant at high temperature. Two types of mechanism for surface profiles and nucleation of PEO film from drying droplets are proposed, providing a theoretical guide for polymer solution application in oil and gas foam flooding technology.

The Role of Kappa and Lambda in Subclassification of B Cell Lymphoblastic Leukemia in Sudanese Patients Using Flow Cytometry

Background: B-cell Acute lymphoblastic leukemia (B-ALL) is a neoplasm of lymphoblasts which are of B-cell lineage typically composed of small to medium sized blast cells, moderately condensed to dispersed chromatin with scanty cytoplasm and inconspicuous nucleoli, involving the bone marrow and/or blood. Methods and materials: This is a prospective cross-sectional study in which 50 blood and/or bone marrow samples of newly diagnosed patients (B-ALL) were tested for immunophenotyping. All samples were prepared for surface and cytoplasmic markers including kappa and lambda human antibody for 10 minutes in dark place and then run by the Flow Cytometer. Results: 64% of the study populations were males and 36% were females. Cases were classified according to immunophenotype and the age into different subtypes and showed the following frequencies: Pro B-ALL (8%), early pre B-ALL (56%), common B-ALL (16%), Pre-B-ALL (14%) and Mature B-ALL (only 6%). Surface immunoglobulin was positive in 10% and negative in 90% of all patients, showing 100% positivity in mature B-ALL and totally negative in other subtypes. While cytoplasmic immunoglobulin was positive in 16% and negative in 84% of all patients and was positive in 100% of Pre-B-ALL and in 50% of mature B-ALL. Surface kappa was more expressed in mature B-ALL than lambda giving a ratio of 2:1, while cytoplasmic kappa:lambda was 6:1 in Pre-B-ALL. Conclusion: Kappa and lambda have important role in B-ALL classification which necessitates their presence in immunophenotyping of B-ALL.

流式细胞分选仪研究进展与展望

流式细胞技术一直是生物细胞研究的重要手段,近年来流式细胞分选仪在细胞富集、筛选后再利用等方面的应用使得对能够满足高通量、高细胞信息提取、高细胞活性要求的流式细胞分选仪的需求增加。本文阐述基于光学探测方法的流式细胞分选仪结构、工作原理及其发展现状出发;提出在光学流式细胞分选仪研究领域出现无标记化和微流控芯片化两种新的发展趋势。在两种发展趋势下,无标记成像方法、微流控片上分选系统和片上压电致动器集成分选等新兴技术越来越多地应用到光学流式细胞分选仪的研发设计中,本文从技术特性、性能优势、应用前景等方面对这些技术的研究进展以及面临的挑战进行了总结和展望,供流式细胞分选技术的使用者及研究者参考。

基于流式细胞术分选绵羊分泌型IgA包裹的消化道细菌

本研究旨在建立1种基于流式细胞术(flow cytometry,FCM)的反刍动物消化道内容物中分泌型免疫球蛋白A(secretory immunoglobulin A,sIgA)包裹细菌的分选方法。随机选用4只健康、体况良好的成年肉用绵羊,采集回肠内容物制备菌液。对菌液上样浓度、细菌染料4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole,DAPI)最佳工作浓度和抗IgA抗体最佳使用浓度等进行优化。结果表明,内容物菌液最佳浓度为2.5~5 mg·mL^(-1),DAPI最佳工作浓度为5μg·mL^(-1),抗IgA抗体最佳使用量为5μg。重复性试验表明,该方法相对偏差小于5%,具有良好的稳定性和可重复性。本试验建立的sIgA包裹细菌分选方法选择性强、样品用量少、操作简单,可为反刍动物消化道sIgA包裹细菌的组成及功能等相关研究提供技术支撑。

高脂饮食及昼夜颠倒对小鼠脾脏免疫功能的影响

探究高脂饮食以及昼夜节律颠倒对于小鼠脾脏免疫功能的影响。将6~7周龄雄性C57BL/6J小鼠分为正常膳食组(CON)和高脂膳食组(HFD),每组根据进食时间又分为白天进食(D)和夜间进食(N),总计4组,每组10只,即:颠倒饮食组(DC)、白天进食高脂组(DH)、正常节律正常饮食组(NC)和高脂进食组(NH)。小鼠饲养18周后,取脾脏组织及血液分别进行B淋巴细胞分离、流式细胞检测。结果显示:与夜间正常膳食组小鼠脾脏系数0.37相比,颠倒饮食组小鼠脾脏指数为0.4,高脂进食组小鼠脾脏指数为0.41,指数增加,流式细胞检测结果显示正常节律正常饮食组小鼠脾脏B细胞水平显著高于颠倒饮食组小鼠脾脏B细胞水平(P<0.001),正常节律正常饮食组小鼠脾脏B细胞水平也显著高于高脂进食组小鼠脾脏B细胞水平(P<0.01),提示昼夜颠倒饮食以及高脂饮食均会导致小鼠免疫系统发生紊乱。因此,颠倒饮食和高脂饮食小鼠B淋巴细胞水平均显示节律性波动,但和正常小鼠相比,波动性较为紊乱或颠倒。这些结果表明,高脂饮食以及颠倒饮食均会导致B淋巴细胞水平的降低和免疫功能的损害,对免疫系统产生不利影响。

