Raman spectroscopy of a single living cell
Raman spectroscopy of a single living cell Raman spectroscopy of a single living cell
Raman Spectroscopy of Institut de Ciències Fotòniques, Barcelona, Spain.
- Page 2 and 3: a Single Living Cell Gajendra Prata
- Page 4 and 5: Optical Tweezers in action Optical
- Page 6 and 7: To realize this we have a dual beam
- Page 8 and 9: Future plans of our Lab I.) As a fi
- Page 10 and 11: NIR Raman Spectra for a range of op
- Page 12 and 13: II.) We are also eager to study the
- Page 14 and 15: Images of a Prostate Cancer cell be
<strong>Raman</strong> Spectroscopy <strong>of</strong><br />
Institut de Ciències Fotòniques,<br />
Barcelona,<br />
Spain.
a Single Living Cell<br />
Gajendra Pratap Singh<br />
Gajendra.Pratap@upc.es
<strong>Raman</strong> Scattering<br />
The <strong>Raman</strong> effect arises when a photon is incident on a molecule and interacts<br />
with the electric dipole <strong>of</strong> the molecule. It is a form <strong>of</strong> electronic (more<br />
accurately, vibronic) <strong>spectroscopy</strong>, although the spectrum contains vibrational<br />
frequencies. In quantum mechanics the scattering is described as an excitation to a<br />
virtual state lower in energy than a real electronic transition with nearly<br />
coincident de-excitation and a change in vibrational energy. The scattering event<br />
occurs in 10 -14 seconds or less.<br />
Energy level diagram for <strong>Raman</strong> scattering; (a) Stokes <strong>Raman</strong> scattering (b) anti-Stokes<br />
<strong>Raman</strong> scattering.
Optical Tweezers in action<br />
Optical Tweezers use light to<br />
manipulate microscopic<br />
objects as small as a <strong>single</strong><br />
atom. The radiation pressure<br />
from a focused laser beam is<br />
able to trap small particles.<br />
Laser beam<br />
Lens<br />
Microscopic particle<br />
Net mechanical force produced by a focused nonhomogeneous beam<br />
moves a sphere to the focus<br />
K. Dholakia et al, Physics World, 2002<br />
In the biological sciences, these instruments have been used to apply forces<br />
in the pN-range and to measure displacements in the nm range <strong>of</strong> objects<br />
ranging in size from 10 nm to over 100 µm.
<strong>Raman</strong> Tweezers in Biology and Medicine<br />
<strong>Raman</strong> <strong>spectroscopy</strong> is very useful in Biology because the analysis <strong>of</strong> optical<br />
spectra <strong>of</strong> a <strong>single</strong> <strong>cell</strong> reveals information about species, structures, and<br />
molecular conformations within the <strong>cell</strong>.<br />
But an individual <strong>cell</strong> in a liquid solution moves continuously due to Brownian<br />
motion. Hence, combining <strong>Raman</strong> Spectroscopy with Optical Tweezers makes it<br />
possible to study <strong>Raman</strong> spectrum <strong>of</strong> a <strong>single</strong> <strong>living</strong> <strong>cell</strong>. The trap is optical and<br />
there is no chemical attachment. So, the chemical composition <strong>of</strong> the <strong>cell</strong> remains<br />
unaltered to a large extent.<br />
<strong>Raman</strong> Spectra <strong>of</strong> Human Skin<br />
Measured in Vivo<br />
Reference :<br />
``Non-Invasive <strong>Raman</strong> Spectroscopic Detection <strong>of</strong><br />
Carotenoids in Human Skin´´<br />
T. R. Hata, T. A. Scholz, I. V. Ermakov, R. W. Mc-<br />
Clane, F. Khachik, W. Gellermann, and L. K. Pershing,<br />
J. Invest. Dermat. 115, 441 (2000).
To realize this we have a dual beam optical<br />
trap system capable <strong>of</strong> forming two traps<br />
simultaneously in the focal plane <strong>of</strong> an<br />
inverse microscope. An NIR beam is used for<br />
optical trapping because it inflicts minimum<br />
damage to the biological samples. To view<br />
the trapped particles, a CCD camera and a<br />
monitor are used. A semiconductor laser (785<br />
nm) and a tunable femto- second laser will<br />
be used to excite the <strong>Raman</strong> spectrum. A<br />
monochromator and a CCD detector collect<br />
the scattered light.
