Jacopo Biasetti, Kaushik Sampath, Angel Cortez, Alaleh Azhir, Assaf A Gilad, Thomas S Kickler, Tobias Obser, Zaverio M Ruggeri, Joseph Katz
{"title":"Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions.","authors":"Jacopo Biasetti, Kaushik Sampath, Angel Cortez, Alaleh Azhir, Assaf A Gilad, Thomas S Kickler, Tobias Obser, Zaverio M Ruggeri, Joseph Katz","doi":"10.1155/2017/8318906","DOIUrl":null,"url":null,"abstract":"<p><p>Tracking cells and proteins' phenotypic changes in deep suspensions is critical for the direct imaging of blood-related phenomena in <i>in vitro</i> 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.</p>","PeriodicalId":47063,"journal":{"name":"International Journal of Biomedical Imaging","volume":"2017 ","pages":"8318906"},"PeriodicalIF":3.3000,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1155/2017/8318906","citationCount":"3","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"International Journal of Biomedical Imaging","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1155/2017/8318906","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2017/11/6 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"ENGINEERING, BIOMEDICAL","Score":null,"Total":0}
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.
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