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 Laboratory Experimental Clinical Chemistry, Academic Medical Center, Amsterdam, The Netherlands 1

Introduction to cell-derived vesicles cells release vesicles: spherical particles with phospholipid bilayer specialized functions clinically relevant van der Pol et al., Pharmacol Rev (2012) 2

Introduction to cell-derived vesicles vesicles are studied mostly by flow cytometry mechanism causing detection incompletely understood 3

Introduction to flow cytometry fluorescence channels electronics and 488-nm laser computer side scatter detector (SSC) forward scatter detector (FSC) smallest detectable polystyrene bead is 200 nm n = 1.61 image adapted from www.semrock.com 4

Problem diameter of vesicles is <300 nm, n = ~1.4 against expectations, vesicles are detected by flow cytometry 5

Goals 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 titration 6

Methods – optimize settings flow cytometer cell vesicle vesicle d = 500 nm d = 50 nm 7

Goals 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 titration 8

Results – scattering power of polystyrene beads SSC SSC × 1.3E6 = 9

Results – scattering power of silica beads SSC SSC × 1.3E6 = 10

Goals 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 titration 11

Results – scattering power vs. diameter SSC * 10 nm * van Manen et al., Biophys J (2008) 12

Results – scattering power vs. diameter SSC * 10 nm * van Manen et al., Biophys J (2008) 13

Goals 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 titration 14

Results – scattering power vs. diameter SSC * 10 nm * van Manen et al., Biophys J (2008) 15

Results – multiple vesicles as single count C = 7 ∙ 10 6 ml -1 C = 9 ∙ 10 5 ml -1 SSC SSC 89-nm silica beads at urine filtered with 220-nm filter concentration 10 10 beads ml -1 10 vesicles ml -1 concentration ≥ 10 16

beam volume ≈ 54 pl At a concentration of 10 10 vesicles ml -1 , >800 vesicles are simultaneously present in the beam. 17

Results – counts from mixtures of beads 18

Results – counts from mixtures of beads 19

Results – counts from mixtures of beads 20

Results – counts from mixtures of beads 21

Results – counts from mixtures of beads 22

Results – counts from urinary vesicles 23

Results – counts from urinary vesicles 24

Conclusion 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 van der Pol et al., J Thromb Haemost (2012) 25

Outlook vesicle detection increase sensitivity of flow “cytometry” reduce detection volume increase irradiance maximize collection angle shorter wavelength employ other techniques Confocal Raman microspectroscopy * * Proc SPIE 8591-11, Wednesday 1:50 pm room 309 26

Acknowledgements Anita Böing Anita Grootemaat Chi Hau Guus Sturk Henk van Veen Marianne Schaap Martin van Gemert Rienk Nieuwland Ton van Leeuwen 27