The Measurement of Diffusion and Perfusion in Biological Systems Using Magnetic Resonance Imaging

Phys. Med. Biol. 45 (2000) R97R138.
Printed in the UK PII: S0031-9155(00)99102-4

TOPICAL REVIEW
The measurement of diffusion and perfusion in biological
systems using magnetic resonance imaging
David L Thomas|, Mark F Lythgoe, Gaby S Pell, Fernando Calamante
and Roger J Ordidge
Department of Medical Physics and Bioengineering, University College London,
Shropshire House, 1120 Capper Street, London WC1E 6JA, UK
RCS Unit of Biophysics, Institute of Child Health, University College London Medical School,
30 Guilford Street, London WC1N 1EH, UK
Division of Medical Physics, Nathan Kline Institute for Psychiatric Research,
140 Old Orangeburg Road, Orangeburg, NY10962, USA
E-mail: thomas@medphys.ucl.ac.uk
Received 12 November 1999
Abstract. The aim of this review is to describe two recent developments in the use of magnetic
resonance imaging (MRI) in the study of biological systems: diffusion and perfusion MRI.
Diffusion MRI measures the molecular mobility of water in tissue, while perfusion MRI measures
the rate at which blood is delivered to tissue. Therefore, both these techniques measure quantities
which have direct physiological relevance. It is shown that diffusion in biological systems is a
complex phenomenon, inuenced directly by tissue microstructure, and that its measurement can
provide a large amount of information about the organization of this structure in normal and diseased
tissue. Perfusion reects the delivery of essential nutrients to tissue, and so is directly related to
its status. The concepts behind the techniques are explained, and the theoretical models that are
used to convert MRI data to quantitative physical parameters are outlined. Examples of current
applications of diffusion and perfusion MRI are given. In particular, the use of the techniques to
study the pathophysiology of cerebral ischaemia/stroke is described. It is hoped that the biophysical
insights provided by this approach will help to dene the mechanisms of cell damage and allow
evaluation of therapies aimed at reducing this damage.
Contents
1. Introduction R98
2. Imaging of diffusion using MRI R98
2.1. Diffusion theory: random molecular motion R98
2.2. NMR and diffusion R99
2.3. Diffusion in biological systems R102
2.4. Implementation of the NMR diffusion technique R108
3. Imaging of perfusion using MRI R110
3.1. What is perfusion? R110
3.2. Measurement of perfusion using bolus tracking methods R110
3.3. Measurement of perfusion using arterial spin-labelling methods R114
3.4. Advantages and disadvantages of MR perfusion techniques R121
3.5. Other techniques for measuring perfusion-related parameters R122
| Address for correspondence: RCSUnit of Biophysics, Institute of Child Health, University College London Medical
School, 30 Guilford Street, London WC1N 1EH, UK.
0031-9155/00/080097+42$30.00 2000 IOP Publishing Ltd R97
R98 D L Thomas et al
4. Applications of diffusion and perfusion imaging R123
4.1. Diffusion-weighted imaging in biological systems R123
4.2. Perfusion imaging R128
5. Conclusion R131
References R131
1. Introduction
Diffusion and perfusion magnetic resonance imaging (MRI) are two of the most recently
developed and rapidly evolving areas of biomedical imaging, with applications ranging
from diagnosis of hyperacute and chronic disease to the study of the microvascular and
haemodynamic changes associated with functional cerebral activation. The aim of this review
is to describe how the development of these techniques has enabled physiology and function
(particularly of the brain) to be imaged directly using MRI. This compares to previous MRI
approaches that have traditionally used contrast which is dependent on the relaxation times T
1
and T
2
. Although qualitatively useful, T
1
- and T
2
-weighted images are inuenced by a large
number of factors, making direct interpretation of a change in contrast in these images difcult.
Even when quantitative T
1
and T
2
mapping approaches are taken, the values are dependent
on the static eld strength of the MR magnet in which they are measured, which complicates
the comparison of results from different centres working at different eld strengths. The
development of diffusion and perfusion MRI has opened up the possibility to study the brain
in ways which were previously unachievable, with results that are universally comparable.
MRI is now a well established modality in both clinical and experimental investigations
of the body. In this review, we will assume familiarity with the basic principles of MR data
acquisition and image formation. Those requiring a comprehensive description of nuclear
magnetic resonance (NMR) and MRI theory are referred to the relevant references (e.g.
Stark and Bradley 1998, Gadian 1995). This review takes the following format: after this
introduction, sections 2 and 3 describe the theoretical and practical issues relevant to the design
and implementation of diffusion and perfusion imaging respectively. In section 2, diffusion
is described mathematically as a fundamental molecular phenomenon, and the sensitivity of
NMR to this phenomenon via the manipulation of magnetic eld gradients is introduced. The
complexity of diffusion in biological systems is explored, and MR pulse sequences which
have been designed to investigate this are described and evaluated. In section 3, perfusion is
dened and two methods for measuring perfusion (exogenous contrast agent bolus tracking
and arterial spin labelling) are described in detail and compared. The review concludes with
section 4 which describes the range of applications in which all these techniques have so far
been used.
