detection of microparticles by flow cytometry · detection of microparticles by flow cytometry ......
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Detection of microparticles by flow cytometry
Edwin van der Pol
Biomedical Engineering and Physics Laboratory Experimental Clinical Chemistry Academic Medical Center, Amsterdam, The Netherlands
May 21st, 2013
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vesicles are studied mostly by flow cytometry mechanism causing detection incompletely understood
Microparticles and flow cytometry
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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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optimize 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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optimize 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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optimize 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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optimize 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.”
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2 405 200
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0.1
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Anita Böing Anita Grootemaat Chi Hau Chris Gardiner Frank Coumans Guus Sturk Henk van Veen Marianne Schaap Martin van Gemert Paul Harrison Rienk Nieuwland Ton van Leeuwen
Acknowledgements
More on vesicle detection: edwinvanderpol.com