1

CHAPTER ONE
1: Introduction
Nuclear Magnetic Resonance Field-Cycling (NMR FC) relaxometry is a technique used
to study the spin-lattice relaxation rates, T
1
of samples as a function of magnetic field
strength or Larmor frequency. Studies of this nature provide information on molecular
dynamics of such samples.

Initially, relaxometry studies involved shuttling the sample between different fixed
magnetic field strengths to obtain the respective relaxation signals from the different field
strengths. However, commercial advances in instrumentation have allowed for a rapid
variation of the magnetic field experienced by the sample while it is fixed in position.
The technique of rapidly varying the field strengths experienced by the sample is called
Fast Field Cycling (FFC).

NMR FC relaxometry shows promise in the early diagnosis of cancer, Alzheimers and
muscle-wasting diseases.
[1, 2]
This is because these conditions can be probed by
relaxation mechanisms involving mainly hydrated proteins whose relaxation rates are
measured noninvasively, in vivo.
[1, 2, 3]
Relaxometry finds applications in the study of
liquid crystal dynamics, hydration of paramagnetic metal ions and organometallic
complexes and dynamics of proteins.
[4, 5, 6]
The latter being the major application in this
study: to measure protein concentration from the dynamics of proteins.
The strength of NMR FC relaxometry lies in the fact that it is fast, non-destructive and
the equipment is able to access information (with sensitivity and resolution) that is
inaccessible to the NMR spectrometer.
[4]

2

This chapter introduces the subject matter and also presents the plan of the project report.
Chapter 2 discusses the background theory in related publications to clarify the
underlying concepts of relaxometry.

Chapter 3 describes the equipment used, sample preparation, pulse sequence selection
and the general methodology involved in obtaining AR
1
. The results of experiments are
presented along with discussions in Chapter 4.

Chapter 5 concludes the report with a brief outline of future work. Spreadsheets of data
from the experiments and other relevant material inappropriate for insertion into the main
report are placed in Appendices.

1.1 Project Activity Schedule and Materials
The project activity outline included: literature search and self-study (research),
preparation of samples, experimentation, data processing and writing up. The research
stage involved a search for related publications and study to build up a good
understanding of the technical aspects and operation of the instrument, and a general idea
of the study area.

Data obtained in each experiment were processed immediately before the next
experiment. All activities carried out during the experiments, progress of work, details of
meetings and resolutions with supervisors of the project were recorded in a logbook. This
record of activities were, indeed, very helpful during the writing up stage.

The resources for the project were bovine serum albumin (BSA), phosphorus buffer
saline (PBS), agarose, copper (II) sulphate (CuSO
4
), NMR tubes, a FFC NMR
3

relaxometer, a MR scanner, a computer and student versions of Matlab, Microsoft Excel
and Word.

1.2 The Literature Review Process
The search keywords: relaxometry, protein concentration, relaxation phenomena,
relaxation processes, protein dynamics, nuclear quadrupole resonance, field-cycling and
NMR relaxometry were used in various search engines for related publications.

The search engines used were: Scopus, ISI Web of Knowledge, the Aberdeen University
library catalogue and the Internet. Some articles were also obtained in hardcopies from
my supervisor, Dr. Gareth Davies.

The relevant publications for this project have been duly referenced, where appropriate,
within this report. To the best of my knowledge, therefore, no such work has been
directly published elsewhere.
1.3 Significance
This study was aimed at determining the veracity of the proportional relationship between
the magnitude of quadrupole peaks and protein concentration in protein gel samples; and
also what were the effects on this relationship with the addition of non-protein gel
components or contrast agents. The sensitivity, to protein changes, of the NMR FFC
relaxometer used was also assessed; thus, providing first-hand information to enable a
further study of protein dynamics in future.


4

CHAPTER TWO
2: Background Theory and Literature Review
This chapter discusses the background of the project and how it fits into related cases of
study to solve problems in the study area. An emphasis is placed on the basic concepts of
relaxometry, sourced from related literature.

2.1 About the Project
This project exploits
1
H dipole-
14
N quadrupole cross relaxation to measure an effect that
should be proportional to the immobilised protein content of a gel. The goal was to use
the field-cycling technique to rapidly, electronically vary the magnetic field experienced
by the protein sample in the FFC NMR relaxometer at a switching time less than the T
1

relaxation constant of the sample so that the cross-relaxation effects can be explored at
specified field strengths of 16 mT, 49 mT and 65 mT, where quadrupole peaks are
observed in the dispersion profile.
[1, 2, 5, 7, 8, 9, 10]
However, for sensitivity and signal
homogeneity reasons, only field strengths of 49 mT and 65 mT were explored.

From the R
1
dispersion plots, a further processing technique was developed to extract and
compute the size of the quadrupole peaks, called change in relaxation rate, AR
1
, due to
quadrupole processes. AR
1
has previously been shown to be proportional to the
concentration of protein in each sample.
[1]


Finally, based on the effect of a contrast agent on the direct relationship between AR
1
and
the concentration of protein in a given sample, a physiologically representative phantom
(with realistic T
1
and protein concentration characteristics) was produced, imaged and its
image analysed to assess the agreement between the MR imaging and the relaxometry
5

data sets. In the end, the sensitivity (to protein concentration changes) of the newly
acquired departmental Stelar SMARtracer FFC relaxometer used for the project was
assessed for the first time.

The next lines of discussion below present the background theory of field-cycling and its
applications in NMR relaxometry, with particular emphasis on protein concentration
measurements.

2.2 Nuclear Magnetic Resonance
Nuclear Magnetic Resonance (NMR) is a process that involves the physical interaction
between radio waves at specified resonance frequencies and certain nuclei in a magnetic
field, the nature of the resonant interaction being dependent upon the magnetic quantum
mechanical properties of the nuclei.
[11]
After its discovery in 1938, it was further
developed by F. Bloch and E.M Purcell, independently, in 1946 for measurement of spins
and magnetic moments of nuclei.
[12]
Since this period, NMR has been applied to the study
of the chemical and physical properties of molecules (NMR Spectroscopy), biological
and medical applications (MR Imaging) and the structure and dynamics of solids, liquids
and gases (NMR Relaxometry).
[13, 14]

The resonant interaction of the nucleus with the magnetic field can be explained by two
models: the Quantum Mechanical and Classical Models.
[11]


The Quantum Mechanical Model explains that the ability of the nucleus to interact with a
magnetic field is due to its intrinsic spin that causes it to apparently precess about its axis.
The relative number of protons and neutrons present within a given nucleus determines
the total (intrinsic) spin, I, of the nucleus, where I is an integer or half integer value. This,
6

therefore, means that for a nucleus to interact with a magnetic field, I 0 necessarily;
which further means that such a nucleus possesses angular momentum, p, along the axis
of the spin related to the intrinsic spin, I, by:
[11]

p = I (2.1)
where = h/2t, h is Plancks constant = 6.626 x10
-34
Js.
[13]


The charge in the nucleus creates an effective current loop by rotating about its axis, thus
giving rise to a resultant magnetic dipole moment, , given by:
[11]

= p = I (2.2)
where is a constant called the gyromagnetic ratio, whose value is characteristic of a
given nucleus. For instance, the
1
H nucleus, which is the nucleus of interest in most NMR
experiments (including this project) due to its relative abundance of 99.98 % and greatest
NMR sensitivity, consists of a single proton and spin, I, value of . For protons,
= 26.75 X 10
7
rads
-1
(/2t = 42.58 MHzT
-1
). It is this dipole moment of the nucleus that
makes it behave like a little bar magnet (Figure 2.1), capable of physical interaction with
an external magnetic field.
[11]



Figure 2.1: A spinning nucleus has a net spin, I and a magnetic dipole moment, along its axis, analogous
to a bar magnet. After reference 14

Direction of
magnetic field
Bar magnet
Spinning proton
7


The quantum mechanical model further specifies that the magnetic dipole moment of the
nucleus can only have (2I + 1) orientations in the magnetic field, corresponding to
(2I +1) discrete energy levels. Thus, the
1
H nucleus with I = has two quantised states:
spin up and spin down. In a magnetic field, therefore, it tends to align with or against
the field corresponding to low and high energy states respectively (Figure 2.2). The
energy difference between the two quantum states is proportional to the external
magnetic field, B
0
:
[11]

AE = B
0
/ I = B
0
(2. 3)












Figure 2.2: Orientation of the nuclear spins in an external magnetic field, B
0
the antiparallel state is
slightly higher in energy than the parallel state. After reference 14

Application of electromagnetic (em) radiation of suitable frequency, called the Larmor
frequency, u
L
, causes transitions between the two energy states. This Larmor frequency
is related to the energy difference, AE, between the two states by:
[11]

AE = h u
L
= e
L
(2.4)
e
L
= AE / = B
0
(2.5)
B
o

Parallel
Antiparall
el
Parallel State
Antiparallel
State
AE
8

where u
L
is the frequency in hertz (Hz) and e
L
is the angular frequency in radians per
second (rads
-1
) of the electromagnetic radiation to cause the transition from the lower to
higher energy states.

The populations of spins in the lower, N
L
, and higher, N
U
, energy states are described by
the Boltzmann distribution:
[11]

N
L
/ N
U
= exp (e
L
/ k T) (2.6)
where k is Boltmanns constant and T is the absolute temperature.

Thus, only radiation of frequency, e
L
, can cause this transition to occur and so the
resonance effect the origin of the name of the phenomenon of Nuclear Magnetic
Resonance.

The observable spins (after the application of the em radiation) in a NMR experiment are
those spins in the lower energy state, referred to as the fractional excess, given by:
[11]

(N
L
N
U
) / N
U
= e
L
/ k T = B
0
/ k T (2.7)

Thus, equation (2.7) suggests that to increase sensitivity to the NMR signal detection of
the equipment (imager or relaxometer), we must either decrease T (impractical in
patients, though possible in the samples used in this project) or increase B
0
.


The Classical Model rather permits spin orientation in all directions in the external
magnetic field, where the magnetic moment, , of each spin is being aligned with the
magnetic field, B
0
, by experiencing a turning force, L, given by:
[11]

L= B
0
(2.8)
9

Due to the angular momentum, p, of the individual spins, the turning force, L, rather
causes them to precess about B
0
, similar to a gyroscope in a gravitational field, at a rate
of change of momentum given by:
[11]

dp / dt = L = B
0
(2.9)
d / dt = B
0
= e
L
(since p = / ) (2.10)
where e
L
= - B
0
(2.11)
Equations (2.10) and (2.11) infer that the nucleus spins at a rate of e
L
rads
-1
about the
direction of the applied field, where the minus (-) sign gives the direction of rotation.
This frequency of precession is equal to the frequency of resonance of equation (2.5) and
is called the Larmor frequency.
Ideally, in a NMR experiment, it is the overall effect of the individual magnetic moments
of all the nuclei of interest in a sample that is observed but not a single magnetic moment.
The combined observable magnetic moment (viewed as a large magnetic dipole moment)
is called bulk magnetisation, M and is given by:
M = (2.12)
At equilibrium, M has no transverse component to B
0
due to phase incoherence among
the many different making up M. However, due to the preference for these many to
align with B
0
, the result is that M has a longitudinal component along the B
0
direction, of
magnitude M
0
, called the equilibrium magnetisation, which is equal to the spin excess
detected as explained by the quantum model (Figure 2.3).