2. Imaging of diffusion using MRI
2.1. Diffusion theory: random molecular motion
The classic description of diffusion relates the macroscopic ux density, J, to an established
concentration gradient, C in the following manner:
J = DC (2.1)
where the proportionality constant, D, is known as the diffusion coefcient (units:
[length
2
/time], usually [mm
2
s
1
]). This is known as Ficks law (Crank 1975). The time
dependent form of this equation allows a determination of D from the measured concentration
Measuring diffusion and perfusion using MRI R99
prole of a tracer. In these experiments, microscopic displacements are of the order of mm
as the observation period is over a period of minutes. In contrast to this classical approach to
diffusion measurements, the NMR method directly monitors the net molecular displacements
of an ensemble of spins. The dependence of this displacement on the measurement duration
is dened by the Einstein relationship
r
2
) = 6Dt (2.2)
where t is the observation (i.e. diffusion) time and r
2
) is the mean squared displacement of
an ensemble of spins in three dimensions and corresponds to the variance of the displacement
distribution. The proportionality constant in this relationship is D, the diffusion coefcient.
The diffusion coefcients almost linear dependence on temperature (Le Bihan 1995d) reects
the origins of the mean displacement in the random thermal (Brownian) motion of molecules.
The fundamental relationship (equation (2.2)) followed Einsteins insight concerning the
incorporation of the diffusion coefcient into the conditional probability distribution that
characterizes molecular displacement. This distribution is denoted P(r
2
, r
1
,
D
) and is the
conditional Gaussian probability of nding a given spin initially at point r
1
between positions
r
2
and r
2
+ dr
2
after a time interval
D
(Einstein 1926). For free diffusion in three dimensions,
the distribution is given by
P(r
2
, r
1
,
D
) =
1
_
(4D
D
)
3
exp
_
(r
1
r
2
)
2
4D
D
_
. (2.3)
This expression acts as the diffusion propagator. The Gaussian nature of this distribution
underlies the quantication of the diffusion coefcient with NMR.
2.2. NMR and diffusion
The effect of diffusion on an ensemble of magnetized spins can be understood in terms of the
phase, , of the magnetization in the transverse plane of the rotating reference frame, and its
behaviour in an applied magnetic eld gradient. A gradient pulse encodes the static spins in
each voxel with a spatially varying resonant frequency and, therefore, with a characteristic
phase dispersion that can be rephased in a relatively easy manner. For diffusing spins, this
intravoxel phase dispersion becomes more complicated and incoherent as the spins randomly
translate with varying velocities along the direction of the applied gradient. The variance of
the distribution of intravoxel phase dispersion is magnied (in a similar way to the conditional
displacement distribution (equation (2.3))) even though the average phase remains zero. The
overall signal from the voxel is attenuated due to destructive interference of the ensemble of
phase-dispersed spins, and this attenuation is a function of the diffusion coefcient. This can
be contrasted to the effect of bulk, coherent motion of the sample which results in a global
phase shift without signal attenuation.
The mechanism of diffusion weighting can be understood by consideration of the phase
changes during the simple bipolar pulsed gradient experiment (Stejskal and Tanner 1965). In
gure 1(a), Gis the gradient amplitude, is the time interval between gradient pulses and is
the duration of the individual gradient pulses. The general expression for the phase evolution
of a spin at position z
1
along the z-axis, after a time , is given by
=
_

0
Gz
1
dt = Gz
1
. (2.4)
The initial 90
radio-frequency (RF) pulse creates coherent transverse magnetization and the

subsequent gradient pulse induces a spatially dependent phase shift. For the simple case of a
mobile two-spin system depicted in gure 1(b) that initially extends over a distance a along
Figure 1. Schematic representation of the StejskalTanner pulsed gradient experiment and its effect
on diffusing molecules. (a) The pulse sequence, with its timing and gradient variables. (b) The state
of the transverse magnetization in the rotating frame at four stages during the diffusion experiment
(iiv) for diffusing spins, in the case of a simple two-spin system which begins with positions
z = {a/2, a/2] and diffuses to z = {a/2+
1
, a/2+
2
] (depicted at the bottom). The transverse
magnetization before (i, iii) and after (ii, iv) each of the two diffusion gradient pulses is represented.
