9
small animals; iii) structural features, allowing the integration with other imaging systems
to form a multimodal system, where one technique is complementary to the other.
As a counterbalance, optical imaging has intrinsic drawbacks that prevent from its
clinical use. Indeed, the interaction optical radiation-tissue is characterized by the
absorption and the strong scattering by tissues with the consequent loss of the information
carried by the light (5). The penetration depth of the light in the tissues depends on the
wavelength and on the irradiation geometry, besides on tissues optical properties, and it
has a maximum (<10 cm) in the so called optical window, the spectral interval between the
600 nm and 900 nm, where the absorption and the scattering effects are strongly reduced.
Here also the fluorescence of biological tissue components, called autofluorescence or
endogenous fluorescence, has a minimum. This limit in the penetration depth affects
optical planar imaging and it can be exceeded only by the tomographic imaging
techniques that, however, we don’t discuss in this thesis. Moreover, in the fluorescence
imaging, the detected signal is a diffuse light signal whose intensity shows a strong
dependence on the depth of the investigated target in the tissue, so the quantitative
determinations are difficult to infer. It is also noteworthy to remark that, since any
exogenous fluorochrome doesn’t have a 100% affinity with the target, some quantity of
fluorochrome will be present also in the non-target tissue generating the so called
background fluorescence. The autofluorescence and fluorescence background signals
lower the optical contrast of the fluorescence labeled target with respect to the
surrounding region. Hence, some instrumental and/or data processing procedures are
required to cancel these contributions and to improve the optical contrast.
This thesis is an experimental study on the in vivo detection of subcutaneous human
tumors implanted on small animals by a laser induced FRI technique using a
hematoporphyrin (HP) compound as an exogenous optical contrast agent (6). It is widely
reported in literature that the HP compounds accumulate preferentially in tumor tissues,
although the uptake mechanisms of HP compounds in tumors are not completely
understood. The absence of specificity in the targeting is a limit of HP labeling with
respect to other fluorescent tumor markers with high specificity, since doesn’t allow for
the monitoring of the molecular and/or genetic pathways of the disease. On the other
hand, the HP tumor unspecific uptake is an advantage, since it makes HP suitable to target
efficiently different type of tumors for diagnostic purposes. In particular, the early
diagnosis is a basic medical requirement to increase the patient survival probability,
especially if the tumor has a high degree of malignancy. Indeed, the final aim of our
investigation is to test the capability of our HP-FRI system to detect tumor in the early
stage of its growth.
In the first chapter the principal features of the interaction of the optical radiation
with biological tissue, which constitutes the basis to understand the physical background
of the FRI technique, is illustrated. In the second chapter an overview of the FRI
techniques is presented, so defining the framework where is collocated this work. In the
third and fourth chapter the experimental apparatus and procedures are described and the
obtained results are discussed. In particular, in the third chapter, we report on the
preliminary tests on the HP-FRI system and on the first HP-FRI measurements, concerning
the in vivo detection in mice of subcutaneous human thyroid carcinomas of different
malignancy degree and the combination of our optical imaging system with a radionuclide
imaging system in order to exploit the complementarity of the two different modality
imaging. In the fourth chapter, the HP-FRI measurements acquired with a high sensitivity
10
CCD camera in the spectral region of interest that allowed for in vivo early detection in
mice of tumors with a high degree of malignancy are described. Finally, the appendix at
the end of the Chapter 3 is devoted to the CCD technology and characteristics, since they
are relevant for the performed FRI measurements.
11
Chapter 1
Interaction radiation - biological tissues
1.1 Introduction
In this first chapter of the thesis the fundamentals of tissue optics, as the theoretical
background of our experimental work in the optical imaging in vivo on small animals, will
be recalled. Here we will just run over the definition of the optical parameters that
characterize the interaction between the radiation and the biological medium versus the
wavelength of the radiation that travels the tissues. We want focus our attention on the
scattering and absorption that dominate the interaction light-tissue and determine the
limits of optical imaging in vivo.
1.2 Property of light
Let us recall briefly the general properties of light and molecules before the specific
tissue-light interactions are described (7).