10








Figure 2.3: Equilibrium magnetisation, M
0
, aligned with B
0
. After reference 13

In order to detect this bulk magnetisation, it is caused to precess about B
0
by the
application of a second magnetic field, B
1
, to excite the NMR nucleus. B
1
is generated by
the passage of an oscillating current at the Larmor frequency, e
L
, through a coil which
creates an oscillating magnetic field, B
1
, perpendicular to B
0
. The B
1
field displaces M
away from B
0
and causes it to execute a spiral path (Figure 2.4). When B
1
is switched off,
M continues its precession about B
0
, describing a cone at a flip angle, o, to B
0
. o depends
on the strength of the B
1
field and how long, t
p
, it acts:
[11]

o = B
1
t
p
(2.13)

Figure 2.4: Spiral path executed by the bulk magnetisation, M
0
following a 90
0
RF pulse viewed from the
laboratory frame of reference. From reference 12
B
o

x
y
z
M
o

11

At a given amplitude and time of the B
1
field, it is possible to tip M through 90
0
(where
B
1
is called a 90
0
pulse) or completely invert M to the B
0
axis (where B
1
is called an
inversion pulse or 180
0
pulse). The frequencies of B
1
used in NMR experiments
typically fall within the radiofrequency bandwidth (1 500 MHz) of the spectrum and so
B
1
is often referred to as a radiofrequency (RF) magnetic field and its pulses are RF
pulses.
[11]
RF pulses of 180
0
and 90
0
shall be explored in this project.
As long as the magnetisation, M, is flipped away from the B
0
axis, its transverse
component generates an oscillating magnetic field. This is then detected as a small
voltage (which also oscillates at e
L
) by electromagnetic induction into an RF coil, tuned
at e
L
.
In the presence of a 90
0
RF pulse, this oscillating transverse component of M, and for that
matter, the induced signal in the RF coil, is maximum. At the end of the application of the
RF pulse, the signal decays gradually due to the relaxation of M, which causes it to return
to its equilibrium position, M
0
, parallel to the axis of B
0
(Figure 2.5). This detected
decaying signal in the absence of the RF pulse is called Free Induction Decay or FID.
[11]




Figure 2.5: An FID after the application of a 90
0
RF pulse. After reference 13
Signal
FID
RF Pulse
90
0

12

It is usual to describe the behaviour of M in a Rotating Frame of Reference for ease of
visualisation, especially when more than two RF pulses are involved.
[11]
In this reference
frame, the x and y axes are replaced with x
/
and y
/
synchronised with the rotation of the
nuclear magnetic moments, .
2.3 Relaxation Phenomena
Nuclear relaxation processes involve the thermal equilibration of the spin systems with
respect to longitudinal or transverse magnetisation components, multiple-quantum spin
coherences and longitudinal dipolar, quadrupolar or scalar order.
[15]
The effect of
relaxation is that it causes a gradual decay away of the FID detected in a NMR
experiment due to energy exchange between spins and between spins and their
surroundings. These interactions are called spin-spin relaxation and spin-lattice
relaxation, with time constants T
2
and T
1
respectively.
[11]
These two relaxation
processes together cause the magnetisation vector to return to its equilibrium position
parallel to the axis of B
0
. Thus, the T
1
and T
2
relaxation time constants are valuable
parameters for studies of molecular dynamics,
[11]
and so in this project, the T
1

components of the protein samples are used in the study of their molecular dynamics. A
parameter of interest, called the relaxation rate or R
1
, is evaluated as the reciprocal of
the T
1
values (1/T
1
) in order to quantify the amount of polypeptides in the samples.
Conversely, the relaxation rate R
2
is also 1/T
2
.
Spin-spin relaxation effect is caused by dipole-dipole interactions between neighbouring
nuclear magnetic moments in an externally applied B
0
field. These interactions result in
variations in the precessional rates of the individual spins and hence loss of phase
13

coherence in the detected transverse magnetisation, M
XY
with time constant T
2
(Figure
2.6). Ordinarily, magnetic field inhomogeneities experienced by the sample cause
additional signal decay or pseudo-relaxation with time constant T
2inhom
. Therefore, the
net loss of phase coherence is described by another time constant, T
2
*
, defined as:
[11]

1 / T
2
*
= 1 / T
2
+ 1 / T
2inhom
(2.14)
where 1/T
2inhom
= AB
0
; with AB
0
defined as the variation of the applied magnetic field
strength over the region of the sample.

Figure 2.6: Loss of phase coherence between the magnetic moments,
i
, due to spin-spin relaxation causes
an exponential decay of the transverse magnetisation, M
XY
, with a time constant T
2
. From reference 11

To measure both the T
2
and T
1
relaxation times, a collection of RF pulses, with
predefined time intervals to control the reception and characteristics of the NMR signal is
used. This set of RF pulses is called a pulse sequence. There are several pulse
sequences available for the measurement of the T
2
and T
1
relaxation time constants but
usually, T
1
is measured by an inversion recovery pulse sequence whereas T
2
is
measured by a spin echo pulse sequence.
14


Figure 2.7: Measurement of T
2
in the spin-echo experiment (shown in the rotating frame of reference,
x
/
, y
/
, z). After reference 11

The spin echo pulse sequence employed in the measurement of the T
2
relaxation time
comprises a 90
0
pulse, followed after an echo time TE/2, by a 180
0
pulse (Figure 2.7).
The 90
0
pulse flips the magnetisation vector, M, onto the y
/
axis (a), where the
component vectors making up M are caused to dephase with respect to one another due to
inhomogeneity of the applied magnetic field (b). Following the application of the 180
0

pulse, after the waiting time of TE/2, the fan of magnetisation vectors is flipped over to
the y
/
axis (c). The initial dephasing process after the 90
0
pulse is now exactly reversed
and the vector sum of the rephrasing magnetisation vectors reaches a maximum at time
TE, at the centre of an echo signal (d). In this case, instead of the initial FID after the 90
0

pulse, it is rather the spin echo signal after the 180
0
pulse that is acquired (e), the
amplitude, S
2
, of which is given by:
[11]

S
2
= S
1
exp (-TE / T
2
) (2.15)
TE/2

15

where S
1
is the initial amplitude of the FID generated by the 90
0
pulse. The value of T
2

can, therefore, be calculated from equation (2.15) above.

Spin-lattice relaxation effect is caused by the interactions of the spin system with its
surroundings, resulting in energy loss from the spins to their surroundings. The energy
loss process is an exponential decay towards the equilibrium value, M
0
, of the
longitudinal component of the magnetisation vector, M
z
, with a time constant T
1
. Once
spin-lattice relaxation has returned the magnetisation vector to its equilibrium value, M
0
,
there cannot be any transverse magnetisation component.
[11]
Hence, T
2
is always less
than or equal to T
1
. In fact, both T
1
and T
2
processes occur simultaneously with the only
restriction being that T
2
is less than or equal to T
1
.
[13]














Figure 2.8: Return of the bulk magnetisation to its equilibrium magnetisation, M
0
, due to spin-lattice
relaxation with time constant T
1
. After reference 14
z
x
y
x
z
y
y
x
z z
x
y
M
M
M
16

The inversion recovery pulse sequence used for measuring the T
1
relaxation time of a
sample comprises a 180
0
pulse, followed after an inversion time TI, by a 90
0
pulse (Figure
2.9). The 180
0
pulse inverts the magnetisation onto the z axis such that its M
z

component has an initial value of M
0
(b). During the inversion time, TI, the
magnetisation recovers towards its equilibrium value of +M
0
along the z axis (c).
Following the application of the 90
0
pulse, any M
z
component recovered (along the z
axis) is converted into transverse magnetisation, M
xy
(d). The magnitude and sign of the
resulting FID is indicative of how far the magnetisation had recovered along the z axis
during TI.











Figure 2.9: The inversion recovery experiment, viewed from the rotating frame of reference x
/
, y
/
, z
/
. The
180
0
RF pulse is followed by a 90
0
RF pulse after an inversion time TI (longer than both pulse lengths).
After reference 11
Signal
90
0

180
0

RF
(a) (b) (c) (d)
B
1

(c)
(d)
B
1

(b)
-M
0

(a)
M
0

z
x
/

y
/

TI
17

The value of T
1
in an inversion recovery experiment is calculated by curve fitting to a
multi-point TI data set (Figure 2.10) using the general equation describing the signal
strength, S proportional to the magnitude of M
z
, just before the 90
0
pulse:
[11]

S o M
z
(TI) = M
0
[1 2 exp (-TI / T
1
)] (2.16)
where M
0
is the equilibrium magnetisation (equivalent to the proton density in the
sample).

Figure 2.10: Plot of M
Z
versus time in the inversion recovery pulse sequence. At t = 0, the inversion pulse
ends; at t = TI at the end of the inversion pulse, the 90
0
pulse is applied, converting M
Z
into transverse
magnetisation and generating an FID proportional to M
Z
(TI). Three curves are shown for samples with
different T
1
values: the sample with the shortest T
1
value appears brightest in an MR image since its
magnetisation recovers fastest to a large positive value. After reference 12

It is worth mentioning that the measured T
1
and T
2
values are influenced by a number of
extrinsic (operator-controlled) and intrinsic (sample property) factors.
[11]

Molecular motions (tumbling) in solutions result in time varying magnetic fields and so
cause spin relaxation. This is because time varying fields at the Larmor frequency cause
transitions between the spin states and hence, a change in the longitudinal magnetisation,
B
r
i
g
h
t
n
e
s
s

18

M
z
. Only those rotational frequencies among the distribution of molecules at the Larmor
frequency affect T
1
. Since the Larmor frequency is proportional to B
0
, T
1
will therefore
vary as a function of magnetic field strength with the result that T
1
becomes inversely
proportional to the density of molecular motions at the Larmor frequency.
[11, 13]

Moreover, the motional frequency distribution is dependent upon the temperature and
viscosity of the solution. Hence, T
1
will again vary as a function of temperature as shown
in Figure 2.11.

Figure 2.11: Plot of number of molecular motions versus frequency at different temperatures: at the Larmor
frequency indicated by u
0
, T
1
(280 K) < T
1
(340 K). From reference 13

This makes keeping the temperature of samples constant essential in the experiments
within this project. Thus, the temperature of each sample did not vary by enough to cause
a significant influence on T
1
at a given field strength. The viscosity, however, does vary
significantly among samples (depending on the concentration) and so a resulting
influence on T
1
.
[13]
A highly viscous solution would have a shorter T
1
than a less viscous
solution. This also required that samples be given long enough time to denature
19

considerably to gels to give high signal-to-noise ratio in the measurements of their
relaxation rates. Figure 2.12 shows spectral density functions of the variation of Larmor
frequency with sample viscosity.