1,2
represents the phase shift caused by the application of the diffusion gradients. To simplify
the situation, T
2
relaxation is ignored. The echo forms at a time t = TE from the application of
the 90
RF pulse, and has an amplitude which depends on the phase coherence remaining between
the two spins. For stationary spins,
1
=
/
1
and
2
=
/
2
so that the application of diffusion
gradients has no effect on the echo amplitude.
the z-axis of the magnet (i.e. spin 1 and 2 at z = {a/2, a/2]), the overall phase dispersion,
A
, across the spin system in the presence of a z-gradient, G, is given (from equation (2.4))
by
A
=
1
+
2
=
Ga
2
+
Ga
2
= Ga (2.5)
where
1
and
2
are the phase shift accrued by spin 1 and spin 2 respectively due to the
application of G. The phase shifts are reversed by the subsequent 180
RF pulse. If the spins

diffuse during the interval between the diffusion gradients, by the time of the second gradient
pulse, the spin positions will have changed to {a/2+
1
, +a/2+
2
] where
1
and
2
represent
random displacements in the z-direction. The total phase dispersion,
B
, is then given by
B
=
/
1
+
/
2
= G
_
a
2

1
_
+ G
_
a
2
+
2
_
= G(a +
2

1
). (2.6)
The net dephasing during the experiment, , is therefore given by
=
B

A
= G(
2

1
). (2.7)
For static spins,
1
=
2
= 0, and the second gradient pulse simply refocuses the effect of the
rst pulse. However, a systemof mobile spins experiences a net dephasing (gure 1(b)). For an
ensemble of spins, it can be shown that the resultant signal attenuation is a function of the phase
dispersion and the conditional probability expression P(r
2
, r
1
,
D
) of equation (2.3) (Le Bihan
1995a). The signal attenuation is, therefore, directly related to the diffusion coefcient. With
the assumption of a Gaussian form for the probability term, the attenuation is given by the
StejskalTanner equation (Stejskal and Tanner 1965):
ln
_
S(G, , )
S(0)
_
=
2
G
2
2
_

3
_
D (2.8)
Figure 2. (a) T
2
-weighted image showing regions of grey matter, white matter and CSF-lled
ventricles obtained in vivo from the cat brain. (b) Diffusion ellipsoid image constructed from the
effective diffusion tensor, estimated in each voxel for the ROI enclosed by the black rectangle.
(Image kindly provided by Dr Peter Basser, reprinted with permission.)
where S(G, , ) and S(0) are the amplitudes of the diffusion-weighted and non-diffusion-
weighted magnetizations respectively. The effects of gradient strength and timing can be
conveniently grouped together in the so-called b-factor (units: [time/length
2
]) so that the
general expression becomes
S(b) = S(0) exp(bD). (2.9)
As will be discussed in the next section, this expression is only valid for a homogeneous, non-
restricted (isotropic) medium such as free water in which the mobility shows no directional
dependence, and the effects of imaging gradients have also been neglected. The signal
attenuation can be calculated for any combination of diffusion gradients with the use of
the diffusion-modied Bloch equations (Torrey 1956). The general expression of the signal
attenuation of the signal in an isotropic medium is given by
ln
_
S(b)
S(0)
_
= D
_
TE
0
k(t
/
) k(t
/
) dt
/
(2.10a)
where k is the k-space vector (Ljunggren 1983):
k(t ) =
_
TE
0
G(t
/
) dt
/
(2.10b)
so that
b =
_
TE
0
k(t
/
) k(t
/
) dt
/
. (2.10c)
Maps of the diffusion coefcient can be obtained by acquiring images over a range of diffusion
weightings (i.e. different b-factors) and tting to equation (2.9) on a pixel-by-pixel basis
(see gure 2). From equation (2.8), it can be seen that stronger diffusion sensitization can
be achieved in one of two ways: (i) an increase in the amplitude of the gradient pulses, or
(ii) an increase in the diffusion time,
D
(i.e. the dephasing duration) which incorporates the
length of the diffusion sensitizing gradients () and the interval between them (). For the
StejskalTanner experiment,
D
(/3).
Table 1. Typical values of the ADC in free water and in human brain with approximate standard
deviations. Cerebrospinal uid (CSF) and grey matter (GM) values from (Turner et al 1990).
White matter (WM) values are obtained from Le Bihan et al (1993) in which diffusion-sensitizing
gradients were placed parallel and orthogonal to the direction of the bre axes.
Substance Diffusion coefcient [mm
2
s
1
]
Water (room temperature) (2.30 0.02) 10
3
CSF (2.94 0.05) 10
3
Grey matter (0.76 0.03) 10
3
White matter ortho. (0.45 0.03) 10
3
para. (0.95 0.03) 10
3
2.3. Diffusion in biological systems
The contrast in diffusion-weighted images is dependent on the diffusive mobility of the
substance under study. For the same degree of diffusion weighting, the signal from free water
or cerebrospinal uid (CSF) will be highly attenuated compared to the signal fromthe restricted
water in the tissue compartments (see table 1). The decreased diffusion coefcient of tissue
water with respect to free water offers an indication of the greatly conned environment within
biological systems. As table 1 shows, a single axis diffusion gradient measurement provides
values of D that vary according to the alignment of the gradient with the bre direction.
Diffusion in biological tissues is a complex phenomenon that can only be approximated by
the classic treatment described thus far. The sophisticated, heterogeneous microstructure of
tissue modies the concept of free diffusion due to the presence of internal structure and
barriers. Exchange must be considered between the multiple compartments. Furthermore, the
diffusion of water molecules becomes an orientation-dependent (anisotropic) phenomenon.