Light is dualistic in nature. It can be described as an electromagnetic wave or as a beam
of massless particles - a photon. The two aspects are equally valid and one or the other is
chosen to describe a specific phenomenon. The wavelength, λ, or the frequency, ν, are used
to describe the radiation when the wave nature is considered, and the energy, E, is used
when light is regarded as a stream of photons. These quantities are easily connected by the
speed of light, c, and the Planck constant, h.
E = h ⋅ν = h ⋅ c/ λ [ J ]
c = λ ⋅ν [m/s]
In the experiments described in this thesis, light in the visible and near infrared
range has been used. The light fluence rate, φ (mW/cm2), the light energy per second
travelling through the unit cross section area, is another important parameter we will use
in the following.
1.2.1 Energy levels in molecules
The structure of energy levels for molecules is more complicated than for atoms,
since vibrational and rotational motion of nuclei is to take into account besides to the
electron motion. In Figure 1.1 (7), the schematic picture of electronic levels together with
vibrational and rotational levels is shown. The electronic levels are determined by the
atomic and electronic configurations of the molecule, whereas the vibrational and
rotational levels are determined by the three-dimensional positions of its atomic assembly,
and the molecule’s quantized vibrational/rotational characteristics are generated by the
movements of the atomic nuclei of the molecules.
12
The simplest calculation of vibrational energy levels for a diatomic molecule
involves the approximation of the potential by a parabola as in a harmonic oscillator. This
results in a simple expression relating vibration frequency ν and energy E
E = (v + ½ )ћν
with ν=√k/µ, where µ is the reduced mass, k is the elastic constant and v=1, 2,...
Thus, the vibrational levels are equidistant and there will always be a non-zero lowest
vibrational energy.
Figure 1.1. Energy states in a molecule.
The degree of complexity of energy levels increases for polyatomic molecules where
the number of modes of vibration increases. There are two types of vibrational modes:
stretching modes, where the distances between the atoms or groups of atoms are altered;
and bending modes, where the angles between atoms or groups of atoms are altered.
Molecules in general can rotate around three different axes. For the simple case of a
diatomic molecule, it rotates around the mass centre and the rotation energy can be
calculated as
E = ћ2J(J +1)/2I
with J = 0, 1, 2, ... and I is the moment of inertia with respect to the rotation axis. If
the bond between the atoms is not completely rigid, the molecular bond will be stretched
out in higher rotational states and the moment of inertia I will increase, leading to a
downshift of higher levels. In polyatomic molecules the theory becomes much more
complex, and also here symmetry aspects are important. In large biomolecules, the
rotational structure, in the energy level diagram, is often lost and the energy levels are
smeared out into a band.
The configuration of the energy levels of a molecule determines many of the
properties of that molecule. For instance, the energy separation between the electronic
states determines the visible wavelengths that can be absorbed and thus the colour of the
molecule. The transitions between the levels are the origin of many interesting phenomena
like laser action, phosphorescence and fluorescence.
13
The energy separations between close-lying electronic levels often corresponds to
visible wavelengths. Vibration transitions occur in the infrared range, while transitions
between rotational levels in the same vibrational mode fall in the far infrared range.
1.2.2 Transitions between levels
Light impinging on a molecule can, if the energy corresponds to the energy
separation between two levels, be absorbed and an electron is transferred to a higher level.
The excited state of a molecule can be an electronic, a vibrational or a rotational level,
where a multitude of vibrational and rotational levels are associated with each electronic
level. The molecule only remains in this state for a short period of time given by the
lifetime of that state. There are selection rules set by quantum mechanics for radiative
transition within vibration and rotational levels. For vibration, ∆v = ±1 must be fulfilled
and for rotation ∆J = ±1. On the way back to the ground state, none or several intermediate
levels may be involved.
Figure 1.2: Jablonski diagram of a fluorescent molecole. Electronic levels are S, singolet states, and T,
triplet states. Vibrational levels are represented by the nonlabeled horizontal bar, contained within the
electronic levels. Rotational levels are not shown. A denotes the resonant absorption of a photon, S is the
resonant scattering, and IC internal conversion. All “wavy” arrows denote internal conversion. F is
fluorescence and P phosforescence. IX represents intersystem crossing and ET energy transfer to another
molecule.