高校开放性高效流式平台的管理实践

配备多种流式仪是生命科学学科建设与高端实验平台发展的趋势,流式实验的特性为流式平台的管理带来挑战。以南开大学生命科学学院的流式平台为例,通过全方位协调,打造高效的开放性流式平台,并对流式平台的管理方式和运行效益进行了分析,以期为其他高校流式平台的管理提供参考。

Temporal change of plankton size structure preserved by Lugol's solution:a FlowCAM study

Plankton size structure is crucial for understanding marine ecosystem dynamics and the associated biogeochemical processes.A fixation step by acid Lugol’s solution has been commonly employed to preserve plankton samples in the field.However,the acid Lugol’s solution can bias the estimation of size structure and the preserved plankton size structure can vary with time.Here,we explore the impact of sample storage time on the size-structure of the plankton community preserved by Lugol’s solution.Two short-term experiments and one long-term experiment were conducted to explore the change of plankton community size structure with the storage time:covering from a week to a month,and to nearly seven months based on particle-size data obtained by continuous Flow Cytometer and Microscope(FlowCAM)measurements.We found a linear change of plankton size with the storage time in short-term periods(less than 3 months)with a decrease of the slope but an increase of the intercept for the normalized biomass size spectrum(NBS S).However,there were opposite trends for NBSS with increasing slope but decreasing intercept after3 months.The potential causes of the distinct patterns of the NBSS parameters are addressed in terms of the interplay between particle aggregation and fragmentation.We found large changes in plankton biovolume and abundance among different size classes,which may indicate a distinct effect of acid Lugol’s solution on various plankton size classes.The mechanism driving temporal change in the size-structure of the Lugolfixed plankton community was further discussed in terms of particle aggregation and fragmentation.Finally,we emphasize that the effect of storage time should be taken into account when interpreting or comparing data of plankton community acquired from samples with various storage durations.

Micro-flow structure at regime transition from bubbling to turbulent fluidization in a fluidized bed

Gas-solid fluidized beds have found extensive utilization in frontline manufacturing,in particular as low-velocity beds.The fluidization status,the bubbling or turbulent flow regime and the transition in between,determine the system performance in practical applications.Though the convoluted hydrodynamics are quantitively evaluated through numerous data-processing methodologies,none of them alone can reflect all the critical information to identify the transition from the bubbling to the turbulent regime.Accordingly,this study was to exploit a coupling data processing methodology,in the combination of standard deviation,power spectrum density,probability density function,wavelet transform,and wavelet multiresolution method,to jointly explain the micro-flow structure at the regime transition from bubbling to turbulent fluidization.The transient differential pressure fluctuation was measured for the evaluation in a fluidized bed(0.267 m i.d.×2.5 m height)with FCC catalysts(d_(p)=65μm,ρ_(p)=1780kg/m^(3))at different superficial gas velocities(0.02–1.4 m/s).The results show that the onset of turbulent fluidization starts earlier in the top section of the bed than in the bottom section.The wavelet decomposition displays that the fluctuation of differential pressure mainly concentrates on the sub-signals with an intermediate frequency band.These sub-signals could be synthesized into three types of scales(micro-scale,meso-scale,and macro-scale),representing the multi-scale hydrodynamics in the fluidized bed.The micro-scale signal has the characteristic information of bubbling fluidization,and the characteristic information of turbulent fluidization is mainly represented by the meso-scale signal.This work provides a systematic comprehension of fluidization status assessment and serves as an impetus for more coupling analysis in this sector.

不同倍性鱼的血细胞和精子DNA含量比较

The flow cytometer was used to compare the DNA content in the blood cells and sperm in the new type of tetraploid (G 1×4n) and in red crucian carp used as the control. The new type of tetraploid (G 1×4n) was produced by mating the diploid female gynogenetic progeny (first generation,G 1) of the tetraploid hybrid of red crucain carp × common carp, with the male of the tetraploid hybrid. Chromosome spreads from the kidney tissue in the new type of tetraploid were also examined. The results from the flow cytometer indicated that the mean DNA content of the diploid sperm produced by the new type of tetraploid was twice that of the haploid sperm generated by the diploid red crucian carp, and the mean DNA content of the blood cells in the tetraploid was twice that in red crucian carp. Examination of chromosome spreads from the kidney tissue also indicated that the examined samples of the G 1×4n were tetraploids with 200 chromosomes, which was in agreement with the results of the DNA content measurements from the sperm and the blood cells. The present study proved that the new type of tetraploids was able to produce normal diploid sperm, which fertilized diploid eggs from the new type of tetraploid to produce viable offspring. The flow cytometer was proved to be one of the accurate, secure and simple methods to distinguish the different ploidy fish in blood cells and sperm by measuring their DNA content .

流式细胞仪鉴定猕猴桃倍性技术研究

利用流式细胞仪(Flow cytometer,FCM)对不同倍性猕猴桃DNA的相对含量进行测定,建立一套简易、快速的鉴定猕猴桃倍性方法。以二倍体"红阳"猕猴桃为对照,‘81-5'、‘皖翠'猕猴桃为试材,通过筛选最优鉴定叶片部位;改良解离液配方;提高滤膜的目数;过滤次数;增加离心次数等优化鉴定技术。结果表明,露地栽培条件下,茎尖下初展叶是倍性鉴定的最佳部位;两次离心;解离液中2%PVP-30;500目滤膜抽滤两次可以有效解决仪器堵管现象,有效去除杂质峰并获得清晰样品峰。通过优化后的鉴定技术可实现同时测定2个以上倍性的猕猴桃混合样品。