A non-Gaussian form of the conditional probability function (equation (2.3)) is, therefore,
predicted, and the use of a scalar diffusion measurement that is independent of experimental
factors becomes unrealistic. For these reasons, the diffusion parameter measured in biological
systems is termed the apparent diffusion coefcient (ADC) in order to acknowledge these
considerations which are described in more detail in the following sections.
2.3.1. Multiple compartments. Tissue water is contained within multiple compartments,
with the most obvious division being the intra/extra-cellular environments. Another proposed
division describes the water residing in a thin layer around the cell membrane and is restricted
due to its proximity to the cellular surface (Helmer et al 1995). In the intracellular environment,
the existence of compartmentalization has been demonstrated (e.g. between the cytoplasm
and the nucleus) during investigations of single neurones (Schoeniger et al 1994). Water is
always in a state of continuous exchange between the various compartments. The observed
signal attenuation in the diffusion experiment, therefore, depends on the rate of exchange and
the diffusion (measurement) time,
D
. In the limit of slow exchange with the water spins
remaining within their compartments during the diffusion time, this will be reected by a
multi-exponential form of the signal attenuation. An expression for the signal behaviour in a
two-compartment system (e.g. intra/extra-cellular compartments) is then given by
S(b) = S
0
[f
1
exp(b
1
ADC
1
) + f
2
exp(b
2
ADC
2
)] (2.11)
where f
1
and f
2
are the volume fractions within each of the two compartments (so that
f
1
+f
2
= 1); ADC
1
and ADC
2
are the apparent diffusion coefcients in the two compartments.
In contrast to this, in the limit of fast exchange with complete redistribution of water
between the compartments during the diffusion time, the signal attenuation will followa single
exponential behaviour. The observed apparent diffusion coefcient for a two-compartment
system is then approximated by
ADC = f
1
ADC
1
+ f
2
ADC
2
. (2.12)
Instead of the existence of either slow or fast exchange within the system, an intermediate
regime is most likely. In investigations of the multi-compartmental nature of tissue, it is also
important to take into account the absolute water concentrations and, more signicantly, the
different T
2
relaxation times (von Meerwall 1982, Buckley et al 1999) within the different
compartments, since this affects signal intensity at a given echo time. The intracellular T
2
is expected to be signicantly smaller than the corresponding extracellular value due to the
differing water mobilities in the two compartments. This will affect the determination of the
compartmental volume fractions. In most practical situations, the biexponential behaviour of
signal attenuation described by equation (2.11) is not observed, and it is therefore common
practice to t data to the single exponential decay model and to quote a volume-averaged
ADC.
2.3.2. Restricted diffusion. The free diffusive motion of water in tissue is restricted by the
presence of natural barriers such as cell membranes. The diffusion time of the experiment
determines the experimental sensitivity to the diffusion restriction. For a short diffusion time,
most water molecules do not have time to reach the barriers, and diffusion will be relatively
free. With increasing diffusion times, an increasing number of molecules will strike the
boundaries of the restricted systemand the diffusion displacement will deviate froma Gaussian
behaviour. The effect of the restriction on the diffusive displacement will strongly depend on
the morphology and the degree of permeability of the barriers and obstacles.
In biological systems, the diffusive displacement will deviate from its expected linear
relationship with the diffusion time,
D
(predicted by equation (2.2)) and will plateau at a level
corresponding to the size of the restricting volume. An experimental determination of the
degree of restriction can, therefore, be obtained by examining the diffusion time dependence
of the diffusion experiment. Restriction is indicated by the decrease of the ADC with an
increasing diffusion time. If diffusion is restricted in the classical sense (i.e. by impermeable
barriers), the diffusion time dependence of the ADC would be expected to be observed even at
relatively long values of
D
(i.e. 1 s) (Stejskal and Tanner 1965). Experimental evidence for
such a phenomenon is lacking for diffusion times above 20 ms (i.e. for diffusion displacements
of 5 m which are of the order of the size of certain tissue microstructures) (Moonen et al
1991, Le Bihan et al 1993). However, evidence of restriction at lower diffusion times (<20 ms)
in brain tissue has been reported (Le Bihan et al 1993, Niendorf et al 1994). This apparent
restriction implicates the presence of either permeable barriers (Stanisz et al 1997, Le Bihan
1995c) or a modied explanation of the concept of restriction (see next section).