Figure 1.3. Schematic of energy transfer from photos
molecules. The molecule absorbs a photon with a electron jump up excited states empty (S
20 nsec three phenomena can be occur: the first is the radiative decay with fluorescence emission,
is energy conversion in heat; the third is state change of the electron which modifies only the state spin (T
T2 triplet states). This later event is statistically dominant and produces other events: radiative decay with
phosphorescence emission and energy transfer to oxygen molecules, that so they produce radicals.
On the one hand, elastic resonant radiation
emitted at the same wavelength as the incoming one. On the other hand, one of several
inelastic scattering processes may ensue. In brief, the molecule can return to its ground
state through one of the following pathways (Figure 1.2)
• resonant radiation of identical wavelength, compared to the incoming photon.
• Internal conversion (IC
transferred to the lowest lying
state furnishing energy
• Intersystem crossing:
including emission of light is forbidden, if an atom, with a singlet ground state, is in
its lowest lying triplet state, it can only release the excess energy due to other
interactions, for instance collisions. Such a transition is called i
Thus the triplet state is a metastable state with a long lifetime. In a so
Jablonski diagram (Figure 1.2), the transition from state S
state, the molecule can stay for long times since the transition to the ground state S
is forbidden. If it returns to the ground state, the emission is called
phosphorescence.
• Fluorescence: when the molecule returns to any of the levels
fluorescence light can be emitted. The wavelength of the fluorescence will always
be longer than the excitation light. Fluorescence light in solids and liquids is
broadbanded due to the strong interaction between molecules and due to
number of vibration and rotation levels
of lower energy, i.e. greater wavelength.
• Energy transfer to a neighboring molecule
relevant example is the transfer of
cytotoxic oxygen species, such as singlet oxygen (
destroy e.g. lipid bilayer membranes (Figure 1.3)
utilized in selectively destroying tissue, e.g. neoplastic lesions in a process called
ensitive substance (hematoporphyrin) to oxygen
can occur, resulting in a photon being
(7):
): due to interaction with other molecules, the electron is
vibrational and rotational state within the excited
to molecular motion generating heat.
since the transition between a singlet and
nter
1 to T
which involves the re-emission of a photon
, causing a chemical reaction. A clinically
energy to oxygen, potentially resulting in
1O2), which can react with and
(8). This phenomenon can be
14
1 e S2). After about
the second
1 e
a triplet state
-system crossing.
-called
1 is shown. In this
0
in the ground state,
the large
15
photodynamic therapy (PDT). In PDT, a photosensitizing substance is either
applied topically, i.v. or i.p. injected or administered orally, followed by
illumination of the treatment area, using an appropriate wavelength. The desired
result is tumor necrosis (9).
• Molecular dissociation after excitation to repulsive states.
In conclusion, the deexcitation modes non-radiative, collision, internal conversion and
inter-system crossing, compete with fluorescence emission.
1.3 Fundamentals of tissue optics
The term tissue optics encompasses modelling of the light transport in tissues,
measurement of tissue optical transport parameters, and development of models which
can explain the optical properties of tissue and their dependence on the number, size and
arrangement of the tissue elements. An exact modelling of the inhomogeneous and turbid
tissue is not presently feasible. The tissue is therefore generally represented as an
absorbing bulk material with scatterers randomly distributed over the volume. Further, it
is usually assumed to be homogenous, even though this is not a true representation.
Hence, absorption and scattering are the two physical phenomena affecting light
propagation in biological tissue (Figure 1.4). Although both are important, scattering is the
dominant mechanism (10). Even for sub-millimetre sections of tissue, injected photons are
likely to be scattered several times before they reach the boundary. As a consequence a
coherent, collimated input laser beam will be effectively incoherent and isotropic after
traversing a few millimetres of tissue.
