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Detection of microparticles by flow cytometry Edwin van der Pol May - - PowerPoint PPT Presentation
Detection of microparticles by flow cytometry Edwin van der Pol May - - PowerPoint PPT Presentation
Detection of microparticles by flow cytometry Edwin van der Pol May 21st, 2013 Biomedical Engineering and Physics Laboratory Experimental Clinical Chemistry Academic Medical Center, Amsterdam, The Netherlands 1 Microparticles and flow
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Introduction to flow cytometry
image adapted from www.semrock.com 488-nm laser electronics and computer fluorescence channels side scatter detector (SSC) forward scatter detector (FSC)
smallest detectable polystyrene bead is 200 nm n = 1.61
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Light scattering and the refractive index
Polystyrene bead Silica bead Vesicle 100 nm n = 1.61 n = 1.45 ncore = 1.38 nmembrane = 1.48
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diameter of vesicles is <300 nm against expectations, vesicles are detected by flow cytometry
Problem
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- ptimize detection settings
measure light scattering power of beads describe measurements by Mie theory determine size of smallest detectable single vesicle investigate role of multiple particles in detection volume by dilution series prospects
Goals
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Methods – optimize flow cytometer settings
cell microparticle d = 500 nm exosome d = 50 nm results based on BD FACSCalibur
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- ptimize detection settings
measure light scattering power of beads describe measurements by Mie theory determine size of smallest detectable single vesicle investigate role of multiple particles in detection volume by dilution series prospects
Goals
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Results – scattering power of polystyrene beads
SSC SSC
× 1.3E6 =
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Results – scattering power of silica beads
SSC SSC
× 1.3E6 =
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- ptimize detection settings
measure light scattering power of beads describe measurements by Mie theory determine size of smallest detectable single vesicle investigate role of multiple particles in detection volume by dilution series prospects
Goals
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Results – scattering power vs. diameter
* van Manen et al., Biophys J (2008) Konokhova et al., J Biomed Opt (2012)
SSC 10 nm
*
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Results – scattering power vs. diameter
SSC 10 nm
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Results – scattering power vs. diameter
SSC 10 nm
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- ptimize detection settings
measure light scattering power of beads describe measurements by Mie theory determine size of smallest detectable single vesicle investigate role of multiple particles in detection volume by dilution series prospects
Goals
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SSC C = 7∙106 ml-1
Results – multiple vesicles as single count
89-nm silica beads at concentration 1010 beads ml-1 urine filtered with 220-nm filter concentration ≥ 1010 vesicles ml-1
SSC C = 9∙105 ml-1
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beam volume ≈ 54 pl At a concentration of 1010 vesicles ml-1, >800 vesicles are simultaneously present in the beam.
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Results – counts from mixtures of beads
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Results – counts from mixtures of beads
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Results – counts from mixtures of beads
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Results – counts from mixtures of beads
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Results – counts from mixtures of beads
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Results – counts from urinary vesicles
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Results – counts from urinary vesicles
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vesicle detection by flow cytometry
scattering power related to diameter and refractive index for single beads and vesicles single event signal attributed to scattering from multiple vesicles
Conclusion
van der Pol et al., J Thromb Haemost (2012)
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calibration should be based on experiments and theory
size distribution refractive index
flow cytometry is good increase sensitivity
Prospects of vesicle detection by flow cytometry
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Sensitivity should be increased
“A flow cytometer is unable to detect the smallest vesicle as long as you can detect cells with it.”
2 2 405 200 20 0.1
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