2.3.3. Tortuosity and hindered diffusion. In order to retain the concept of classical restricted
diffusion by impermeable barriers and maintain consistency with the experimental ndings
mentioned above (i.e. diffusion time independence of the ADCwhen
D
is long), an alternative
hypothesis has been suggested. In the presence of impermeable obstacles, extracellular water
will remain unrestricted if the molecules are able to diffuse around the restricting volume. The
mean path between two points is thereby lengthened so that the measured ADC for a xed
D
is lowered with respect to a corresponding measurement in the free medium. This aspect
of hindered diffusion can be quantied by the concept of tortuosity that has its origins in the
study of solid-boundary porous media (Mitra et al 1993, Latour et al 1993). In the limit of
long diffusion times, each water molecule in the extracellular space has sampled an equivalent
volume of the restricting systemand the measured ADCwill not deviate further with increasing
D
. The tortuosity coefcient, , can then be dened as
ADC =
D
0
2
(2.13)
where D
0
is the diffusion coefcient observed in the absence of obstacles (i.e. in the free
medium). Tissue water can diffuse over unlimited distances (cf restricted diffusion) as
there is no real barrier to diffusion. The ADC will, therefore, only show a diffusion time
dependence at short values of
D
when the hindered and free path lengths are similar. This
would occur at the shorter
D
values (<20 ms) that have been reported in studies of the
time dependence (Le Bihan et al 1993, Niendorf et al 1994). The deviation of the ADC
at very short diffusion times is not dependent on the size of the restricting volume and
can be used to provide information on the local structure of the medium. In this manner,
evaluation of the surface-to-pore-volume ratio, S/V, in porous media (Mitra et al 1993)
with the use of NMR, has been extended to the study of cerebral tumours (Helmer et al
1995). Hindered diffusion and tortuosity provides an explanation for the non-monoexponential
behaviour of the signal attenuation without requiring distinct compartmentalization of tissue
water (Helmer et al 1995, Pfeuffer et al 1998) but instead dependence on the underlying
geometrical structure. Measurements of the tortuosity have classically been obtained with the
use of ion-sensitive microelectrodes to follow the diffusion of a purely extracellular marker
(e.g. TMA) (Nicholson and Philips 1981). The correspondence of these data with MRI results
from observable water, which is probably in continuous exchange between compartments,
remains to be claried.
2.3.4. Anisotropy. In addition to the dependence of the experiment on its time scale,
the direction of the measurement also plays a crucial role in the evaluation of ADC. The
diffusion of water in biological systems is an orientation-dependent (anisotropic) phenomenon.
The scalar measurement of effective diffusivity described in section 2.1 is essentially a
one-dimensional measure of diffusive mobility and cannot, therefore, describe the three-
dimensional translational mobility of water within tissue. The most obvious practical
demonstration of anisotropy is the dependence of the ADC measurement in white matter
(Moseley et al 1990a) and skeletal muscle (Cleveland et al 1976) on the relative angle
between the applied diffusion gradient and the grain or bre tract axis (see table 1). Diffusion
displacement in these media is greater when measured with the diffusion sensitization gradient
in a direction parallel to the bre-tract direction as demonstrated by a higher ADC value.
This corresponds to a macroscopic degree of structural anisotropy. Orientation-dependent
restriction is, therefore, the underlying cause of anisotropy in these cases, but is not a requisite
characteristic since permeable structures with a degree of spatial organization can also display
anisotropy, such as in the grey matter of the cerebral cortex (Lythgoe et al 1997).
Anisotropy implicates the correlation of water mobility along different directions so
that diffusion gradients in orthogonal directions can interact with each other. Accurate
ADC measurements, therefore, requires the consideration of all gradientswhether diffusion,
imaging or background gradientsand in all directionswhether parallel, oblique or
perpendicular to each other. In this respect, diffusion cannot be considered a vector quantity.
Instead, diffusive transport can be characterized by nine diffusion coefcients grouped in an
effective second-rank tensor, D, so that
D =
_
D
xx
D
xy
D
xz
D
yx
D
yy
D
yz
D
zx
D
zy
D
zz
_
. (2.14)
The conditional probability distribution (equation (2.3)) is also modied by incorporation of the
tensor description (Basser et al 1995, Basser and Pierpaoli 1996). The off-diagonal elements
of the tensor (e.g. D
xy
, D
zy
) reect correlations between displacements in perpendicular
directions. This can be understood by consideration of the physical structure of anisotropic
media in which translational mobility will be biased along particular directions but away
from others. These off-diagonal elements can be as large as the diagonal components. Since
diffusion must be described by real values and the tensor can be shown to be Hermitian,
the tensor elements are symmetric (i.e. D
ij
= D
ji
) (Basser et al 1995). The tensors six
independent terms and S(0) can be extracted from a series of measurements with diffusion
gradients appliedina range of directions (diffusion-tensor MRI). Aminimumof sevenweighted
images are required. The expression for the signal attenuation in an anisotropic mediumcan be
shown to be provided by the following matrix-based expression which evaluates to a nine-term
sum of products (cf equation (2.10)):
ln
_
S(b)
S(0)
_
=
_
TE
0
k(t
/
)
T
D k(t
/
) dt
/
(2.15)
where
T
denotes the transpose of the matrix. This equation can be simplied to provide an
equivalent expression for equation (2.9)
ln
_
S(b)
S(0)
_
=
3
i=1
3
j=1
b
ij
D
ij
(2.16)
where b
ij
represents the components of the matrix of b-factors (the b-matrix) and D
ij
are the
components of the diffusion tensor.