Figure 2.12: Spectral density functions of Larmor frequency variation with viscosity. From reference 13

Furthermore, variable fields which agitate the energy levels of the spin states dephase the
transverse magnetisation, M
xy
, with the result that the number of molecular motions less
than or equal to the Larmor frequency becomes inversely proportional to T
2
. Thus,
relaxation times get longer with increasing B
0
as there are fewer relaxation-causing
frequency components available in the random molecular motions.
[13]

In addition to the above, the exchange of spin states between two spins affects T
2
but not
T
1
. T
1
is not affected because spin distribution between the lower and upper states is not
changed but phase coherence of the transverse magnetisation, M
xy
, is lost during the
process of spin exchange which consequently affects T
2
.
20

Lastly, the exchange of nuclear species between two or more molecules in a mixture by a
process called chemical exchange affects both T
1
and T
2
relaxation times. T
1
is affected in
this case because energy is transferred from one nucleus to another.
[13]
For instance, if
there are more nuclei in the upper state of specimen A and a normal Boltzmann
distribution in B, the exchange process will force the excess energy from A to B
with the effect that T
1
becomes smaller. This phenomenon has been observed in this
project from a mixture of the protein samples with some contrast agents. The chemical
exchange process reduces the T
1
relaxation time of the samples and so increased
relaxation rates are observed from them. T
2
is also affected because phase coherence of
the transverse magnetisation is not preserved during a chemical exchange process.
[13]

2.4 Nuclear Quadrupole Resonance
Nuclear Quadrupole Resonance (NQR) is a physical phenomenon involving the nucleus
interacting with an external field similar to NMR discussed in section 2.2. In NMR, it has
been shown that the energies of nuclei with a magnetic dipole moment are split by an
external magnetic field in order to allow resonance absorption of energy that is related to
the difference between the ground state energy and the excited state. In NQR, however,
the energies of nuclei with an electric quadrupole moment are split by an electric field
gradient that has been created by the electronic bonds in the local environment.
[6]
The
nuclei are then set in a static inhomogeneous electric field, allowing the absorption of
energy from a radiofrequency field.
[16]
Since unlike NMR, NQR occurs in an
environment without a static (or direct current) magnetic field, it is sometimes called
zero-field NMR. Many NQR transition frequencies rely greatly on temperature.
[6]

21

Any nucleus with more than one unpaired nuclear particle (the nuclear particles being
protons or neutrons) will have a quadrupolar charge distribution. Examples of such
nuclei are nitrogen-14 (
14
N), chlorine-35 (
35
Cl) and copper-63 (
63
Cu). The resonance
interaction of this quadrupole with an electric field gradient supplied by the non-uniform
distribution of electron density (from bonding electrons) results in the NQR effect. This,
thus, makes the technique of NQR very sensitive to the kind of bonding surrounding the
quadrupolar nucleus.
[6]

For an illustration of the NQR effect, in the simplest case of
35
Cl in solid Cl
2
, NQR is
involved in the precession of the angular momentum, I (and the nuclear magnetic dipole
moment, ) of the nucleus, shown in Figure 2.13, as a flat ellipsoid of rotation around the
symmetry axis (taken to be the z axis) of the Cl
2
molecule fixed in the crystalline solid.
[16]










Figure 2.13: Interaction of
35
CI nucleus with the electric field of a Cl
2
molecule. After reference 16

I
I
z


Cl
2

Z
eQ

Z
22

The constant angle, u, (of the precession) between the nuclear axis and symmetry axis of
the molecule results from the torque exerted by the inhomogeneous molecular electric
field on the nucleus of electric quadrupole moment, eQ. Classically, the resonant
absorption occurs when the frequencies of the RF field and the precessing motion of the
angular momentum are equal.
The resonance interaction in NQR is considerably larger compared with the chemical
shift measured in NMR but can be approximated to zero (or will not virtually occur) in
the liquid phase. Therefore, NQR spectra can only be necessarily measured for samples
in a fairly solid phase. This condition is remarkably both a strong point and a weakness of
the NQR phenomenon.
[6]
Samples used in this project, therefore, had to be cooked over
prolonged periods to ensure complete denaturation to gels in order to measure the NQR
effect in them.
So far, NQR spectra have been observed in the approximate Larmor frequency range of 1
to 1000 MHz
[16]
and in this project, a range of 1.5 to 3.5 MHz is used to probe the NQR
effect in the protein samples. These samples have
14
N-
1
H groups (in which the backbone
H atoms are strongly coupled to the spins of adjacent N atoms)
[12]
that serve as
relaxation sinks in order to enhance proton relaxation due to their interactions with the
14
N quadrupolar nucleus.
[2, 5]
This leads to reductions in the proton spin-lattice relaxation
time, T
1
, called quadrupole dips (where 1/T
1
is called a quadrupole peak), which
occur at three NMR frequencies corresponding to the
14
N nuclear quadrupole transitions.
Since
14
N has I = 1, this implies that (2I + 1) or 3 energy transition states are available in
the
14
N nucleus, thus, resulting in electrical asymmetry in the peptide nitrogen and hence,
the three (3) distinct transitions in the
14
N system to which the protons of the protein may
23

couple. The transition frequencies are observed at field strengths of 65 mT, 49 mT and 16
mT (or 2.8 MHz, 2.1 MHz and 0.7 MHz, respectively, for the proton Larmor
frequencies).
[2, 4, 5, 7]
Thus, at these stated magnetic field strengths, protein and water
proton spin-lattice relaxation rates become sensitive to the concentration of rotationally
immobilised peptide nitrogen due to field dependent heteronuclear cross relaxation
coupling between protein proton and nitrogen-14 spins that have been carried to the water
by proton homonuclear cross relaxation.
[1]

Measurement of the water proton spin-lattice relaxation time or a signal amplitude that is
proportional to it (in this case, the size of the quadrupole dip or peak) may provide a
noninvasive measure of peptide bond concentration to reveal immobilised protein content
in most tissues.
[1]
The quadrupole dip effect was studied to a greater extent in the early
to mid 1980s and dips were measured in hydrated proteins and various biological
samples. Kimmich et al carried out the first in vivo demonstration of the effect in living
leeches.
[8]
This was followed by the work of Lurie in the 1990s in which quadrupole dips
were measured in vivo in human muscle and brain using a whole-body sized field-cycling
relaxometry and imaging system.
[2, 10]
Among other subsequent works, quadrupole dips
have been measured in collagen fibres,
[8]
calf lens
[9]
and multiple sclerosis plaques.
[12]

In this study, however, the quadrupole peak effect shall be measured in vitro in protein
samples prepared by the author.
2.5 Field-Cycling NMR Relaxometry
In normal NMR and MRI, the magnetic field strength, B
0
is fixed and very stable
throughout the signal measurement and imaging processes respectively. In Field-Cycling
24

NMR, however, the B
0
field is switched rapidly to different values during the pulse
sequence. If the B
0
field switching time is less than the T
1
relaxation time of the sample,
the technique is called Fast Field Cycling or FFC.
The process of varying the field strength, B
0
experienced by the sample can be achieved
by either moving the sample through a magnetic field gradient (i.e. shuttling the sample
rapidly between different magnetic fields) or by keeping the sample fixed in position
while varying the magnetic field it experiences.
[5, 17, 18, 19]
This latter option of field-
cycling application is what is being employed in this project by the use of an instrument
called the FFC NMR relaxometer. The technique is called field-cycling (FC) NMR
relaxometry. This is a NMR technique used to determine the longitudinal relaxation time
(T
1
) over a range of magnetic field strengths at a fixed temperature. However, the
boundaries of this range are not specific (Figure 2.14): the lower limit depends on the
local fields while the upper limit is mainly determined by technical choices.
[4]


Figure 2.14: Time (t) and angular frequency (e) scales covered by various NMR techniques. The
ranges indicated refer to proton resonance. From reference 5

T
1
is particularly suitable in relaxometry studies because T
1
in tissues is strongly
dependent on field strength (gets longer with increasing field strength) while T
2
is hardly
influenced by changes in field strength.
[7, 11]

25

More generally, FC NMR relaxometry is used to study the behaviour of nuclear
relaxation phenomena dependent on internal and/or external parameters, among which
are molecular and supramolecular structures, temperature, viscosity, pH, the influence of
paramagnetic and ferromagnetic agents and the magnetic field strength.
[7]
The theory of
the technique is based on the physical aspects of nuclei relaxation to the ground state
after being excited by an RF pulse.
[3]

While the conventional NMR approach studies molecular motions at constant frequency
by varying the temperature in biological systems, NMR relaxometry does exactly the
opposite. This is advantageous because when the temperature is varied, the sample
suffers from severe limitations arising from freezing or denaturation of the sample
material. Therefore, the only possible way to study the protein motions by relaxation
spectroscopy is to vary the Larmor frequency this is called Nuclear Magnetic
Relaxation Dispersion (NMRD).
[9]
Variation of the Larmor frequency means variation of
the external flux density, B
0
(according to equation 2.5). The range of variation, however,
is limited in conventional NMR spectrometers due to the fixed flux density provided by
the magnet in use.
[15]

The dispersion depends on the type of motion and on the observed nucleus. The most
informative
1
H NMR spectroscopic frequency range for rotational motions is centered
around 10 MHz for small motions (molecular weight, M
w
~ 10
4
Da) and smaller
frequencies for larger proteins and protein aggregations
[20]
(e.g. BSA with M
w
~ 66 10
3
Da). However, at these low fields, resolution is low and so, practically, only one signal
can be detected. Exploring the low frequency range, on the other hand, is beneficial in
isolating the typical relaxation features associated with molecular processes characterised
26

by very long correlation times like molecular surface dynamics and collective effects.
The range, thus, significantly enhances the spatial domain usually explored by the
relaxation techniques. This advantage of low field relaxometry has been exploited in this
project, where only the abundant water protons (with high S/N) can be conveniently
studied under such low sensitivity.
[20]

For the purpose of analysis and good understanding of NMR relaxation experiments with
biological materials, it is important to discuss the relevant interactions and fluctuations at
the molecular level in such materials. This is necessary because spin-lattice relaxation is
generally due to spin couplings modulated by the fluctuations within the system.
[8]

The interaction of water with biopolymers in general is considered to be the main source
of relaxation in biological tissue, and is therefore of paramount importance for the
interpretation of both relaxation data and MR images.
[5]
This interaction is paramount as
water molecules have sufficiently rapid motion such that on the time scale of a relaxation
time, they have thousands of encounters with the cellular constituents; the collective,
average influence of the constituents on the water protons is manifested in 1/T
1
.
[9]

Fundamentally, three (3) competitive mechanisms contribute to the spin-lattice relaxation
of this hydration water:
[5]

I. Restricted rotational diffusion of water molecules about axes perpendicular to the
local surface. This process can be described by an exponential correlation
function with a correlation time not much longer than that in bulk water.
27

II. Reorientation mediated by translational displacements (RMTD) along the,
somewhat, rough and coiled surface of the protein. This depends on both the
surface topology and the effective diffusivity along the surface.
III. Tumbling of the protein molecule as well as its hydration shell. An exponential
function can be used as a correlation function to describe this process.
However, restricted rotational diffusion is feasible only at high frequencies ( 10 MHz).
The low frequency dispersion is influenced by the RMTD mechanism of water and
tumbling of the hydrated protein molecule. The later mechanism is only possible if the
protein molecules are not prevented by mutual sterical hindrance at high protein
concentrations. Aside tumbling of the protein molecule itself, if not immobilised, spins
are relaxed by backbone fluctuations and side-group motions.
Only proteins in aqueous solutions undergo tumbling and side-group motions; the latter is
only possible at frequencies greater than or equal to 10
8
Hz (or 100 MHz).
[21]
Since the
protein samples used in this project were denatured to gels, and the maximum frequency
range explored was from 0.5 to 8.0 MHz, the only two possible relaxation mechanisms
were RMTD and backbone fluctuations.
Surprisingly, the R
1
dispersion profile is qualitatively the same regardless of the presence
of a bulk-like water phase. The same dispersion features occur even at water contents as
low as 25% by weight, where the hydration cells are just saturated and where protein
molecules are immobilised. In the absence or relatively low content of water (such that
water proton signals are negligible) protein internal motions become relevant in the spin-
lattice relaxation dispersion of protons.
[5]
This is when backbone fluctuations dominate
28

in the low frequency field-cycling window. A direct indication of backbone fluctuations
are
14
N-
1
H and (if deuterated water is used)
2
H-
1
H quadrupole dips originating from
relaxation sinks formed by quadrupole nuclei in the amide groups
14
N and (after
deuteron exchange)
2
H. The additional quadrupole interactions experienced by these
nuclei cause a much tighter coupling to the lattice and consequently produce enhanced
(faster) spin-lattice relaxation. This phenomenon reveals itself as quadrupole dips in the
T
1
dispersion (Figure 2.15).
[5, 9]


