Refractive index determination of single sub micrometer vesicles in - - PowerPoint PPT Presentation

▶

$refractive index determination of single sub micrometer$

May 30, 2023 464 likes •781 views

Refractive index determination of single sub micrometer vesicles in suspension using dark field microscopy Edwin van der Pol 1,2 Frank Coumans 1,2 , Anita Bing 2 , Auguste Sturk 2 , Rienk Nieuwland 2 , and Ton van Leeuwen 1 February 1st, 2014

SLIDE 1

1

Refractive index determination of single sub micrometer

vesicles in suspension using dark‐field microscopy

Edwin van der Pol1,2

1Biomedical Engineering and Physics; 2Laboratory Experimental Clinical Chemistry,

Academic Medical Center, Amsterdam, The Netherlands

February 1st, 2014 Frank Coumans1,2, Anita Böing2, Auguste Sturk2, Rienk Nieuwland2, and Ton van Leeuwen1

SLIDE 2

2 Academic Medical Center

Biomedical Engineering and Physics Laboratory Experimental Clinical Chemistry

European Association of National Metrology Institutes (EURAMET)

The European Metrology Research Programme (EMRP) is jointly funded by the EMRP participating countries within EURAMET and the European Union

Acknowledgements

University of Oxford

Chris Gardiner

University of Birmingham

Paul Harrison

NanoSight Ltd.

Patrick Hole Andrew Malloy Jonathan Smith

SLIDE 3

3

cells release vesicles: spherical particles with phospholipid bilayer specialized functions clinically relevant

Introduction to extracellular vesicles

van der Pol et al., Pharmacol Rev (2012)

SLIDE 4

4

measure the refractive index to distinguish vesicles from lipoproteins and protein aggregates

Determine refractive index to identify vesicles

vesicles (1.36 ≤ n ≤ 1.45 for d > 500 nm)* lipoproteins (n = 1.45‐1.60) protein aggregates (n = 1.53‐1.60)

* Konokhova et al., J. Biomed. Opt. (2012)

SLIDE 5

5

Refractive index to relate scatter to diameter

flow cytometry is widely used to detect vesicles refractive index provides scatter to diameter relation

SLIDE 6

6 ?

Refractive index of vesicles is unknown

refractive index of vesicles is unknown detection range is unknown

SLIDE 7

7

determine the refractive index of sub micrometer vesicles in suspension

distinguish vesicles from lipoproteins and protein aggregates provide insight in the vesicle detection range of flow cytometry

Goal

SLIDE 8

8

btain particle diameter d by tracking the Brownian

motion of single particles (Stokes‐Einstein equation) measure scattering power P derive particle refractive index n(P,d) from Mie theory

Methods ‐ single particle tracking

SLIDE 9

9

Commercial instrument

Nanosight NS‐500

microscope

bjective

NA = 0.4 glass laser beam power = 45 mW wavelength = 405 nm particles in solution EMCCD +

figure adapted from Nanosight Ltd, UK

Methods ‐ setup

SLIDE 10

10

intensity corrected for camera shutter time and gain minimum tracklength 30 frames discard scatterers that saturate the camera

Methods ‐ data acquisition and processing

SLIDE 11

11

Polystyrene beads (n=1.63)

Thermo Fisher Scientific, USA

Silica beads (n=1.45)

Kisker Biotech, Germany

Human urinary vesicles

differential centrifugation protocol from metves.eu

Methods ‐ samples

cells vesicles

SLIDE 12

12

measure light scattering of beads describe measurements by Mie theory derive particle diameter from Brownian motion validate technique with a beads mixture determine the refractive index of vesicles

Methods ‐ approach

SLIDE 13

13

Results ‐ scattering power versus diameter

f polystyrene beads

SLIDE 14

14

Results ‐ scattering power versus diameter

f polystyrene beads described by Mie theory

SLIDE 15

15

Results ‐ scattering power versus diameter

f polystyrene and silica beads

SLIDE 16

16

Results ‐ scattering power versus diameter of a mixture of polystyrene and silica beads

SLIDE 17

17

Results ‐ Scattering power versus diameter of a mixture of polystyrene and silica beads

SLIDE 18

18

Results ‐ refractive index and size distribution of a mixture of polystyrene and silica beads

SLIDE 19

19

Results ‐ refractive index and size distribution of a mixture of polystyrene and silica beads

n silica n polystyrene diameter (nm) Expected 1.45±0.02 1.63±0.02 206±18 Measured 1.45±0.02 1.67±0.04 213±25

SLIDE 20

20

measure light scattering of beads describe measurements by Mie theory derive particle diameter from Brownian motion validate technique with a beads mixture determine the refractive index of vesicles

Methods ‐ approach

SLIDE 21

21

Results ‐ scattering power versus diameter of urinary vesicles

SLIDE 22

22

Results ‐ size and refractive index distribution of urinary vesicles

SLIDE 23

23

Results ‐ refractive index versus diameter for urinary vesicles

SLIDE 24

24

Results ‐ refractive index versus diameter detection limits for urinary vesicles

SLIDE 25

25

Results ‐ refractive index versus diameter detection limits for urinary vesicles

SLIDE 26

26

Results ‐ refractive index versus diameter detection limits for urinary vesicles

SLIDE 27

27

Results ‐ refractive index versus diameter detection limits for urinary vesicles

SLIDE 28

28

single particle tracking can be used to determine the refractive index of single sub micrometer particles median refractive index of urinary vesicles is 1.36 with 90% of the vesicles between 1.35 and 1.41

Conclusions

SLIDE 29

29

Discussion ‐ urinary vesicles contain mainly water

image courtesy of Issman et al., PLoS ONE (2013) * van Manen et al., Biophys. J. (2007)

thickness = 5 nm n membrane = 1.46 *

n core = 1.34

SLIDE 30