TITLE OF POSTER IN CAPITALS

(ARIAL NARROW 96PT BOLD)First name last name authors, underline presenting author

Name Chair, MIRA Univesity of Twente (Arial narrow 48pt regular)

Identification and characterization of cells and

extracellular vesicles (EVs) by SEM-RamanAgustin Enciso Martinez1, Afroditi Nanou1, Frank Timmermans1, Aufried Lenferink 1, Edwin van der Pol 2, Leon W.M.M. Terstappen1, Cees Otto1

1 Department of Medical Cell BioPhysics, MIRA institute, University of Twente, Enschede, The Netherlands;2 Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, The Netherlands

Introduction

Methods

I

Conclusion

Results

Optical forcesLight – matter interaction

Flow

Size

Exclusion

Chromato-

graphy

Glass slide with beads/EVs in suspension

3 Optical trapping Rayleigh scattering4

1 2 3 4

PC3 derived EVs

Platelet derived EVs

Chemical information

of cells – Raman

spectroscopy

Morphological and size information – SEM

II

Goal: to correlate and using an integrated Raman-

SEM microscope

I II

1 Cell culture on

slides overnight

(LnCap), WBCs

isolated from WB

2 Cell fixation,

dehydration in

increasing

concentrations of

ethanol and after

drying with HMDS

3

4

SEM overview

Raman

measurements5 SEM imaging

Data analysis

and SEM-Raman

correlation

6

Distance μm

Dis

tan

ceμ

m

Wavelength: 785 nm Laser power: 7.2 mWIntegration time: 5 s/pixel Step size: 500nm

LnCap cells

White blood cells

Wavelength: 647 nm Laser power: 70 mWIntegration time: 38 ms

2

Tumor-derived extracellular vesicles (EVs), i.e. exosomes and microparticles, have been proposed to play a key role in tumor development andmetastasis. Their presence in body fluids makes them potential biomarkers for cancer disease. Within the framework of the Cancer-ID program,we will develop novel technology to determine EV IDentity. Optical tweezers have allowed us to study single vesicles and to distinguish individualtrapping events from accumulative clusters. In addition, Raman tweezers allowed us to perform single and bulk EV characterization. Anotherapproach for EV characterization is the use of a scanning electron microscope integrated with a Raman microscope. This discloses size andchemical information of cells by correlated SEM imaging and hyperspectral Raman microscopy as a step towards SEM-Raman EV characterization.

We have observed optical trapping of EVs in the laser focus of a Raman microscope. The light scattered of trapped EVs gave rise to different timetraces that can be used as a signature to distinguish individual trapping events from accumulative cluster events. We confirmed the presence ofindividual EVs in the laser focus by a step response in the light scattering signal when an EV is trapped. By correlating the light scattering signalwith the Raman signal we were able to perform single and bulk characterization of EVs. For that purpose, we have automated a measuringsystem that acquires both signals during EV trap and release. Furthermore, we have developed a method to present cell samples to anintegrated Raman-SEM microscope. In preliminary studies, we have obtained Raman microscopy images and SEM images of cells and overlaidthem. This allowed us to perform chemical and morphological characterization of cells as a step towards SEM-Raman EV characterization.

1 EV

collection

* Butler, Holly J et al., 2016

A) Optical tweezers B) SEM – Raman characterization

A) Optical tweezers B) SEM – Raman characterization

*http://experimentationlab.berkeley.edu/OTZ

Dis

tan

ceμ

m

Distance μm

Raman scattering

Peaks:

Peaks:

Peaks:

Lipid band

Lipid band

Raman peaks:

Raman peaks:

a)

b)

No trapping Trapping

Time [s]

Inte

nsity

Time [s]

Inte

nsity

(counts

/ 38m

s)

Inte

nsity

(counts

/ 38m

s)

1 2 3 4

Inte

nsity

(counts

/ 38m

s)

Inte

nsity

(counts

/ 38m

s)

Time [s]