Figure 2.15: Origin of quadrupole dips in the case of
14
N-
1
H amide groups (a) Magnetic field dependence
of the
1
H (u
H
) and the three
14
N (u
N
(1)
, u
N
(2)
, u
N
(3)
) resonance frequencies in amide groups. (b) Proton
spinlattice relaxation dispersion; the quadrupole dips arise at the resonance crossings of
1
H and
14
N. From
reference 5

29

Thus, the condition for the occurrence of quadrupole dips or peaks is that molecular
motions are restricted such that motional averaging is incomplete on the time scale of the
experiment. This is true for dry or hydrated (but rotationally immobilised) proteins as
rotational reorientation of the peptide environment on the time scale of hundreds of
nanoseconds will average the nuclear electric quadrupole interaction to zero.
[1]

Specifically for protein backbone fluctuations, spin-lattice relaxation is universally
subject to a power law after elimination of potential contributions from side-group
motions:
[5, 21]

T
1
o u
L
b
(2.17)
which is expressed as a rate:
R
1
o u
L
-b
; (where R
1
= 1/T
1
) (2.18)
where the exponent b is usually in the range 0.65 to 0.85 at room temperature and
decays to lower values upon temperature reduction.
[5]
Kimmich and Nusser have
reported that b changes at about 200 K from a constant value above this temperature to
values decreasing with decreasing temperatures. This change, they reported, is a
manifestation of a transition from ergodic (at high temperature) to nonergodic
(associated with frozen states) behaviours in the time scale of the experiments. The
conclusion, therefore, is that interpretation of water relaxation data on the basis of
intramolecular protein dynamics alone is misleading.
[21]

Hence, for a complete description of the relaxation data referring to protons bound to
protein backbones, the two rate contributions below are essential:
[8]

30

1/T
1
= R
1
= R
p
+ R
q
(2.19)
where R
p
refers to homonuclear dipolar coupling between protons (i.e. proton-proton
interaction) and R
q
refers to heteronuclear dipolar interaction between protons and non-
resonant nuclei (i.e. the quadrupole peaks).
2.6 Technical Aspects of FC NMR Relaxometry
In a typical FFC multi-block experiment, a high magnetic field, called the polarization
field, B
0
P
is applied to pre-polarize the sample in order to increase signal intensity;
thereafter, the sample is allowed to relax in a second field, called the relaxation field,
B
0
E
which can be set to any value, including zero. The field switching time, Swt,
determines the shortest measurable T
1
and so a faster and more flexible electronic method
for switching the B
0
E
field is required. Therefore, a low-inductance, air-coil magnet is
used in the relaxometer (Figure 2.16) with power supplies capable of switching the field
electronically to any desired value in a matter of milliseconds while maintaining the high
field stability and homogeneity, at the same time, required by NMR. The magnetic flux
density relevant for relaxation is different from that during signal detection. While the
relaxation field may be varied over several orders of magnitude, the detection field, B
0
D

is kept fixed at the highest possible value by tuning the RF probe to this particular
detection field at which the signal, an FID generated by the application of a 90
0
pulse, is
detected.
[4, 5]




31













Figure 2.16: A block diagram of the FFC NMR Relaxometer. The Cooling System and Magnet Power
Supply and Switching Control Circuitry are the units specifically designed for field-cycling purposes.
5

After reference 4

The Fourier transform of this detected FID gives the magnetisation, M (t), within the
sample after relaxation for a period, t, in B
0
E
.
[13]
The magnetisation decays exponentially
as a function of t, starting from the equilibrium magnetisation at B
0
P
towards B
0
E
. For a
sample containing a single type of spin, the relaxation towards the equilibrium value is
monoexponential with a rate constant R
1
at B
0
E
described by the equation:
[15]

M (t) = M
E
+ [M
P
M
E
] exp [-t R
1
(B
0
E
)] (2.20)
where M
E
and M
P
are the amounts of magnetisation present in the sample during the
relaxation and polarisation periods, respectively. The relaxometer then reduces the data in
each t-block to a single value, S (t) proportional to M (t) in the curve for a number of B
0
E

values (Figure 2.17).
[15, 17]
Pre-amplifier
Temperature Control
RF Unit
Control
Acquisition
Magnet Power
Supply and
Control Circuits
for Switching
Cooling System
Workstation
Data System
Software
Solenoid
Magnet
Cooling
enclosure
Probe
32


Figure 2.17: A monoexponential plot from a relaxation experiment using the Stelar SMARtracer FFC
relaxometer

In practice, the FFC experiment can be carried out by a number of available pulse
sequences. Four of these were explored in this project: Non-polarised (NP), Pre-polarised
(PP), Non-polarised/Pre-polarised (NP/PP) hybrid and Inversion Recovery (IR) pulse
sequences.






Figure 2.18: The Non-Polarised (NP) Pulse Sequence. After reference 17
Pre-polarisation
Relaxation Detection
B
B
0
E

B
0
D

T
x

RD Swt
1

t
Swt
2
Swt
0

Acq
33

The NP sequence (Figure 2.18) is appropriate for T
1
relaxation measurements at
comparatively high relaxation fields (usually above a few MHz).
[17]
It involves the
preparation of a rather null initial longitudinal magnetisation by allowing the sample to
relax in a null field for a period, RD (recycle delay) which triggers off the sequence. After
a switching time, Swt
1
, the magnet is switched to B
0
E
, and is kept constant for the variable
period t during which the samples magnetisation grows towards the equilibrium
magnetisation. The magnet is then switched to B
0
D
followed by a 90
0
RF pulse (after a
switching time, Swt
2
) to generate an FID. The field is then switched off and the entire
sequence is repeated.






Figure 2.19: The Pre-Polarised (PP) Pulse Sequence. After reference 17
The PP sequence (Figure 2.19) is appropriate for T
1
relaxation measurements at low
fields, possibly, down to zero.
[17]
In this case, the initial magnetisation is prepared by a
high polarisation field, B
0
P
over a considerable long polarisation time, t
P
this results in a
high initial magnetisation value. After a switching time, Swt
1
the magnet is switched to
B
0
E
and is kept constant over a variable period t during which the magnetisation decays
B
0
D

T
x

Acq
B
0
P

B
0
E

Swt
0
t
P
Swt
1

t

Swt
2
Swt
0

B
Pre-polarisation Relaxation Detection
34

towards the equilibrium value. From this stage onwards, the processes involved are
exactly the same as in the NP sequence.
The NP/PP hybrid sequence is a combination of the NP and PP sequences in such a way
that a switching field value is specified (before acquisition) at which the relaxometer
switches from the NP (at high fields) to PP (at low fields) pulse sequences.







Figure 2.20: The Inversion Recovery (IR) Pulse Sequence. After reference 17
The IR sequence (Figure 2.20) involves pre-polarising the sample in the B
0
P
field, then
switching to B
0
D
where the first 180
0
RF pulse is applied to invert the pre-polarised
magnetisation. The magnet is then switched to B
0
E
where the sample is allowed to relax
for a variable period t. The field is then switched to a final B
0
D
field where a 90
0
RF
pulse is applied to generate the FID.
[17]

B
T
x

Acq
B
0
P

B
0
E

B
0
D

t
P

t

180
0
90
0

I
n
v
e
r
s
i
o
n

Pre-polarisation Relaxation Detection
35

Optimum performance of a typical FFC NMR relaxometer (especially in keeping the
initial magnetisation constant for all the t-blocks) is limited by the following crucial
technical factors irrespective of the pulse sequence in use.
[15]

Signal-to-noise ratio (S/N): According to Curies law, M
P
o B
0
P
, where M
P
is the
magnetisation reached in the polarisation field, B
0
P
. The induction signal increases
proportionally to B
0
D
, whereas noise is proportional to the square-root of the carrier
frequency (i.e. B
0
D
). Therefore:
S / N o (B
0
D
)
1/2
M (t) (2.21)
The magnetisation after the evolution period follows:
M (t) B
0
P
exp [-t / T
1
(B
0
E
)] (2.22)
So that S/N is limited by:
S / N o (B
0
D
)
1/2
B
0
P
exp [-t /T
1
(B
0
E
)] (2.22)
Therefore, field levels B
0
D
and B
0
P
must be as high as technically possible for maximum
S/N.
Thermal Stability: The B
0
P
field is applied as long as required for attaining thermal
equilibrium, typically, a period t
P
~ 5T
1
(B
0
P
). The B
0
D
field is applied within a very short
interval, t
D
required for obtaining an FID. Thus:
t
P
>> t
D
(2.23)
36

Since the polarisation period is long, and the magnet coils are mostly resistive, joule
heating, and thus thermal drifts of the field, becomes unavoidable during the field cycle.
It is therefore necessary to restrict the B
0
P
field to a level still compatible with thermal
stability while the B
0
D
field is kept as high as the magnet power supply permits.
Field Homogeneity: Homogeneity within the sample should correspond to the stability of
the B
0
D
field. B
0
D
must therefore be reproducible within an accuracy of 10
-5
, as the field
homogeneity in the sample does not necessarily need to be better than 10
-5
provided that
a suitable magnet design is used.
[15]
The commonest cause of inhomogeneous field in a
sample is the sample extending beyond the homogeneous bounds of the RF coil. This can
be avoided by confining the sample to the volume of the RF coil.
[13]
This is achieved in
practice by filling an NMR tube with a sample to a height not greater than the length of
the RF coil, and positioning the tube with the sample in the centre of the coil.








37

CHAPTER THREE
3: Methodology
In the previous chapters, the problem has been defined and background concepts in the
problem domain discussed in relation to other publications. This chapter is a first-step
presentation of how the main aim of the project will be accomplished.
3.1 The FFC NMR Relaxometer and Accessories
Measurements of the relaxation rates of the protein samples were done in the relaxometer
shown in Figure 3.1 the principle of operation has been discussed in section 2.6. It
comprises a multifunctional NMR console for carrying out NMR measurements.
[23]


Figure 3.1: Stelar SMARtracer FFC relaxometer and Accessories

38

The console consists of four parts: a computer, RF power transmitters/receiver,
temperature/flow-rate control unit and the main magnet (Figure 3.2).