EXPERIMENTAL SET UP
Future plans <strong>of</strong> our Lab<br />
I.) As a first step we would like to understand and resolve the controversy<br />
reported in spectra for <strong>living</strong> and dead yeast <strong>cell</strong>s . <strong>Raman</strong> spectra<br />
characteristic <strong>of</strong> the nucleus, mitochondrion and septum have already been<br />
identified during <strong>cell</strong> division <strong>of</strong> a fission yeast <strong>cell</strong>, and we would like to lead<br />
this investigation further to understand the <strong>cell</strong> signaling events taking place<br />
inside the <strong>cell</strong> during <strong>cell</strong> division or under varying environmental conditions.<br />
Enhanced resonance effects can be created in an optical trapping environment<br />
by co-trapping <strong>cell</strong>s with nanometer sized metal clusters. This will make the<br />
<strong>Raman</strong> signature more explicit.<br />
Reference :<br />
C.A. Xie and Y.Q. Li, “<strong>Raman</strong> spectra and optical trapping <strong>of</strong> highly refractive and<br />
nontransparent particles”, Applied Physics Letters, 81, 951-953 (2002).
Near-infrared <strong>Raman</strong> spectra and images <strong>of</strong><br />
a <strong>single</strong> <strong>living</strong> yeast <strong>cell</strong> and a dead yeast<br />
<strong>cell</strong> in solution. A significant difference can<br />
be seen in the <strong>Raman</strong> spectra <strong>of</strong> the <strong>living</strong><br />
and the dead <strong>cell</strong>s.
NIR <strong>Raman</strong> Spectra for a range <strong>of</strong> optically<br />
trapped biological specimens.<br />
A: a <strong>single</strong> healthy cerevisae yeast <strong>cell</strong>(also in<br />
the inset image);<br />
B: a yeast <strong>cell</strong> from the same sample after<br />
overnight bleaching;<br />
C: a mammalian <strong>cell</strong> and D: bacteria.<br />
The <strong>Raman</strong> signature from the dead <strong>cell</strong>s shows<br />
that the spectra A and B are similar, which is<br />
contrary to the literature findings where a<br />
complete loss <strong>of</strong> signal was reported for the<br />
dead <strong>cell</strong>s.
Images <strong>of</strong> Yeast Cells in our lab :<br />
Single beam trap at work:<br />
Dual beam trap at<br />
work
II.) We are also eager to study the effects <strong>of</strong> various drugs on Neurons and<br />
record the molecular interactions taking place inside and outside the <strong>cell</strong> through<br />
<strong>Raman</strong> Spectroscopy. We will try growing them on polystyrene beads and<br />
experiment with them after trapping the beads.<br />
Images <strong>of</strong><br />
Neurons in<br />
our lab<br />
Laser<br />
Neurons from rat<br />
hippocampus were<br />
obtained in<br />
collaboration with Dr.<br />
Eduardo Soriano at the<br />
Parc Cientific de<br />
Barcelona.
III.) Another experiment in our agenda is regarding the <strong>cell</strong>s <strong>of</strong> the immune<br />
system. We are determined to know more about the programmed <strong>cell</strong> death<br />
(Apoptosis) through our <strong>Raman</strong> Spectroscopy as we can trap two <strong>cell</strong>s in our<br />
dual beam Optical Tweezers and bring them near to each other to see how they<br />
interact and what molecular interactions lead to the ultimate end.<br />
DIE<br />
Apoptosis !!!<br />
<strong>Raman</strong> Spectrum<br />
IV.) DNA properties attract us too. Since the <strong>Raman</strong> Spectra for the bases<br />
constituting the nucleic acids have already been identified, we would like to<br />
experiment a bit further.<br />
A<br />
T<br />
C<br />
G<br />
<strong>Raman</strong> Spectrum
Images <strong>of</strong> a Prostate Cancer <strong>cell</strong> being moved<br />
by a trapped polystyrene bead in our lab.<br />
These polystyrene beads can be coated with an<br />
antibody and then they can attach specifically<br />
to the surface <strong>of</strong> the <strong>cell</strong>. The interaction can be<br />
studied by <strong>Raman</strong> Spectroscopy.
V.) The Prostate cancer <strong>cell</strong>s are now under investigation in our lab. We are<br />
anxious to see if their growth rate remains the same under our Optical<br />
Tweezers !!!<br />
<strong>Raman</strong> Spectrum<br />
Trapped Healthy Cell<br />
<strong>Raman</strong> Spectrum<br />
Trapped Prostate Cancer Cell<br />
OR<br />
<strong>Raman</strong> Spectrum database<br />
????????<br />
Prostate Cancer Cells will be provided in collaboration with Dr. Timothy M<br />
Thomson at the Centre d´Investigació i Desenvolupament, Barcelona.