Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions.

IF 3.3 Q2 ENGINEERING, BIOMEDICAL International Journal of Biomedical Imaging Pub Date : 2017-01-01 Epub Date: 2017-11-06 DOI:10.1155/2017/8318906
Jacopo Biasetti, Kaushik Sampath, Angel Cortez, Alaleh Azhir, Assaf A Gilad, Thomas S Kickler, Tobias Obser, Zaverio M Ruggeri, Joseph Katz
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引用次数: 3

Abstract

Tracking cells and proteins' phenotypic changes in deep suspensions is critical for the direct imaging of blood-related phenomena in in vitro replica of cardiovascular systems and blood-handling devices. This paper introduces fluorescence imaging techniques for space and time resolved detection of platelet activation, von Willebrand factor (VWF) conformational changes, and VWF-platelet interaction in deep suspensions. Labeled VWF, platelets, and VWF-platelet strands are suspended in deep cuvettes, illuminated, and imaged with a high-sensitivity EM-CCD camera, allowing detection using an exposure time of 1 ms. In-house postprocessing algorithms identify and track the moving signals. Recombinant VWF-eGFP (rVWF-eGFP) and VWF labeled with an FITC-conjugated polyclonal antibody are employed. Anti-P-Selectin FITC-conjugated antibodies and the calcium-sensitive probe Indo-1 are used to detect activated platelets. A positive correlation between the mean number of platelets detected per image and the percentage of activated platelets determined through flow cytometry is obtained, validating the technique. An increase in the number of rVWF-eGFP signals upon exposure to shear stress demonstrates the technique's ability to detect breakup of self-aggregates. VWF globular and unfolded conformations and self-aggregation are also observed. The ability to track the size and shape of VWF-platelet strands in space and time provides means to detect pro- and antithrombotic processes.

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深悬液中血小板活化和血管性血友病因子构象变化的时空分辨检测。
在深度悬浮液中追踪细胞和蛋白质的表型变化对于在体外复制心血管系统和血液处理装置中直接成像血液相关现象至关重要。本文介绍了用于深度悬浮液中血小板活化、血管性血血病因子(VWF)构象变化和VWF-血小板相互作用的时空分辨荧光成像技术。标记的VWF、血小板和VWF-血小板链悬浮在深比皿中,照明,并用高灵敏度EM-CCD相机成像,曝光时间为1ms。内部后处理算法识别和跟踪移动信号。采用重组VWF- egfp (rVWF-eGFP)和fitc偶联多克隆抗体标记的VWF。使用抗p -选择素fitc偶联抗体和钙敏感探针Indo-1检测活化血小板。每张图像检测到的平均血小板数量与通过流式细胞术确定的活化血小板百分比呈正相关,验证了该技术。暴露于剪切应力下rVWF-eGFP信号数量的增加证明了该技术检测自聚集体破裂的能力。还观察到VWF的球状和未展开构象和自聚集。在空间和时间上跟踪vwf -血小板链的大小和形状的能力为检测促血栓和抗血栓过程提供了手段。
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来源期刊
CiteScore
12.00
自引率
0.00%
发文量
11
审稿时长
20 weeks
期刊介绍: The International Journal of Biomedical Imaging is managed by a board of editors comprising internationally renowned active researchers. The journal is freely accessible online and also offered for purchase in print format. It employs a web-based review system to ensure swift turnaround times while maintaining high standards. In addition to regular issues, special issues are organized by guest editors. The subject areas covered include (but are not limited to): Digital radiography and tomosynthesis X-ray computed tomography (CT) Magnetic resonance imaging (MRI) Single photon emission computed tomography (SPECT) Positron emission tomography (PET) Ultrasound imaging Diffuse optical tomography, coherence, fluorescence, bioluminescence tomography, impedance tomography Neutron imaging for biomedical applications Magnetic and optical spectroscopy, and optical biopsy Optical, electron, scanning tunneling/atomic force microscopy Small animal imaging Functional, cellular, and molecular imaging Imaging assays for screening and molecular analysis Microarray image analysis and bioinformatics Emerging biomedical imaging techniques Imaging modality fusion Biomedical imaging instrumentation Biomedical image processing, pattern recognition, and analysis Biomedical image visualization, compression, transmission, and storage Imaging and modeling related to systems biology and systems biomedicine Applied mathematics, applied physics, and chemistry related to biomedical imaging Grid-enabling technology for biomedical imaging and informatics
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