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Single versus coincidence detection of cell-derived vesicles by flow cytometry
Edwin van der Pol1,2
1Biomedical Engineering and Physics; 2Laboratory Experimental Clinical Chemistry,
Single versus coincidence detection of cell-derived vesicles by flow - - PowerPoint PPT Presentation
Single versus coincidence detection of cell-derived vesicles by flow - - PowerPoint PPT Presentation
Single versus coincidence detection of cell-derived vesicles by flow cytometry Edwin van der Pol 1,2 Martin van Gemert 1 , Auguste Sturk 2 , Rienk Nieuwland 2 , and Ton van Leeuwen 1 February 3rd, 2013 1 Biomedical Engineering and Physics; 2
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vesicles are studied mostly by flow cytometry mechanism causing detection incompletely understood
Introduction to cell-derived vesicles
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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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diameter of vesicles is <300 nm, n = ~1.4 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 titration
Goals
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Methods – optimize settings flow cytometer
cell vesicle d = 500 nm vesicle d = 50 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 titration
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 titration
Goals
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Results – scattering power vs. diameter
* van Manen et al., Biophys J (2008)
SSC 10 nm
*
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Results – scattering power vs. diameter
SSC 10 nm
* van Manen et al., Biophys J (2008)
*
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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 titration
Goals
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Results – scattering power vs. diameter
SSC
*
10 nm
* van Manen et al., Biophys J (2008)
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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 ≥ 10
10 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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increase sensitivity of flow “cytometry”
reduce detection volume increase irradiance maximize collection angle shorter wavelength
employ other techniques
Confocal Raman microspectroscopy*
Outlook vesicle detection
* Proc SPIE 8591-11, Wednesday 1:50 pm room 309
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