Figure 3.2: The PC-NMR Console

The pulse sequence timing and parameter settings of all measurements (and hardware)
are controlled by a powerful NMR software package (Section 3.2) installed on a Pentium
M, 1.6 GHz, 512 MB ram, HD 60 GB single board computer with 4 USB and Ethernet
ports. A high-resolution LCD monitor is connected to the computer for a visual output of
all measurements.
RF pulses are delivered from three (3) independent RF transmitter channels, each being
programmable from DC to 90 MHz. However, only one of these is chosen as the default
39

transmitter channel; while the other two can be configured, if desired, as supplementary
channels or even as DC outputs for driving external gradients.
NMR signals from samples, after the RF excitation of their spins, are directly acquired by
a digital receiver. Signals detected are usually of low intensity and if not amplified, could
be heavily corrupted by noise. Hence, a preamplifier amplifies these detected signals by
the digital receiver before they are recorded and further displayed on the monitor.
The receiver (or probe) can be tuned, electronically, to signals within a range of 500 kHz
to 90 MHz with a maximum spectral width of 10 MHz. However, it has been tuned for
signal acquisition at a fixed spectral width of 7.2 MHz, ensuring a constant S/N in all
measurements. This fixed detection field (or receiver frequency) enables phase sensitive
detection by the probe, making signal accumulation possible in four separate buffers:
real and imaginary and phase and absolute.
Temperature of the sample whose relaxation rate is being measured is very important as
far as the accuracy of the measurement is concerned. Therefore, the console is further
equipped with a variable temperature control (VTC) and flow-rate control unit. These
two controls respectively set fixed temperature and flow-rate of air (which ideally should
be nitrogen gas) driven through an air flux heater to a temperature sensor just below the
placement depth of the sample tube (in the magnet bore). This conditioned air is drawn-in
(through an inlet valve) from the surroundings, sieved and pumped out (through a tube
connecting its outlet valve to the air flux heater) by an electrically operated air pump.
Finally, the main magnet whose B
0
field is being cycled is an air-cored resistive solenoid
through which an electrically controlled AC is passed to generate the required fields in a
40

typical field-cycling experiment. Joules heat in the magnet is transferred through a high
specific heat capacity liquid (called galden) in heat exchangers to continuously running
tap water. The galden is circulated in the heat exchangers by an electric pump. This
ensures that the magnet is operated near room temperature; in order to avoid considerable
drifts in the field homogeneity. Thermistors are connected to the pipes conveying the
water between the tap and the magnet heat exchangers to monitor the amount of heat
produced from the magnet during operation.
3.2 Software
The NMR software package installed on the computer of the relaxometer is called
AcqNMR (Figure 3.3). It comprises a huge library of pulse sequences and parameters
for most NMR and NQR experiments. It has an option to be Run online, which is best
to select at start up before it is initialised. This option allows saving the results (for
reasons of backups) to a storage space on the network server of Aberdeen University.
There is a multi-page control tab where the acquisition parameters are entered. Most
editable entries in this window essential for the measurements are in the Main
Parameter sheet. These editable items include: sample type, experiment (i.e. pulse
sequence type), data acquisition file, maximum T
1
, polarisation time, polarisation field
and relaxation filed. However, since the relaxation field is usually a range, and each pulse
sequence has a unique, specified evaluation format, another window, the NMRD Profile
Wizard, under the Actions command (on the Windows main menu bar) is used for
these specifications; other parameters are also specified here. The profile Wizard
enables the required acquisition parameters in the program window to be synchronised
to the NMRD profile parameters before the sequence is run.
41


Figure 3.3: The main program window of the AcqNMR software

Within the profile window, the user is also expected to provide a user-specified file name
and location to which the acquisition data will be saved. Files are saved in *.sef (stelar
export file) and *.sdf (stelar data file) formats, usually accessible in Notepad but can
be exported to MS Excel for further editing. The .sdf document contains all the
acquisition parameters used by the system for a particular experiment while the .sef
document contains the acquired data (arranged in columns) from the experiment.
Once all parameters are set and the Execute button on the profile wizard is hit, the
software triggers all other hardware of the relaxometer to start work; except the galden
and air pumps which run continuously, independently of the software. However, the
temperature of the air pumped into the system and the number of scans at any given time
42

are displayed by the software on the immediate mode bar, just above the multi-page
control tabs.
For complete information on the relaxometer and software, the reader is referred to
references 23 and 24.
3.3 The Field-Cycling MRI System
The phantom was imaged in a home-built, whole-body sized MR scanner (Figure 3.4)
which operates on field compensation method of field-cycling: B
0
D
field generated by the
subtraction of variable field strengths from a fixed field by the use of two coaxial
magnets.
[25]

Figure 3.4: The Field-Cycling MRI System

The outer, primary magnet (constructed from ferrite) generates a fixed, vertically oriented
B
0
D
field of 59 mT. This is a whole-body sized permanent magnet with an approximate
43

mass of 2600 kg and a clear bore of 65 cm. Gradient coils are constructed into this
magnet.
The inner, secondary magnet (made from sheets of copper conductor), when energised,
generates an antiparallel variable magnetic field to the primary magnets field; thus,
reducing the net field at the sample. It is a resistive saddle-shaped coil with an outer
diameter of 64 cm and is well-fitted into the primary magnet, creating a free bore
diameter of 52 cm.
The composite support structure of the magnet and the ferrite are non-conducting and so
there is no requirement for eddy current compensation during field-cycling. The
laboratory is equipped with an air-conditioning unit to maintain an ambient temperature
of 22 0.5
0
C as the ferrite magnet has a relatively large temperature coefficient
(-0.2%
0
C
-1
). The temperature within the ferrite magnet structure itself is stabilised by a
continuously operating fan that circulates air around it. The inner coil is also water cooled
on its outer surface, and a network of platinum resistance thermometers is coupled to it
for monitoring its temperature during operation.
The transmit/receive coil used was a split-solenoid RF coil assembly made from a 6 mm
diameter copper pipe, length 12 cm and diameter 32 cm, tuned to 2.5 MHz. A series of
trimmer capacitors are attached to the coil to allow for resonance matching to be done
with the sample in place. This coil assembly is housed within a cylindrical RF shield.
All operations of the scanner are controlled by a commercial MRI console through
software on an IBM-compatible PC. Gradient and field-cycling waveforms are generated
from the console through four programmable 12-bit digital-to-analogue converters.
44

Programmable TTL logic waveforms are also generated to enable/disable external
hardware. The console is also capable of RF pulse synthesis (for NMR excitation), pre-
amplification of the NMR signals generated and phase-sensitive detection of the signals.
The console is also capable of image transformation and display.
3.4 Sample Preparation and Handling
The protein aggregate used for the entire project was bovine serum albumen (BSA). It
was prepared with either one or two of the following agents: agarose, phosphate buffer
saline (PBS) and copper sulphate (CuSO
4
). While the BSA and agarose were in dried
forms, the PBS was diluted according to suppliers assay 1:3 parts of deionised water.
Separate CuSO
4
solutions of the required molarities were prepared by dissolving a
weighed mass of CuSO
4
crystals in 1 litre of deionised water.

Figure 3.5: From left to right: a volumetric flask of CuSO
4
solution, BSA (in white container), PBS, the
composite phantom and in front, NMR tube containing BSA gel
45

Now that the agents used in preparing the samples for the relaxometry measurements
have been presented, the sample preparation procedure shall now be discussed.

Three different types of BSA gels were prepared: BSA in PBS, BSA and agarose in
PBS and BSA in CuSO
4
solution. The use of PBS was because it easily dissolves BSA
and also provides similar conditions to proteins in human tissues. All samples were
prepared in glassware thoroughly washed with a powerful decontaminant, rinsed with
deionised water and air-dried.

Preparation of BSA in PBS
The required percentage by weight of BSA was weighed (in grams) on an electronic
balance and carefully transferred into a small beaker of 5 cm
3
of dilute PBS. This BSA-
PBS co-mixture was gently warmed in a water bath at a temperature of 40
0
C while
stirring with a spatula. The temperature was slowly raised to 80
0
C, while stirring
continued. This continued for approximately 10 minutes to achieve complete dissolution
of the BSA in the PBS. 1 ml of the resulting solution was syringed into a NMR tube,
which was further heated (with a smaller beaker covering its open end to prevent
significant vapour loss from the contents) in the water bath. The temperature was raised
to 95
0
C and heating continued for 30 minutes. During this period, the BSA-PBS co-
mixture had denatured to gel.

Preparation of BSA and Agarose in PBS
The required percentages by weight of BSA and agarose were weighed (in grams) on an
electronic balance, separately, and transferred gently into the same beaker of 5 cm
3
of
PBS. The BSA-agarose-PBS mixture was then gently warmed, while stirring with a
46

spatula, in a water bath at 40
0
C. The temperature was slowly increased to 95
0
C while
stirring continued. A total heating time of 10 minutes, approximately, was allowed for a
complete mixture to be attained. 1 ml of the resulting solution was syringed into a NMR
tube which was heated (with the open end covered with a smaller beaker) at 95
0
C
continuously for 35 minutes, during which period the contents have transformed into gel.

Preparation of BSA in CuSO
4
Solution
The required percentages by weight of BSA were added to 5 cm
3
of CuSO
4
solution in a
small beaker and gently warmed, while stirring with a spatula, in a water bath at 40
0
C.
The temperature was slowly raised to 80
0
C. An approximate period of 10 minutes was
allowed to attain a uniform mixture. 1 ml of the resulting solution was syringed into a
NMR tube which was then further heated (with a smaller beaker covering the open end)
in the water bath at 90
0
C for 30 minutes. During this period, the contents of the tube had
changed to a gel.

Sample Handling

In each of the above cases, after the gel had been formed, the sample tube was removed
from the water bath, wiped dry with a tissue paper, covered with a tight-fitting cork at the
open end and labelled by contents name and preparation date. This labelled tube was
then refrigerated overnight.
Before an experiment with a sample (from the fridge), it was warmed for a period of 10
minutes at room temperature (25
0
C) before placement in the relaxometer; then left for
another waiting period of 10 minutes to ensure complete equilibration of its temperature
just before data acquisition.
47

The sample volume of 1 ml was crucial so that the sample did not extend beyond the
homogeneous bounds of the RF coil in order for it to experience a homogeneous
magnetic field. The necessity for a homogeneous filed also required that the sample
remained fixed, without movement of any sort, within the relaxometer (Figure 3.6)
throughout the time scale of the experiment.

Figure 3.6: Sample positioning during a relaxation measurement in the relaxometer. The clamp and load
provide a firm grip and stable position of the sample tube against expulsion by the air pressure around it.

3.5 Pulse Sequence Selection and Implementation
Ideally, the same T
1
relaxation rate measures should be obtained regardless of the pulse
sequence used. However, it was necessary to find the most appropriate pulse sequence
that could show least errors in the relaxation measurements, consistency and robustness
to RF pulse fluctuations (due to temperature drifts for example). For this reason, the four
pulse sequences discussed in section 2.6 were each assessed on these bases to arrive at
the best choice of pulse sequence.
48

Experiments were performed with an initially prepared BSA sample of 20% by weight.
The same parameters were set for each measurement except for the pulse sequence type
which was varied; the RF pulse duration was deliberately varied (by calculated
percentage offsets) when experiments to test pulse sequence robustness to RF pulse
fluctuations were to be carried out. Each pulse sequence was applied in four different
experiments; their order of selection for application was done by randomised trials. The
parameters that were kept constant for each pulse sequence were:
Sample temperature (
0
C): 25.0 0.2
T
1
Maximum value (s): 0.27
Repetition delay (s): 0.6
Polarisation field (MHz): 7.9996
Polarisation time (s): 0.6
Detection field (MHz): 7.1999
Relaxation time (s): 0.0027
Number of t-blocks: 8
Number of averages: 3

The relaxation field values were chosen over two ranges: 0.5 to 8.0 MHz and 1.5 to 3.7
MHz. All other parameters on the software of the relaxometer are either default or
hardware-specific control/configuration parameters and so were not changed. It was also
possible to type-in the names of the experimenter and sample.
The following were the results obtained with measurements on the 20 % BSA sample
with the four pulse sequences. Each pulse sequence experiment was repeated four times,
49

with three repetitions per experiment. Therefore, each point plotted in each graph is an
average of twelve measurements. This was done to ensure a high signal-to-noise ratio.
Comparison of the Pulse Sequences in the 1.5-3.7 MHz Range
The average standard error in the relaxation rate measurements was evaluated over the
entire Larmor frequency range (in the dispersion profiles shown in Figure 3.7) of all the
sequences. In order of good accuracy, the IR sequence recorded a standard error of
0.02 s
-1
, the NP sequence recorded 0.03 s
-1
, the PP sequence recorded 0.05 s
-1
and
the NP/PP hybrid sequence recorded 0.07 s
-1
.