Figure 1.4 shows schematically what happens to a light photons beam when they incide
on the interface between air and biological medium. If the incidence angle is not normal, a
fraction will be reflected (specular reflectance, see Figure 1.12); a remaining fraction will be
refracted in the biological medium, where photons can be or absorbed by molecules when
the light photon energy equals the energy difference between levels of molecular
electronic states; or backscattered, after photons have carried out multiple scattering
events that report them on the interface (diffuse reflectance); or forward scattered
remerging on the opposite interface after multiple scattering events that have altered their
motion direction (diffuse transmittance); or unaltered transmitted without scattering
events on their walk (ballistic component).
Therefore, the parameters used to characterize the optical properties of the tissue are:
• the absorption coefficient µa,
• single scattering coefficient µs,
• transport coefficient µt = µa + µs
• phase function p(es,es´), from which derive the anisotropy parameter g and the
reduced scattering coefficient µs’ = µs (1 – g).
In the next paragraphs we will illustrate with greater details these two basic
phenomena in the light propagation in tissue, by introducing the corresponding
parameters and by discussing the main characteristics of biological tissues components.
Figure 1.4: Scattering and absorption of light photon
are reflected or refracted on the interface between air and biological medium; the photons refracted are or
absorbed by molecules, or backscattered returning on the interface (diffuse reflectance) or forward scattered
remerging on the opposite interface (diffuse transmittance) or unal
1.3.1 Absorption
The condition where
matches the energy difference between the final (excited) and initial state of a molecule is
called resonance. The photon ca
the initial state to the excited state through a transition. Hence, the probability of
absorption is strongly wavelength dependent.
chromophores.
There are two major types of transition:
transitions (5).
The former are relatively energetic and hence are associated with absorption of
ultraviolet, visible and near-infrared wavelengths. Many biological molecules can absorb
light via electronic transitions. In early biological evolution, the pyrrole molecule was a
chromophore which could absorb sunlight which enabled subsequent synthetic reactions
that produced biological polymers and other proto
pyrroles into a tetrapyrrole ring
photons: porphyrin. Chlorophyll is such a porphyrin. Hemoglobin, vitamin B12,
cytochrome C are also examples of porphyrins in biology. We
contrast agent used in optical imaging in our work belongs to the porphyrin family, as we
will see in the next chapter.
The vibrational transitions interest
variety of bonds which can resonantly vibrate or twist in response to infrared wavelengths
and thereby absorb such photons. Perhaps the most dominant chromophore in biology
which absorbs via vibrational transitions is water (Figure 1.
absorption of water is the strongest contributor to tissue absorption.
s in a biological medium. The incident photons
tered transmitted (ballistic component).
the photon energy of the incident radiation field exactly
n be then absorbed by the molecule which changes from
Molecules that absorb light are called
electronic transitions
-metabolic products. Combining four
yielded an efficient chromophore for collecting solar
remark
the field of infrared spectroscopy that studies the
5). In the infrared, the
16
and vibrational
that the optical
17
Figure 1.5: Scheme of vibrational modes of water molecule together to the corresponding frequency
and wavelength of the transition.
1.3.2 Absorption coefficient
The absorption coefficient is the parameter used to describe the effectiveness of
absorption. To understand the meaning of this parameter, we can use a rough
modelization of the real situation. We can modelize a chromophore as a sphere with a
given radius; this sphere, absorbing incident light, projects a shadow (Figure 1.5). The area
of the shadow represents the effective cross-section (σa [cm2]) and can be smaller or larger
than the geometrical area of the chromophore (A [cm2]), depending on the dimensionless
proportionality constant Qa called the absorption efficiency:
σa = QaA
If a medium containing many chromophores at a concentration described as a
volume density ρa [cm3] is considered, the absorption coefficient µa [cm-1] is the cross-
sectional area per unit volume of medium
µa = ρaσa
Figure 1.6: Schematic description of the absorption of light by a chromophore.
Since µa [cm-1] has the dimension of a reciprocal of a length, the product µaL, where
L [cm] is a photon's pathlength through the medium, is a dimensionless quantity. So the
absorption coefficient µa can be defined from the equation
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