Transcript Confocal Raman Tweezing Spectroscopy for a Nanotoxicology

Confocal Raman Tweezers
for a Nanotoxicology Application
Emanuela Ene
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 Tweezers Setting
Raman
spectro
meter
The CRT system
schematics
Entrance
slit
Confocal
pinhole
4X beam
expander
Dual
axis
AOD
Laser
Microscope
objective
piezo
controlled
The actual CRT system
working with a green 514.5nm Ar+ ion laser
Imaging
system
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.
Simulations for trapping with a Gaussian beam
The tweezing profile in the image plane
The cover glass and the colloidal solution
introduce aberrations
Trap image (tweezing focus)
in the X-Y plane
Water immersed complex microobjects have
been optically manipulated
Cell “stuck” near a
0.8µm PMMA sphere
with 6nm gold
nanoparticles coating
Diffraction rings of trapped objects.
Sub-micrometer coated clusters were optically manipulated near plant
SFM image of a cluster of 0.18μm PS “spheres”
cells; both of the objects stayed in the trap for several hours.
coated with 110nm SWCN.
PMMA = polymethylmethacrylate
Scanning range: 4.56μm
Calibration spectrum
Slide with 1.5mm depression, filled with 5μm
polystyrene (PS) spheres in water. Focus may move
≈ 440 μm from the cover glass.
Backward
scattered
Raman light
Δz≈440μm
Slide
Aqueous
solution
of PS
spheres
(m=1.19)
Incident
laser
beam
Oil immersion Oil layer
(n=1.515)
objective
(NA=1.25)
Cover
glass
(n=1.525,
t=150μm)
Focusing objective and sample
for calibration the CRT system
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.
Confocal Raman Tweezers Spectra from
magnetic particles
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.
1.16μm-sized iron oxide clusters (BioMag 546, Bangs Labs)
with silane (SiHx) coating
Future development
In our nanotoxicity study, CRTS will be used to monitor the chemical and functional changes in nanoparticleembedded 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.
References
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