Download Raman spectroscopy of a single living cell

Raman Spectroscopy of
Institut de Ciències Fotòniques,
Barcelona,
Spain.
a Single Living Cell
Gajendra Pratap Singh
[email protected]
Raman Scattering
The Raman effect arises when a photon is incident on a molecule and interacts
with the electric dipole of the molecule. It is a form of electronic (more
accurately, vibronic) spectroscopy, although the spectrum contains vibrational
frequencies. In quantum mechanics the scattering is described as an excitation to a
virtual state lower in energy than a real electronic transition with nearly
coincident de-excitation and a change in vibrational energy. The scattering event
occurs in 10-14 seconds or less.
Energy level diagram for Raman scattering; (a) Stokes Raman scattering (b) anti-Stokes
Raman scattering.
Optical Tweezers in action
Optical Tweezers use light to
manipulate microscopic
objects as small as a single
atom. The radiation pressure
from a focused laser beam is
able to trap small particles.
Laser beam
Lens
Microscopic particle
Net mechanical force produced by a focused nonhomogeneous beam
moves a sphere to the focus
K. Dholakia et al, Physics World, 2002
In the biological sciences, these instruments have been used to apply forces
in the pN-range and to measure displacements in the nm range of objects
ranging in size from 10 nm to over 100 µm.
Raman Tweezers in Biology and Medicine
Raman spectroscopy is very useful in Biology because the analysis of optical
spectra of a single cell reveals information about species, structures, and
molecular conformations within the cell.
But an individual cell in a liquid solution moves continuously due to Brownian
motion. Hence, combining Raman Spectroscopy with Optical Tweezers makes it
possible to study Raman spectrum of a single living cell. The trap is optical and
there is no chemical attachment. So, the chemical composition of the cell remains
unaltered to a large extent.
Raman Spectra of Human Skin
Measured in Vivo
Reference :
``Non-Invasive Raman Spectroscopic Detection of
Carotenoids in Human Skin´´
T. R. Hata, T. A. Scholz, I. V. Ermakov, R. W. McClane, F. Khachik, W. Gellermann, and L. K. Pershing,
J. Invest. Dermat. 115, 441 (2000).
To realize this we have a dual beam optical
trap system capable of forming two traps
simultaneously in the focal plane of an
inverse microscope. An NIR beam is used for
optical trapping because it inflicts minimum
damage to the biological samples. To view
the trapped particles, a CCD camera and a
monitor are used. A semiconductor laser (785
nm) and a tunable femto- second laser will
be used to excite the Raman spectrum. A
monochromator and a CCD detector collect
the scattered light.
EXPERIMENTAL SET UP
Future plans of our Lab
I.) As a first step we would like to understand and resolve the controversy
reported in spectra for living and dead yeast cells . Raman spectra
characteristic of the nucleus, mitochondrion and septum have already been
identified during cell division of a fission yeast cell, and we would like to lead
this investigation further to understand the cell signaling events taking place
inside the cell during cell division or under varying environmental conditions.
Enhanced resonance effects can be created in an optical trapping environment
by co-trapping cells with nanometer sized metal clusters. This will make the
Raman signature more explicit.
Reference :
C.A. Xie and Y.Q. Li, “Raman spectra and optical trapping of highly refractive and
nontransparent particles”, Applied Physics Letters, 81, 951-953 (2002).
Near-infrared Raman spectra and images of
a single living yeast cell and a dead yeast
cell in solution. A significant difference can
be seen in the Raman spectra of the living
and the dead cells.
NIR Raman Spectra for a range of optically
trapped biological specimens.
A: a single healthy cerevisae yeast cell(also in
the inset image);
B: a yeast cell from the same sample after
overnight bleaching;
C: a mammalian cell and D: bacteria.
The Raman signature from the dead cells shows
that the spectra A and B are similar, which is
contrary to the literature findings where a
complete loss of signal was reported for the
dead cells.
Images of Yeast Cells in our lab :
Single beam trap at work:
Dual beam trap at
work
II.) We are also eager to study the effects of various drugs on Neurons and
record the molecular interactions taking place inside and outside the cell through
Raman Spectroscopy. We will try growing them on polystyrene beads and
experiment with them after trapping the beads.
Images of
Neurons in
our lab
Laser
Neurons from rat
hippocampus were
obtained in
collaboration with Dr.
Eduardo Soriano at the
Parc Cientific de
Barcelona.
III.) Another experiment in our agenda is regarding the cells of the immune
system. We are determined to know more about the programmed cell death
(Apoptosis) through our Raman Spectroscopy as we can trap two cells in our
dual beam Optical Tweezers and bring them near to each other to see how they
interact and what molecular interactions lead to the ultimate end.
Apoptosis !!!
DIE
Raman Spectrum
IV.) DNA properties attract us too. Since the Raman Spectra for the bases
constituting the nucleic acids have already been identified, we would like to
experiment a bit further.
A
T
C
G
Raman Spectrum
Images of a Prostate Cancer cell being moved
by a trapped polystyrene bead in our lab.
These polystyrene beads can be coated with an
antibody and then they can attach specifically
to the surface of the cell. The interaction can be
studied by Raman Spectroscopy.
V.) The Prostate cancer cells are now under investigation in our lab. We are
anxious to see if their growth rate remains the same under our Optical
Tweezers !!!
Raman Spectrum
Raman Spectrum
OR
Trapped Healthy Cell
Trapped Prostate Cancer Cell
Raman Spectrum database
????????
Prostate Cancer Cells will be provided in collaboration with Dr. Timothy M
Thomson at the Centre d´Investigació i Desenvolupament, Barcelona.