The b-matrix weights the relative contributions of the components of the diffusion tensor
to the signal attenuation. The cross-terms, b
ij
where i ,= j, describe interactions of orthogonal
gradient directions that are associated with the off-diagonal elements of the tensor. For
example, the application of the y and z diffusion gradients in a spectroscopy experiment
(i.e. without imaging gradients) provides the following expanded form of equation (2.15):
ln
_
S(b)
S(0)
_
= b
yy
D
yy
b
zz
D
zz
2b
yz
D
yz
. (2.17)
This can be compared to the situation of an isotropic material. In this case, the diagonal terms
of the tensor are equivalent (D
xx
= D
yy
= D
zz
) while the off-diagonal terms vanish i.e. there is
no preferred direction of displacement, so that equation (2.15) becomes ln[S(b)/S(0)] = bD
where b = b
yy
+ b
zz
.
In imaging experiments, the inuence of imaging gradients can be signicant and must
be incorporated into the evaluation of the overall b-matrix in order to accurately characterize
the tensor. Both the direct, self-mediated signal attenuation due to these gradients (self-terms)
and their mathematical interactions with other gradients (cross-terms) must be considered.
Therefore, in an anisotropic material, the contributions of every gradient to the signal
attenuation must be considered. The proportional contribution of a diffusion gradient, G
d
,
and an accompanying background (e.g. imaging) gradient, G
b
, to the overall b-factor in an
anisotropic medium can be written as
b
TOTAL
[G
d
[
2
+ [G
b
[
2
+ G
d
G
b
(2.18)
where the second and third terms are the background gradients self-term and cross-terms
respectively; the variables, , and , include the timing factors ([time
3
]). For acquisitions
with differing degrees of diffusion weighting, it can be seen that the self-termof the background
gradients will cancel since this term will be present in each image. However, the cross-term
remains as it is proportional to the changing diffusion gradient, and if not suitably treated, this
term will introduce a systematic error into the ADC measurement. The additional diffusion
weighting as a result of the imaging gradients will generally lead to an overestimation of
the ADC. It should be noted that the unique sign-dependence of the b-factor cross-term in
equation (2.18) allows the presence of background gradients to be ascertained with the use of
diffusion gradients of alternate sign in two successive experiments. It is important to note that
even in an isotropic medium, the self-terms are relevant and must be considered.
2.3.5. Principal diffusivities. The magnitude of the tensor components depends on the subject
orientation within the laboratory frame of reference dened by the read, phase and slice gradient
directions {x, y, z]. Rotation of the sample will change the individual components and the
tensor is, therefore, non-unique. The sample will, however, possess a unique local orthogonal
coordinate system{
1
,
2
,
3
] (the principal coordinate axes) in which the displacements along
the orthogonal directions appear uncorrelated. The diffusion coefcients along these three
principal directions are termed
1
,
2
and
3
(the principal diffusivities). The principal
coordinate axes anddiffusivities correspondmathematicallytothe eigenfactors andeigenvalues
respectively of the diagonalized diffusion tensor, so that the following relationship applies:
DE = E where E = (
1
[
2
[
3
) and =
_
1
0 0
0
2
0
0 0
3
_
. (2.19)
In macroscopically anisotropic media such as white matter, these principal directions
correspond to the orthotropic directions of the internal structure.
2.3.6. Diffusion ellipsoid. The properties of the diffusion tensor can be visualized in terms of
an effective diffusion ellipsoid (Basser et al 1995). This geometric formulation denes a three-
dimensional surface of constant translational displacement probability for a given diffusion
time. The principal axes of the ellipsoid are the mean effective diffusive displacements along
the principal coordinate axes. The visual interpretation of the formof the ellipsoid corresponds
to the state of a drop of ink after being added to a water-lled container. The dispersion of the
ink droplet in the isotropic medium will be direction independent and the resulting spherical
volume represents the shape of the diffusion ellipsoid in an isotropic material. If the water is
substituted for an anisotropic medium, the spatially distributed dispersion of the droplet will
follow the directional dependence of the tensor. The magnitude of the radii of the resulting
ellipsoid (i.e. its overall size) when compared to the corresponding geometry of the spherical
surface in the isotropic situation reects the general differences in diffusivity. The ellipsoid
representation of the tensor can be determined for each voxel in an image in order to provide
a striking visual indication of the degree of directional dependence and structural similarity
(see gure 2).
2.3.7. Scalar invariants. The orientation dependence of the diffusion tensor can be used
to provide important physiological and morphological information concerning the biological
system under consideration. However, an objective measure of the inherent diffusivity of the
sample is precluded unless the diffusion gradient axes are aligned with the principal coordinate
axes of the object. The coincidence of these two frames of reference is unlikely due to the
unpredictability and variability of the principal axes. However, matrix algebra predicts the
existence of certain invariant, scalar combinations of the elements of the diffusion tensor.
