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]