Transcript 48x36 poster template - Oklahoma State University

Confocal Raman Tweezers for a Nanotoxicology Application
Emanuela Ene and James P. Wicksted
Department of Physics, Oklahoma State University
Raman measurements from optically trapped dielectric and magnetic microparticles, under various visible laser excitation wavelengths, are being studied.
Changes in the Raman spectra for trapped living cells embedded with nanoparticles will be investigated.
Our Confocal Raman Tweezing Setting
Raman spectra from trapped microobjects
Raman
spectrometer
The CRT system schematics
Slide with 1.5mm depression, filled with 5μm polystyrene (PS)
spheres in water. Focus may move ≈ 440 μm from the cover glass.
16
Entrance
slit
Backward scattered
Raman light
Δz≈440μm
Slide
Confocal pinhole
The laser trap’s image
4X beam
expander
Aqueous solution
of PS
spheres (m=1.19)
Dual axis
AOD
Incident laser beam
Laser
Oil immersion objective
(NA=1.25)
Microscope objective
piezo controlled
The actual CRT system
working with a green 514.5nm Ar+ ion laser
Fig. 1
Imaging
system
The CTRS schematics
Oil layer
(n=1.515)
Cover glass
(n=1.525, t=150μm)
Focusing objective and sample
for calibration the CRT system
Fig. 2
Fig. 8
The CRT spectrum collected from a single 5.0μm, polystyrene
sphere ( Bangs Laboratoratories) continuously trapped for more
than eight hours with a Meredith 632.8nm HeNe laser, 5mW in front
of the objective.
The total collection time was 1500s, with 2.0s per each 0.2cm-1 step.
Fig. 9
Fig. 10
OSLO Simulations for a Gaussian Beam
The Confocal Raman Tweezers Spectroscopy (CTRS) has the ability to provide precise characterization of a living cell without physical or chemical contact.
The CRTS allows the analysis of single cells in wet samples, in contrast with the classical micro Raman spectroscopy that utilizes dried samples. In a confocal
setting, the collected signal comes just from a minimum volume around the trapped-excited object.
The biological applications of nanoparticles, from imaging to drugs delivery, have created an increased interest in the past decades. Already in widespread
use, superparamagnetic iron oxide nanoparticles associated with biological molecules are easily for manipulating and attractive for MRI contrast or targeted
molecule delivery. Although used in biological and medical research, there is just little work done in investigating the effects of interactions between these
magnetic particles and the living cells they are attached to.
Fig. 3
Fig. 4
Future development
The tweezing profile in the image plane.
The cover glass and the colloidal solution introduce aberrations(Fig.3);
trap image (tweezing focus) in the X-Y plane (Fig.4).
In our nanotoxicity study, CRTS will be used to monitor the chemical and functional changes in nanoparticle-embedded living cells. Both stability of the trap,
for around eight hours of successive spectra collection, and repeatability are required.1,2,3,4,5,6
For living cells, photodamage effects restrict the range of wavelengths to be used. We intend to employ a tunable 505 to 750nm (Coherent) beam for both
tweezing and Raman excitation. The automatic fast laser beam steering will allow moving the beam focus in 3D to “chase” the cell that will be trapped and
analyzed.
For a photodamage initial evaluation, the life time of the trapped cells will be measured based on the fluorescence signal excited with the tunable laser 8 .
Resonance Raman spectra for individual nanoparticles will be mapped spatially, near resonance, using the same tunable laser.
A living cell embedded with nanoparticles will be monitored via CRTS over a series of different time points and distinguish the death or chemical changes in
the cell.
Microobjects optically manipulated
Cell “stuck” near a 0.8µm PMMA
sphere with 6nm gold
nanoparticles coating
REFERENCES
Fig. 6
Fig. 5
SFM image of a cluster of 0.18μm PS
“spheres” coated with 110nm SWCN.
Scanning range: 4.56μm
Diffraction rings of trapped objects.
Sub-micrometer coated clusters were optically
manipulated near plant cells;
both of the objects stayed in the trap for several hours.
PMMA = polymethylmethacrylate
1. Carls, J.C. et al, Time- resolved Raman spectroscopy from reacting optically levitated microdroplets, Appl. Optics, 29, 1990, pp. 2913-18
2. Cao, Y.C. et al, Raman Dye-Labeled Nanoparticle Probes for Proteins , J. Am. Chem. Soc., 125 (48), 14676 -14677, 2003
3. C. Xie, Y-qing Li, Confocal micro-Raman spectroscopy of single biological cells using optical trapping and shifted excitation techniques, J. Appl. Phys. ,
2003, 93(5), 2982-2986
4. Owen, C.A. et al , In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy, J. Cell. Biochem., 2006, 99, 178-186
5. Volpe, G. et al, Dynamics of a growing cell in an optical trap, Appl. Phys. Lett., 2006, 88, 231106-31108
6. Creely, S.M. et al, Raman imaging of neoplastic cells in suspension, Proc. SPIE, 2006, 6326: 63260U
7. Shaevitz, J.W. , A practical Guide to Optical Trapping, web resource at www.princeton.edu/~shaevitz/links.html
8. Neumann, K.C. et al, Characterization of Photodamage to Escherichia coli in optical traps, Biophys. J., 1999, 77(5), 2856-2863
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Supported by an NSF EPSCoR Grant EPS-047262