Figure 3.7: R
1
Dispersion profiles of the four pulse sequences compared in the 1.5 to 3.7 MHz Larmor
frequency range

These results showed that the IR sequence was most reliable in the range of Larmor
frequencies where the quadrupole peaks were expected whiles the NP/PP hybrid
sequence was the worst.
2.5
3
3.5
4
4.5
5
1.5 2 2.5 3 3.5 4
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
IR/S
NP/S
NP/PP
PP/S



50

Comparison of the Pulse Sequences in the 0.5-8.0 MHz Range
The average standard errors were again evaluated (in the dispersion profiles shown in
Figure 3.8) for the sequences over a wider range of Larmor frequencies, to include
background spin-lattice relaxation processes. In these measurements, the NP/PP hybrid
sequence recorded a standard error of 0.26 s
-1
, the PP sequence recorded 0.28 s
-1
, the
IR sequence recorded 0.44 s
-1
and the NP sequence recorded 1.17 s
-1
.

Figure 3.8: R
1
Dispersion profiles of the four pulse sequences compared in the 0.5 to 8.0 MHz Larmor
frequency range

The error values increased among the sequences due to the wider Larmor frequency
range used but in comparison, the NP/PP sequence was the best in this case because the
signal-to-noise ratio in the measurements were actually averaged from a NP sequence
between 8.0 MHz and a sequence switch over frequency of 2.5 MHz; then PP sequence
from 2.5 MHz down to 0.5 MHz, thus showing an overall improved S/N ratio.
However, by separate application of the two sequences, the PP sequence was better than
the NP sequence in this range though both showed higher errors than when combined.
0
1
2
3
4
5
6
7
8
0.5 5
R
1

(
s
-
1
)
Proton Larmor Frequency (MHz)
IR
NP
NP/PP
PP
51

It is the T
1
relaxation of an inverted magnetisation that is recorded in the IR experiment
and so increasing the range of Larmor frequencies for the measurement (and for that
matter, a longer time of measurement) would mean an availability of a smaller transverse
magnetisation towards the lower frequencies. Therefore, the signal recorded was much
corrupted by noise towards the lower frequencies and the relaxometer even had to abort
the experiment at a Larmor frequency of 0.89 MHz.
When the error difference between the two ranges of Larmor frequency was evaluated to
assess the effect on the observed errors in switching from one range to a relatively larger
one, the following results were obtained: the NP/PP sequence recorded a standard error
change of 0.19 s
-1
, the PP sequence recorded 0.23 s
-1
, the IR sequence recorded
0.42 s
-1
and the NP sequence recorded 1.14 s
-1
.
Based on these error analyses and the understanding of the operations of these sequences,
a decision was arrived at to choose between the IR and PP pulse sequences. This was
because the PP sequence as a stand-alone sequence showed a very close accuracy to the
combined NP/PP sequence which rather was more likely to show larger errors around the
sequence switching over frequency (Appendices C and G) and also put stress on the
switching circuitry. The IR sequence, on the other hand, would be ideal if frequencies
below 1 MHz were not to be explored. Therefore, a further test was carried out to confirm
between which of these two sequences would be robust in situations of RF power offsets.



52

Comparison of the Pulse Sequences in Respect of Tolerance to RF Pulse Offsets
The 90
0
RF pulse height is fixed in the relaxometer and so the duration (4.5 s) was
deliberately mal-tuned to 10%, 20% and 40% in order to compare the resulting
dispersion profile with that obtained from using the standard 90
0
RF pulse duration.
Dispersion profiles over a range of 0.5 to 3.7 MHz of the IR and PP pulse sequences were
then compared.
With a fixed standard error of 1 s
-1
(error bars fitted by MS Excel), the profile for the
IR sequence (Figure 3.9) showed consistent correspondence in the mal-tuned data set
with the standard data set. However, with the 40% RF offset, significant deviations
were recorded over a Larmor frequency range of 0.89 to 2.09 MHz. The operation was
aborted below this range due to T
1
Max overflow error; i.e. a considerable decay away
of the magnetisation to be recorded. Apart from this deviation over this range, the IR was
insensitive to RF offsets.

Figure 3.9: R
1
dispersion profiles with the RF pulse offset compared to the profile with the 90
0
RF pulse in
the inversion recovery sequence experiment.
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
0.5 1 1.5 2 2.5 3 3.5 4
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
-40%
-20%
-10%
90 deg
10%
20%
40%
53

With the same standard error of 1 s
-1
, the PP sequence (Figure 3.10) showed significant
deviations in response to RF offsets of -20% and -40% over a Larmor frequency range of
1.18 to 3.70 MHz, and also slight deviations in response to + 20% offset over a range of
2.0 to 3.70 MHz.

Figure 3.10: R
1
dispersion profiles with the RF pulse offset compared to the profile with the 90
0
RF pulse
in the pre-polarised sequence experiment.

Therefore, the IR sequence showed enough evidence to be reliable and so was the pulse
sequence of choice in the subsequent experiments to measure the relaxation rates.

3.6 Extraction of AR
1
from Background Relaxation Processes
It has been discussed in section 2.5 that the background T
1
relaxation processes in
proteins can be described by a power law:
[5, 21]

R
p
= k u
L
-b
(3.1)
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
0.5 1 1.5 2 2.5 3 3.5 4
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
-40%
-20%
-10%
90 deg
10%
20%
40%
54

Relaxation rates over a range of 0.5 to 6.5 MHz were taken and the resulting dispersion
profiles were curve-fitted to generate an exponential equation of the form of equation 3.1
using MS Excel (Figure 3.11). Relaxivities at 0.5 MHz, 2.1 MHz and 2.8 MHz were not
included in these plots. The reasons were to avoid the significant error to be imposed on
the measurement at 0.5 MHz by the sequence in use and to also exclude the quadrupole
peaks (which occur at 2.1 MHz and 2.8 MHz) from the background plots.

Figure 3.11: Illustration with a background relaxation curve of a 20% BSA sample. The R
2
value
expresses the goodness of the fit


Relaxation rates were measured again over a narrower range (1.5 to 3.5 MHz), with
particular consideration for inclusion of the quadrupole peaks (Figure 3.11).
R
p
= 7.5541u
L
-0.383
R = 0.9977
2
3
4
5
6
7
8
9
10
0 1 2 3 4 5
R
p
(
s
-
1
)
Proton Larmor Frequency (MHz)
55


Figure 3.12: R
1
dispersion profile of the 20% BSA sample showing the quadrupole peaks at 2.1 MHz
and 2.8 MHz Larmor frequencies, resulting from the molecular interaction of water protons with
protein protons of the immobilised peptide (
1
H-
14
N) bonds in the protein molecule; R
1
= R
p
/
+ R
q

The Larmor frequencies (u
L
) of Figure 3.12 were substituted into the equation of the fit
(of Figure 3.11) to generate another set of relaxation rates, R
p
/
these were the
relaxations due to the background processes that added to the quadrupole processes (R
q
)
to constitute the R
1
dispersion of Figure 3.12. Subtraction of R
p
/
from R
1
(i.e. the
relaxation rates for the quadrupole peak data set) gave the values of AR
1
(or R
q
), which
were then plotted versus the u
L
values of the quadrupole peak data set (Figure 3.13).
4
4.5
5
5.5
6
6.5
7
7.5
1.5 2 2.5 3 3.5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
56


Figure 3.13: The quadrupole peaks generated by plotting the relaxation difference (AR
1
) versus the
Larmor frequencies of the quadrupole peak data set. The AR
1
axis has been normalised to 10
-6
to cancel
out the mega (10
6
) unit of the Larmor frequencies so that the areas are in units of per squared second
(s
-2
)

The sizes of the peaks were then obtained by stripping them into smaller trapezia (on
the frequency axis), calculating the area of each trapezium and summing the areas up. In
order to avoid the introduction of significant noise into the computation, the lower tails of
the peaks were ignored; thus, u
L
ranges of 1.8 to 2.5 MHz and 2.5 to 3.0 MHz were
chosen for the peaks at 2.1 MHz and 2.8 MHz respectively.
Therefore, for this 20% sample, the areas under the peaks at 2.1 MHz and 2.8 MHz were
evaluated (Appendix L) to be 0.55 0.03 s
-2
and 0.66 0.05 s
-2
respectively, giving a
total area of 1.21 0.04 s
-2
.



0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
1.5 2 2.5 3 3.5
A
R
1
(
s
-
1
)

/

1
0
-
6
Proton Larmor Frequency (MHz)
a
h
b
An
A
n
= 0.5h (a + b)
A
QP
= A
n

A
QP

57

3.7 Imaging Relaxometry Measurements
The IR sequence was also used with gradients at the detection field to image the
phantom. In this case, an initial 10 ms adiabatic fast passage was used to provide a
uniform inverted magnetisation. The field was then switched to an evolution value of
59 mT for 30 ms and then returned to a detection field of 59 mT where the signal was
read out, after a 70 ms delay, by a 90
0
pulse. K-space data was collected at five evolution
fields: 59 mT, 62 mT, 65 mT, 68 mT and 71 mT. The data were used to generate T
1

colour images in a Matlab program. Dispersion plots were obtained from regions of
interest to compute AR
1
at 65 mT by interpolation of the R
1
values at 59 mT and 71 mT.











58

CHAPTER FOUR
4: Experimental Results and Discussions
This chapter presents the results of the relaxometry measurements in different protein
concentrations and also image analysis of a phantom based on these measurements. The
same constant parameters outlined in section 3.4 were used in the experiments (except
where specified) and the AR
1
values reported therein were evaluated as described in
section 3.5. Ten and thirty points were recorded for the background and quadrupole
curves respectively.
4.1 Correlation between AR
1
and Concentration of BSA
This section presents results of the measured background effects (Figure 4.1) and
quadrupole effects (Figure 4.2) for five different BSA concentrations. The reason was to
determine the relationship between the peak size and the gel concentrations (Table 4.1).

Figure 4.1: Background relaxation rate curves of five BSA concentrations
0
2
4
6
8
10
12
14
0.5 1.5 2.5 3.5 4.5
R
p

(
s
-
1
)
Proton Larmor Frequency (MHz)
5% BSA
10% BSA
15% BSA
20% BSA
25% BSA
59


Figure 4.2: R
1
dispersion profiles of the five BSA concentrations of Figure 4.1


Figure 4.3: Quadrupole peaks of the five BSA concentrations



0
1
2
3
4
5
6
7
8
9
10
1.5 2 2.5 3 3.5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
5% BSA
10% BSA
15% BSA
20% BSA
25% BSA
0
0.5
1
1.5
2
2.5
3
1.5 2 2.5 3 3.5
A
R
1
(
s
-
1
)
/
1
0
-
6
Proton Larmor Frequency (MHz)
5% BSA
10% BSA
15% BSA
20% BSA
25% BSA
60

The areas under the peaks are summarised in Table 4.1.
BSA Concentration
(% by weight)
5 10 15 20 25
Total area under
quadrupole peaks (s
-2
)
0.22 0.01 0.55 0.02 0.84 0.04 1.21 0.04 1.77 0.07
Table 4.1: Correlation between the area under the peaks and protein concentration of the BSA samples

From these results, it was obvious that the area under the quadrupole peaks is directly
proportional to the concentration of polypeptides in the BSA samples. The linear
correlation is shown in Figure 4.4.

Figure 4.4: Linear correlation between the total areas under both peaks and the protein concentrations of
their respective BSA samples. The same correlation is true between each separate peak and the
concentration of the sample (Appendices I and J). R
2
is the goodness of the fit

The gradient of Figure 4.4 was evaluated to be (0.08 0.003) s
-2
(%)
-1
, representing the
sensitivity of the relaxometer to changes in BSA concentration. In other words, the
relaxometer measures relaxation rates to an order of accuracy of (0.08 0.003) s
-2
for
every 1% by weight BSA.