These measurements will possess rotational and translational invariance and thus will not
Figure 3. Calculation of unidirectional (X, Y, Z) ADC maps and maps of the trace of the diffusion
tensor, from three sets of single axis (X, Y, Z) diffusion-weighted images at different b-values. All
images were acquired 2 hours following middle cerebral artery occlusion in the rat. The effect of
tissue anisotropy on lesion delineation (left side of image) is observed in the X, Y and Z ADC
maps. The chequered bars depict the process of image combination (e.g. by tting of the image
signal intensity to the monoexponential decay model, see inset top right) to generate the next level
of information and eliminate confounding image dependences.
depend on the sample orientation within the laboratory coordinate system. The best known of
these invariant quantities is the trace of the diffusion tensor, Trace(D) which is given by
Trace(D) = D
xx
+ D
yy
+ D
zz
=
1
+
2
+
3
(2.20)
where the second and third expressions are the diagonal terms in the laboratory and principal
frames of reference respectively. The invariant is scaled to provide a parameter, D
av
, where
D
av
=
1
3
Trace(D). Figure 3 displays the procedure of obtaining a map of the trace parameter
from the component images. In the example shown, ADC maps are generated by acquiring
three images with different diffusion weighting in each direction (x, y, z). The three ADC
maps are then averaged to create a D
av
map. This parameter represents a comparable and
consistent measure of the mean diffusivity. It can be seen from the preceding discussion
(sections 2.3.5 and 2.3.6) and equation (2.20) that D
av
is proportional to the averaged sum
of squares of the major and minor radii of the corresponding diffusion ellipsoid. It has been
shown that the measurement of the trace improves contrast between normal and ischaemic
tissue which would otherwise have been confounded by the anisotropy-related subjectivity of
the diffusion measurement (van Gelderen et al 1994, Lythgoe et al 1997). It should be noted
that in an imaging experiment, the individual determination of each of the diagonal tensor
elements is a time-consuming process and must include the analysis of the contributions of the
background gradients (Basser 1995). In order to optimize the time efciency and in order to
reduce the sensitivity to background gradients, single shot trace sequences have been devised
(Mori and van Zijl 1995, Wong et al 1995). Simultaneous application of gradient pulses in
multiple directions provides equal diffusion weighting to the elements of the diagonal tensor
while the off-diagonal elements cancel out.
In a similar manner to the previous discussion of invariant measures of intrinsic
diffusivity, the characterization of the degree of anisotropy in a medium also requires a scalar,
rotationally invariant measurement. A variety of such quantities incorporating combinations
of the principal diffusivities have been devised which possess this attribute such as the
lattice anisotropy index (Pierpaoli and Basser 1996). However, a methodology for simple
visualization of the degree of anisotropy (usually with the use of colour coding schemes) is
still being sought.
2.3.8. Background gradients. The importance of the imaging gradients to the accuracy of
the ADC measurement has already been described. The background gradients (i.e. gradients
whose specic purpose is not to provide diffusion weighting) also include a contribution
due to the gradients that are generated within the sample itself. These are generated as a
result of inter/intra-subject differences in the bulk susceptibility. The effect of intravascular
susceptibility variation is especially signicant (Zhong et al 1991). The deleterious effect of
these background gradients on ADC quantitation can be more signicant than for the imaging
gradients since the sample gradients are active during the entire experiment. The resulting
systematic error in the measured ADCcan be either manifested by an under- or over-estimation
of the value (Zhong et al 1991, Does et al 1999) and the diffusion-time-dependent effects of
restriction will also be modied (Zhong et al 1991). Both self-terms and cross-terms will
be generated by the sample gradients but the self-mediated losses are usually eliminated by
multiple acquisitions (see section 2.3.4). The signicance of the cross-terms can be reduced by
optimized positioning of the diffusion gradients in the pulse sequence, such as the placement of
bipolar diffusion gradient pairs around the 180
pulse (Hong and Dixon 1992) or the alternation

of gradient polarity in a multi-echo sequence (Karlicek and Lowe 1980).
2.3.9. Discussion. As the uses of diffusion NMR are being demonstrated in an increasingly
wide variety of applications, the complexity of the relationships between the external structure
of a biological system, and the translational mobility within it, is becoming increasingly clear.
The underlying assumption of a Gaussian form for the conditional probability distribution
upon which much of the preceding theoretical description of diffusion is based, is affected by
the impeding cellular architecture such as membranes, bres and organelles. The experimental
observation of non-monoexponential signal attenuation plots is the most obvious indicator of
the deviation of theory fromreality. However, the appearance of this behaviour in experimental
data can reect any one of the interrelated complications discussed in the preceding sections
restriction, background gradient, multiple compartments, exchange or anisotropyand it is
more likely that a host of these and other factors contribute to this observation. Hence,
a theoretical model of the tissue is ideally required in order to relate the diffusion NMR
measurements to the physical and geometrical properties of the tissue (Szafer et al 1995,
Pfeuffer et al 1998). A complete characterization of tissue at the cellular level is the ultimate
goal of the diffusion technique but requires a more complete understanding of the organization
of biological systems.
