Research Motivation

Sample Preparation:The bone samples were prepared by scientists at Mayo Clinic. The samples under
investigation are that of metastasized cancer into the bone tissue. For comparison,
healthy bone tissue was used as a control. The bones were harvested from
______________ and are cut into small fragments of about ____________. The bone
tissue is fixed onto a microscope slide with ______________.
In the United States, about 3,300 new cases are projected to be diagnosed by the
end of 2016. Of those new cases, about 1,490 deaths from this disease are to be
expected. Cancer begins, and is characterized, with the out of control proliferation
of cells. In regards to bone, cancer can start in the tissue of the bone, or be
metastases into it from other remote cancers in the body. The most common
metastasis originates from tumors in the breast or the prostate. Cancer of the bone
usually affects the long bones that make up the arms and legs which can cause
pain, swelling, fatigue, weight loss, bone fractures, and eventually death. Knowing
this, a microscopic and spectroscopic investigation on the composition of healthy
and tumor bearing bone is set forth to characterize molecular signatures that
determine the mechanical properties of bone.
Raman spectroscopy is an inelastic scattering technique. The process consists
of sending a monochromatic light beam from a laser source onto some target mater
ial (sample to be analyzed); the incident photons excite the electron cloud
within the
molecules in the material, making them jump into a higher virtual quantum energy
state
(see Figure 7). When the molecules are hit by the incident photons they scatter the
light
in different ways; this is the scattered beam. These photons are called the scattere
d
photons and their energy can be higher, lower or equal to the energy of the incident
photons. 28 Fig. 7: Graphical representation of molecular electronic energy levels
and vibrational energy states [26] The energies of the scattered photons are
directly related to the vibrational energy (phonons) states of the molecules
in the material.
This gives valuable
information about the bonding and structure of the material.
If the energy of the scattered photon is equal to the energy/frequency of the
incident photon, this is elastic scattering and it's also called Rayleigh scattering. It
means that the molecules have relaxed to the same vibrational energy state they h
ad before being excited. When the energy of the scattered photon is lower
than the incident photon

energy, the molecules have relaxed to a higher vibrational energy state; a portion of
the
energy from the incident photons was absorbed by the molecules. In this way, whe
n
the scattered photon is detected it contributes to the peaks in the Raman spectrum
called the Stokes lines.
Finally, if the energy of the scattered photons is higher than that of the incident
photons, it means that the molecules have relaxed to a lower vibrational energy stat
e. In
this case the molecules give some of their initial energy to the scattered photons; th
is gives rise to the antiStokes lines. Figure 8 details the Stokes and antiStokes processes in simple diagrams; here
and k are the frequency and wavevector of the incident photon, respectively; ' a
nd k' are the frequency and wave vector of the scattered photon; and and K
are the 29
frequency and wavevector of the associated phonon created or annihilated during t
he scattering process. The selection rules for the firstorder Raman effect are:
= ' ; and k = k' K [27]
It can be observed from Figure 9 that the Stokes line signal is stronger than the antiStokes line. This is because, at room temperature, the proportion of the scattered
photons with higher energy than the incident photons with respect to the scattered
photons with lower energy than the incident photons is extremely small. That is wh
y in
Raman spectroscopy the measurements are generally done on the Stokes lines. Th
e 30 Fig. 8: Raman scattering of a photon with emission or absorption
of a phonon [27] . energy units are normally expressed in terms of wavenumbers (1/
) [cm 1] which are related to the frequency as well as to the energy.
The results of the measurements give the data needed to plot an intensity vs.
wavenumber graph. With the information provided by the graph it is possible to find
what chemical group corresponds to each vibration, giving valuable information abo
ut the bonding and structure of the material. 2.3.2 Confocal Raman Microscopy A
particularly useful mode in Raman spectroscopy is confocal Raman
microscopy. This technique is valuable for providing a higher spatial resolution and
31 Fig. 9: Stokes, antiStokes and Rayleigh lines diagram
allowing the observation and measurement of the undersurface layers of a material.
The results of such processing are called depth profiles. Thus, when the undersurface
layers of a material have different structural and mechanical properties than the sur
face,
confocal Raman microscopy allows the execution of 3D mappings of the sample.
A schematic representation of a confocal microscopy system is shown in Figure
10. Notice that this is not a Raman spectroscopic system, as it is intended to illustr
ate
the concept of confocal microscopy only, without the addition of a spectrometer sys

tem. 32 Fig. 10: Schematic representation of a confocal microscopy

system [28] The principle of confocal microscopy lies in the elimination of the outoffocus
scattered light. The light that scatters from the focal point is received by the objecti
ve pinhole, allowing a very precise, localized, and high definition measurement.
2.3.3 Instrumentation For the Raman scattering measurements the equipment
used was a WITec
Alpha 300 R confocal Raman spectrometer (see Figures 11 and 12). 33
Fig. 11: A WITec Alpha 300 R confocal Raman spectrometer [29] .
The parameters of operation used for the measurement of all the samples in the
Alpha WITec 300 R instrument are detailed in Table 6.