R = 0.9824
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
5 10 15 20 25 30
A
r
e
a

u
n
d
e
r

P
e
a
k
s

(
s
-
2
)
Concentration of BSA (% by Weight)
61

4.2 Effect of Agarose on the Relaxation Processes in BSA

At relatively low concentrations, particularly below 5% by weight, it becomes very
difficult, if not impossible, to de-nature BSA to gel. Therefore, the denaturing process
was enhanced by preparing some low BSA concentrations in a matrix of agarose to help
immobilise the protein molecules. BSA concentrations of 1%, 2% and 5% were prepared,
and separate samples of these concentrations were again prepared with 0.5% agarose in
each. The temperature on the VTC was set to 30
0
C in these and the next block of
experiments because the ambient temperature varied around 28 0.2
0
C during that
period. So it was necessary to set the VTC to a point that could remain stable throughout
the experiments.
Background relaxation curves (Figure 4.5) were subtracted from R
1
dispersion profiles
(Figure 4.6) to generate the quadrupole peaks (Figure 4.7) for these six samples.

Figure 4.5: Background relaxation curves of the three BSA concentrations and their corresponding co-
mixtures with 0.5% agarose (AGR)
0
0.5
1
1.5
2
2.5
0 1 2 3 4 5
R
p
(
s
-
1
)
Proton Larmor Frequency (MHz)
1% BSA+ AGR
1 % BSA
2 % BSA + AGR
2 % BSA
5 % BSA + AGR
5 % BSA
62


Figure 4.6: R
1
dispersion profiles of the three BSA concentrations and their corresponding co-mixtures with
0.5% agarose (AGR)


Figure 4.7: Quadrupole peaks of the three BSA concentrations and their corresponding co-mixtures with
0.5% agarose (AGR)

0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
1.5 2 2.5 3 3.5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
1 % BSA + AGR
1 % BSA
2 % BSA + AGR
2 % BSA
5 % BSA + AGR
5 % BSA
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
1.5 2 2.5 3 3.5
A
R
1
(
s
-
1
)
/
1
0
-
6
Proton Larmor Frequency (MHz)
1 % BSA + AGR
1 % BSA
2 % BSA + AGR
2 % BSA
5 % BSA + AGR
5 % BSA
63

The peaks at 2.1 MHz were very noisy due to the relatively low concentrations measured.
Therefore, areas of the peaks at 2.8 MHz were evaluated for each concentration (Table
4.2). Figure 4.8 shows the results in Table 4.2 graphically.
BSA Concentration (%) 1 2 5
A
BSA
(s
-2
) 0.060 0.004 0.082 0.004 0.131 0.004
A
BSA + 0.5% Agarose
(s
-2
) 0.049 0.004 0.040 0.010 0.158 0.009
Table 4.2: Effect of agarose on the size of the quadrupole peak at 2.8 MHz of BSA; A
BSA
and A
BSA+0.5%
agarose
are the peak sizes of BSA and BSA with 0.5% agarose respectively.


Figure 4.8: Effect of agarose (AGR) on the size of the quadrupole peak at 2.8 MHz of BSA

The results show that for concentrations lower than 5%, the 0.5% agarose rather causes a
greater winding up of the peptide strands in the protein molecules; so preventing an
interaction of water protons with those of the proteins, which are now sealed up in the
agarose matrix. This effect is even greater in the 2% sample because it is easier for that
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
1.0 2.0 3.0 4.0 5.0
A
r
e
a

U
n
d
e
r

p
e
a
k

(
s
-
2
)
Concentration of BSA (% by weight)
BSA + 0.5 % AGR
BSA
64

sample to turn to gel (though it is a low concentration) compared to the 1% and so under
such condition in agarose, the tightening up of the strands would even be greater. The 5%
sample rather showed an increased relaxation rate because the amount of agarose is
comparatively small and so in this case, agarose would only make the denaturing process
(to immobilise the protein molecule) faster and enhance the relaxation processes.
4.3 Correlation between AR
1
and Concentration of Agarose in BSA
The effect of agarose on the peak size was further probed by varying the amount of
agarose in 1% BSA, the sample most difficult to de-nature. Figures 4.9 to 4.11 show the
usual process of extracting the quadrupole peaks.

Figure 4.9: Background relaxation curves of different concentrations of agarose (AGR) in 1% BSA
compared to the background relaxation curve of 1% BSA without agarose

0
0.5
1
1.5
2
2.5
0 1 2 3 4 5
R
p
(
s
-
1
)
Proton Larmor Frequency (MHz)
1% BSA + 0.5%AGR
1% BSA + 1% AGR
1% BSA + 2% AGR
1% BSA
65


Figure 4.10: R
1
dispersion profiles of different concentrations of agarose (AGR) in 1% BSA compared to
the R
1
dispersion profile of 1% BSA without agarose


Figure 4.11: Quadrupole peaks of different concentrations of agarose (AGR) in 1% BSA compared to the
quadrupole peak of 1% BSA without agarose

0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.5 2 2.5 3 3.5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
1% BSA + 0.5% AGR
1% BSA + 1% AGR
1% BSA + 2% AGR
1% BSA
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
1.5 2 2.5 3 3.5
A
R
1
(
s
-
1
)
/
1
0
-
6
Proton Larmor Frequency (MHz)
1% BSA + 0.5% AGR
1% BSA + 1% AGR
1% BSA + 2% AGR
1% BSA
66

The peaks show more scatter of points at their tails, especially those at 2.1 MHz due to
the low concentrations that were probed. Hence, the areas of the peaks at 2.8 MHz were
evaluated. The results are summarised in Table 4.3 and graphed in Figure 4.12.
Concentration of
agarose (% by weight)
0 0.5 1 2
Area under curve (s
-2
) 0.042 0.004 0.033 0.004 0.019

0.006 0.016

0.005
Table 4.3: Effect of varying concentrations of agarose on the size of the peak at 2.8 MHz of 1% BSA


Figure 4.12: Effect of varying concentrations of agarose on the size of the peak at 2.8 MHz of 1% BSA.
R
2
is the goodness of the fit

The results indicate that increasing amounts of agarose reduce the relaxivity of a fixed
concentration of BSA and could, at a certain amount of agarose, null out the relaxation
phenomena observed in BSA. This is possible because a relatively small concentration of
BSA in a dense matrix of agarose is completely wound up tightly such that its protein
protons almost no longer exhibit the quadrupolar resonance effect; hence the very low
relaxation rates measured.
R = 0.8458
0.01
0.02
0.03
0.04
0.05
0 0.5 1 1.5 2
A
r
e
a

u
n
d
e
r

p
e
a
k

(
s
-
2
)
Concentration of agarose (% by weight)
1% BSA without agarose
67

4.4 Effect of CuSO
4
on the Relaxation Processes in BSA

It is only expected that a contrast agent should enhance background relaxation processes
without affecting the size of the quadrupole peaks. Thus, the presence of a contrast agent
(in this case, CuSO
4
) should not affect the quadrupole processes in a protein gel. 10%
BSA was prepared with varying concentrations of CuSO
4
to estimate a concentration of
CuSO
4
that would bring the background relaxation rate of the 10% to that of a 25% BSA
at a Larmor frequency of 2.5 MHz (the detection frequency of the MRI system) without
changing the peak size.

Figures 4.13 to 4.15 show the process of extracting the quadrupole peaks.

Figure 4.13: Background relaxation curves of varying concentrations of CuSO
4
in 10% BSA compared to
the background relaxation curve of 25% BSA without CuSO
4

0
2
4
6
8
10
12
14
0 1 2 3 4 5
R
p
(
s
-
1
)
Proton Larmor Frequency (MHz)
0.25 mM CuSO4
0.5 mM CuSO4
1 mM CuSO4
2 mM CuSO4
4 mM CuSO4
10% BSA
25% BSA
CuSO
4

CuSO
4

CuSO
4

CuSO
4

CuSO
4

68


Figure 4.14: R
1
dispersion profiles of the varying concentrations of CuSO
4
in 10% BSA compared to the R
1

dispersion profile of 25% BSA without CuSO
4



Figure 4.15: Quadrupole peaks of the varying concentrations of CuSO
4
in 10% BSA compared to the peak
of 25% BSA without CuSO
4
. AR
1
of 4mM CuSO
4
in BSA is closer to that of 25% BSA at 2.5 MHz
0
1
2
3
4
5
6
7
8
9
10
1.5 2 2.5 3 3.5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
10% BSA
10%BSA + 0.25mM
10%BSA + 0.5mM
10% BSA + 1mM
10%BSA + 2mM
10% BSA + 4mM
25% BSA
0
0.5
1
1.5
2
2.5
3
1.5 2 2.5 3 3.5
A
R
1
(
s
-
1
)
/
1
0
-
6
Proton Larmor Frequency (MHz)
10% BSA
10% BSA+0.25mM
10%BSA+0.5mM
10%BSA+1mM
10%BSA+2mM
10%BSA+4mM
25% BSA
69

Figure 4.16 compares the peak sizes of the 10% BSA samples with CuSO
4
(blue diamond
markers) and the 10% BSA sample without CuSO
4
(horizontal dashed line).

Figure 4.16: Peak sizes of 10% BSA samples with CuSO
4
compared to the peak size of 10% BSA without
CuSO
4


The results confirm that CuSO
4
really enhances the background relaxation rate of BSA
without changing the size of the peaks.
4.5 Analysis of a Composite BSA-CuSO
4
Phantom

So far, the relaxometry results have shown that the quadrupole peak size is proportional
to the protein concentration of a given sample and that the size does not change in the
presence of contrast agents. This idea was extended to imaging a BSA-CuSO
4
composite
phantom (Figure 4.17). The image intensity was expected to be proportional to the peak
sizes of the protein gels.
[10]

0
0.5
1
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
A
r
e
a

u
n
d
e
r

p
e
a
k
s

(
s
-
2
)
Concentration of CuSO
4
(mM)
70


Figure 4.17: The composite phantom, made up of seven NMR tubes of different sample concentrations

The concentrations of CuSO
4
in the tubes were calculated by adopting the model:
[26]

1/T
1
= 1/T
1
0
+ R[CuSO
4
] (4.1)
where T
1
is the spin-lattice relaxation of BSA in the presence of CuSO
4
(equal to the T
1

of 25% BSA at 2.5 MHz), T
1
0
is the T
1
relaxation time of water or BSA (at 2.5 MHz) in
the absence of CuSO
4
, R is the relaxivity of BSA in 1 mM CuSO
4
and [CuSO
4
] is the
concentration of CuSO
4
.
R
1
dispersion profiles (Figure 4.20) were plotted for regions of interest selected from the
images (Figure 4.19) in a T
1
colour scale range of 0 to 1400 ms for the five evolution
fields (Section 3.7). Figure 4.18 shows the key to the images in Figure 4.19.



