Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions
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 p...
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| Format: | Article |
| Language: | English |
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Wiley
2017-01-01
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| Series: | International Journal of Biomedical Imaging |
| Online Access: | http://dx.doi.org/10.1155/2017/8318906 |
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| author | Jacopo Biasetti Kaushik Sampath Angel Cortez Alaleh Azhir Assaf A. Gilad Thomas S. Kickler Tobias Obser Zaverio M. Ruggeri Joseph Katz |
| author_facet | Jacopo Biasetti Kaushik Sampath Angel Cortez Alaleh Azhir Assaf A. Gilad Thomas S. Kickler Tobias Obser Zaverio M. Ruggeri Joseph Katz |
| author_sort | Jacopo Biasetti |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-bfd9fc4082b840e7be01e1bdacd5e5b1 |
| institution | Kabale University |
| issn | 1687-4188 1687-4196 |
| language | English |
| publishDate | 2017-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | International Journal of Biomedical Imaging |
| spelling | doaj-art-bfd9fc4082b840e7be01e1bdacd5e5b12025-02-03T01:04:43ZengWileyInternational Journal of Biomedical Imaging1687-41881687-41962017-01-01201710.1155/2017/83189068318906Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep SuspensionsJacopo Biasetti0Kaushik Sampath1Angel Cortez2Alaleh Azhir3Assaf A. Gilad4Thomas S. Kickler5Tobias Obser6Zaverio M. Ruggeri7Joseph Katz8Department of Mechanical Engineering, Johns Hopkins University, 3200 N. Charles Street, Baltimore, MD 21218, USADepartment of Mechanical Engineering, Johns Hopkins University, 3200 N. Charles Street, Baltimore, MD 21218, USADepartment of Radiology and Radiological Science, Johns Hopkins School of Medicine, 1550 Orleans Street, Baltimore, MD 21287, USADepartment of Biomedical Engineering, Johns Hopkins University, 3200 N. Charles Street, Baltimore, MD 21218, USADepartment of Radiology and Radiological Science, Johns Hopkins School of Medicine, 1550 Orleans Street, Baltimore, MD 21287, USADepartment of Pathology, Johns Hopkins School of Medicine, 1800 Orleans Street, Baltimore, MD 21287, USADepartment for Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, 20246 Martinistraße 52, 20251 Hamburg, GermanyMERU-Roon Research Center for Vascular Biology, Department of Molecular Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, USADepartment of Mechanical Engineering, Johns Hopkins University, 3200 N. Charles Street, Baltimore, MD 21218, USATracking 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.http://dx.doi.org/10.1155/2017/8318906 |
| spellingShingle | Jacopo Biasetti Kaushik Sampath Angel Cortez Alaleh Azhir Assaf A. Gilad Thomas S. Kickler Tobias Obser Zaverio M. Ruggeri Joseph Katz Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions International Journal of Biomedical Imaging |
| title | Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions |
| title_full | Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions |
| title_fullStr | Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions |
| title_full_unstemmed | Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions |
| title_short | Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions |
| title_sort | space and time resolved detection of platelet activation and von willebrand factor conformational changes in deep suspensions |
| url | http://dx.doi.org/10.1155/2017/8318906 |
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