Figure 4.18: Key to figure 4.19 1: 5% BSA; 2: 15% BSA; 3: 5% BSA + 18.7 mM CuSO
4
;
4: 4.4 mM CuSO
4
; 5: 15% BSA + 3.7 mM CuSO
4
; 6: 10% BSA; 7: 10% BSA + 4.8 mM CuSO
4
1 1
2
3
4
5
6
7
71






Figure 4.19: Colour T
1
field-cycled 64 x 64 MR images of the BSA-CuSO
4
composite phantom
B
o
= 59 mT
(2.5 MHz)


B
o
= 62 mT
(2.6 MHz)

B
o
= 65 mT
(2.8 MHz)

B
o
= 68 mT
(2.9 MHz)

B
o
= 71 mT
(3.0 MHz)

T
1
: 0 630 ms
T
1
: 0 1400 ms
72

Tubes 3, 4, 5 and 7 were each expected to show the same colour while 1, 2 and
6 show an increasing order of intensity. These comparative intensities were expected to
be the same at all fields.
In the 0 to 630 ms colour scale range, the intensities of tubes 1, 2 and 6 agree with
their concentrations at all fields. For instance, at 59 mT, 2 appeared brightest (with T
1
~

225 ms), followed by 6 (with T
1
~ 330 ms) then 1 (with T
1
~ 630 ms). Tubes 4 and
5 have approximate T
1
values of 120 ms and 180 ms respectively while tubes 3 and
7appear rather too noisy.
The 0 to 1400 ms range is rather too long a period (even compared to the T
1
of tube 1
which is the longest among the samples) and so the observable magnetisation in the
transverse plane would have significantly decayed in each sample. In this case, the
sample with the longest T
1
would have the greatest observable spins and would,
therefore, appear brightest; this is the cause of the reversal in the order of intensity of
tubes 1, 2 and 6 at all fields. For instance, at 59 mT, Tube 1 appears brightest (with
T
1
~ 650 ms), followed by 6 (with T
1
~ 380 ms) then 2 (with T
1
~

220 ms). Tubes 4
and 5 had approximate T
1
values of 120 ms and 190 ms respectively while 3 and 7
again appear very noisy.
These results also confirm the unexpected R
1
dispersion profiles obtained for tubes 3
and 7 (Figure 4.20).
73


Figure 4.20: R
1
dispersion profiles of the samples in the composite phantom for T
1
range of 0 to 1400 ms

Each profile in Figure 4.20 was expected to show a peak at 65 mT so that by interpolation
of the measured R
1
values at 59 mT and 71 mT, a background relaxation value (R
p
) could
be obtained. Thus, by subtraction of R
p
from the peak R
1
value at 65 mT in each profile,
AR
1
values would be generated, that must show a linear correlation with the intensity and
concentration of the contents in the corresponding tubes. However, the peak heights were
rather too low for this to be done.
4.6 Comparison of Data from Relaxometry and Imaging
Sample Measured Relaxivity at 2.5 MHz or 59 mT (s
-1
)
FC NMR Relaxometer FC MRI System
5% BSA 1.55 0.03 1.51
10% BSA 2.32 0.02 3.17
15% BSA 4.45 0.01 4.26
5% BSA + 18.7 mM CuSO
4
2.31 1.27 4.69
10% BSA + 4.8 mM CuSO
4
2.90 0.03 4.63
15% BSA + 3.7 mM CuSO
4
6.87 0.06 5.88
4.3 mM CuSO
4
7.04 0.09 8.62
Table 4.4: Comparison of the relaxometry and MR imaging results; from relaxometry at 2.5 MHz,
R
1
(25% BSA) = [8.3 0.18] s
-1
; R
1
(10% BSA + 4 mM CuSO
4
) = [2.17 0.10] s
-1

0
1
2
3
4
5
6
7
8
9
10
59 62 65 68 71
R
1
(
s
-
1
)
B
o
E
(mT)
5% BSA
10% BSA
15% BSA
5% BSA + [CuSO4]
10% BSA + [CuSO4]
15% BSA + [CuSO4]
4.3 mM CuSO4 CuSO
4

[CuSO
4
]
[CuSO
4
]
[CuSO
4
]
74

The discrepancies observed in the imaging data could have been due to the single average
used for the scans to save time. It was also necessary to obtain the relaxometry data for
each sample at the five fields used for imaging for a complete comparison this was also
not possible due to time constraints. However, some comparison was still possible at
59 mT since that was the field at which the concentration of the contrast agent was
estimated.
From Table 4.4, it appears that the relaxometry and imaging data agree for the BSA
samples without the contrast agent. The differences are however significant for samples
containing CuSO
4
. As was expected, the 5% and 10% BSA samples with CuSO
4
show
approximately equal relaxation rates in each separate technique; but these relaxivities
differ greatly when the two techniques are compared (which should not be the case). The
two methods have each also recorded higher relaxivities for the CuSO
4
solution and the
15% BSA with CuSO
4
. Meanwhile, it is only the relaxivity recorded for CuSO
4
with
imaging that has so far been shown to be approximately equal to the relaxivity of 25%
BSA measured by relaxometry.
It was expected that all samples with CuSO
4
and the CuSO
4
solution would show
approximately the same results in the two methods. However, time did not allow further
experiments to be done to arrive at this expectation.



75

CHAPTER FIVE
5: Conclusions
This project was aimed at developing and optimising a robust technique of evaluating the
size of quadrupole peaks resulting from the cross-relaxation of water protons and
macromolecules at specified resonance frequencies. It has been shown previously that the
size of these peaks is proportional to the amount of polypeptides in the protein.
[1]
The
technique should be able to quantify the peak sizes to correlate linearly with the
concentrations of protein samples prepared by the author. It was further aimed at
elucidating the effects on this linear correlation with the addition of non-protein gel
components or contrast agents.
Concentrations of between 1% and 25% by weight of various samples of bovine serum
albumin (BSA) and co-mixtures with agarose and CuSO
4
have been prepared and their
relaxivities measured in the departmental Stelar SMARtracer FFC relaxometer. For each
sample, background relaxation processes have been subtracted from quadrupolar
processes to extract the quadrupole peaks, which have been stripped into trapezia. The
sum of the areas of these trapezia has been evaluated to be the area under the peaks. This
area has been found to vary linearly with the concentration of the samples. The gradient
of the linear plot of this correlation has been calculated to be the sensitivity of the
relaxometer to changes in protein concentration.
It was observed that, generally, peaks at 2.1 MHz were considerably noisy for
concentrations less than 5% by weight. So it was rather better to evaluate areas of peaks
76

at 2.8 MHz for these concentrations. The situation was, however, different in
concentrations above 5%.
Whereas agarose has been found to enhance the background relaxivity of BSA and
reduces its peak size, CuSO
4
has been found to enhance the background relaxivity of
BSA but keeps its peak size constant. This result is important in the clinical setting since
it shows that the measured quadrupole peaks in the R
1
dispersion profile should not be
altered by the presence of contrast agents, in vivo. Thus, protein concentration
measurement by FFC relaxometry or imaging ought to be robust, irrespective of the
tissues inherent T
1
relaxation time. This observation has further been explored by the
production of a BSA-CuSO
4
composite phantom of various concentrations.
It was hoped that good images of the phantom would be obtained by field-cycling
inversion recovery imaging pulse sequence so that R
1
dispersion profiles could be plotted
from regions of interest (ROIs) selected from these images. The images were however
significantly noisy and so the peaks were not well observed in the plots. Moreover, tubes
that contained samples of the same relaxivities did not all show the same intensity as was
expected. Two out of four tubes showed nearly the same intensity while the other two
showed different but high levels of noise.
These inconsistencies could have emanated from the single signal average used in the
images in order to reduce acquisition time even with that, acquisition period was
approximately 52 minutes. Unfortunately, time constraints during the project meant that
it was not possible to repeat the imaging experiments.
77

In spite of these few discrepancies, the entire project proved worthy of providing an
appropriate baseline for future work and subsequent clinical applications.
5.1 Future Directions
Some future work would still have to be done in addition to the advances made in this
project.
The methods of extracting the peaks and evaluating their areas could be developed into
standard software rather than using Excel spreadsheets. This future program should be
able to compute the area of a peak within a user-selected range of Larmor frequencies.
The NP/PP hybrid sequence would be a good choice next to the inversion recovery
sequence used in this project. However, it would be necessary for a future investigation to
study whether there exists some level of inconsistency or noise introduced into the
dispersion profile at the crossover field (Appendix C).
The imaging aspect of the project had not been investigated further due to time
constraints. There is, therefore, the need for developing a sensitive, fast imaging pulse
sequence to produce images of high echo strength and a reasonable number of averages
within a relatively short time. This would, hopefully, provide greater signal-to-noise ratio
in the selected ROIs and hence well observed peaks.



78

BIBLIOGRAPHY
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th
Annual Meeting of the International
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th
Conference on FC NMR Relaxometry, May 26-28, 2005,
Torino, Italy.


11. University of Aberdeen (2008) Deeper Study 3: Magnetic Resonance Imaging Lecture
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th
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81

APPENDICES

Appendix A: Dispersion profile of NP sequence in the 1.5 3.5 MHz range


Appendix B: Dispersion profile of PP sequence in the 1.5 3.5 MHz range

2.5
3
3.5
4
4.5
5
1.5 2 2.5 3 3.5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
2.5
3
3.5
4
4.5
5
1.5 2 2.5 3 3.5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
82


Appendix C: Dispersion profile of the NP/PP hybrid sequence in the 1.5 3.5 MHz range


Appendix D: Dispersion profile of IR sequence in the 1.5 3.5 MHz range

2.5
3
3.5
4
4.5
5
1.5 2 2.5 3 3.5
R
1

(
s
-
1
)
Proton Larmor Frequency (MHz)
2.5
3
3.5
4
4.5
5
5.5
1.5 2 2.5 3 3.5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
83


Appendix E: Dispersion profile of NP sequence in the 0.5 8.0 MHz range


Appendix F: Dispersion profile of PP sequence in the 0.5 8.0 MHz range

2.5
3.5
4.5
5.5
6.5
7.5
0.5 5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
0.5 5
R
1
(
s
-
1
)
Proton Larmor Frequency (Mhz)
84


Appendix G: Dispersion profile of the NP/PP hybrid sequence in the 0.5 8.0 MHz range


Appendix H: Dispersion profile of IR sequence in the 0.5 8.0 MHz range

2.5
3
3.5
4
4.5
5
5.5
6
6.5
0.5 5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
0.5 5
R
1
(
s
-
1
)
Proton Larmor Frequency (MHz)
85


Appendix I: Linear correlation between the areas of the quadrupole peaks at 2.1 MHz and the protein
concentrations of their respective BSA samples.


Appendix J: Linear correlation between the areas of the quadrupole peaks at 2.8 MHz and the protein
concentrations of their respective BSA samples.

R = 0.984
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
5 10 15 20 25 30
A
r
e
a

u
n
d
e
r

P
e
a
k

(
s
-
2
)
Concentration of BSA (% by Weight)
R = 0.9808
0
0.2
0.4
0.6
0.8
1
1.2
5 10 15 20 25 30
A
r
e
a

u
n
d
e
r

P
e
a
k

(
s
-
2
)
Concentration of BSA (% by Weight)
86

Appendix L: Evaluation of the area under the quadrupole peaks of 20% BSA

First peak:
(1.840 - 2.465) MHz


_B
0
E
_____ AR
1
/10
-6
a+b h 0.5*h(a+b)
2.4652 0.571751 1.203446 0.0712 0.042843

2.394 0.631695 1.434973 0.0689 0.049435

2.3251 0.803278 1.741218 0.0666 0.057983

2.2585 0.93794 1.95534 0.0657 0.064233

2.1928 1.0174 2.225772 0.0635 0.070668

2.1293 1.208372 2.290714 0.0602 0.068951

2.0691 1.082342 2.024748 0.0606 0.06135

2.0085 0.942406 1.845343 0.0573 0.052869

1.9512 0.902938 1.592524 0.0565 0.044989

1.8947 0.689587 1.212902 0.0546 0.033112

1.8401 0.523316

Total

0.546432 s
-2


Second peak:
(2.465 - 3.025) MHz


_B
0
E
_____ AR
1
/10
-6
a+b h 0.5*h(a+b)
3.025 0.509654 1.655814 0.0873 0.072276

2.9377 1.14616 2.735918 0.0846 0.115729

2.8531 1.589758 3.252131 0.082 0.133337

2.7711 1.662373 3.102465 0.0809 0.125495

2.6902 1.440092 2.546282 0.077 0.098032

2.6132 1.10619 1.824598 0.0758 0.069152

2.5374 0.718408 1.290158 0.0722 0.046575

2.4652 0.571751

Total

0.660597 s
-2


Overall

1.207028 s
-2