2.4. Implementation of the NMR diffusion technique
2.4.1. Hardware. In order to implement diffusion imaging with a satisfactory degree of
accuracy and precision, a number of practical issues have to be addressed. In addition to the
potential biophysical issues described in the previous sections, system-related factors are also a
signicant consideration. The gradients obviously play a crucial role by inducing the diffusion
weighting and a high degree of gradient stability is sought in order to provide reproducible
ADC values. Any gradient mismatch between a pair of diffusion gradients would induce
artefactual signal loss. A well calibrated and highly linear gradient set with well dened
control of amplitude and direction is required. Errors in the gradient strength can be especially
severe in diffusion imaging due to the eddy currents which arise as a result of the rapid and
powerful pulsing of the gradient coils (Ahn and Cho 1991). Eddy current elds are induced in
the presence of conducting structures such as the cryostat shield and oppose the main magnetic
eld in accordance with Lenzs law of electromagnetic induction. The elds persist, decaying
with multi-exponential characteristics whose time constants are associated with the resistance
and the inductance of the current path and may be of the order of hundreds of ms. For the
diffusionexperiment, this canresult inanimbalance betweenthe diffusion-sensitizinggradients
and, therefore, unintended signal dephasing. The effects of eddy currents can be reduced with
pre-emphasis compensation techniques or, more efciently, by hardware modications such
as actively shielded gradients (Manseld and Chapman 1987).
Another important source of inaccuracies is the sensitivity of the technique to bulk subject
motion even to the extent of physiological pulsations. The resulting phase errors will be
manifested as ghosting and smearing artefacts in the phase-encoding direction of an image
acquired with a 2D-Fourier transform (FT) sequence. Phase discontinuities arise between
successive phase encoding cycles such that the lines of k-space are acquired with an intervening
recovery time (TR) that may be close to the period of the motion. The motion artefacts can be
signicantlyreducedinsuchsequences withthe use of a navigator-echo-basedphase correction
in which an extra non-phase-encoded echo is acquired together with each k-space line (Ordidge
et al 1994, Anderson and Gore 1994). The motion-induced relative phase changes of these
navigator echoes with respect to a reference echo can then be used to back-correct the respective
k-space lines. The use of comfortable subject restraints and single shot MRI methods (see
next section) have virtually eliminated the problem of motion artefacts.
2.4.2. Pulse sequences. Any imaging strategy can conceivably be implemented for diffusion
imaging whether on its own or in combination with a separate diffusion preparation period.
However, quantitative studies may preclude the use of certain techniques due to the difculty
in ascertaining the overall b-factor and signal pathways. Bipolar gradients (i.e. immediate
refocusing) are commonly incorporated in gradient-echo-based methods. Apair of monopolar
gradients (i.e. delayed refocusing) positioned around a refocusing 180
pulse in a spin-echo-
based sequence is an efcient manner of prolonging the diffusion time at the expense of
additional T
2
-mediated signal decay (as shown in gure 1). Stimulated echo sequences
comprise three (usually 90
) RF pulses with diffusion gradients placed after the rst and

third pulses. This scheme allows the investigation of variable diffusion times without as severe
a penalty in T
2
signal loss (Merboldt et al 1991). The signal similarly undergoes delayed
refocusing, but the signal is stored along the longitudinal axis during much of the preparation
period. Signal decay is, therefore, via T
1
relaxation (where usually T
1
T
2
) but signal to
noise (SNR) is reduced by 50% with respect to spin-echo preparation.
Single shot techniques for signal acquisition have become the standard procedure for
diffusion imaging due to the improved SNR per unit time and the elimination of motion
artefacts. An entire set of echoes needed to form an image is collected within a single
acquisition period (25300 ms). High speed fast low angle shot (FLASH) imaging (Lee
and Price 1994, Thomas et al 1998) steady-state free precession (SSFP, also called CE-FAST)
(Le Bihan 1988, Merboldt et al 1989) and high speed stimulated echo (Merboldt et al 1992)
have been implemented. The method of echo-planar imaging (EPI) has become established
as the technique of choice for systems with the appropriate hardware (Turner 1998). Rapid
gradient switching enables the attainment of acquisition times of <100 ms. EPI images are,
however, especially vulnerable to distortion and signal dropout (susceptibility artefact) and to
the misregistration of the fat signal (chemical shift artefact). Various techniques are currently
under development to reduce or eliminate these problems (e.g. see Fischer and Ladebeck 1998).
3. Imaging of perfusion using MRI
3.1. What is perfusion?
This section describes various MRI methods that can be used to measure the parameter known
as perfusion. The term perfusion describes the amount of blood delivered to the capillary beds
of a block of tissue in a certain period of time. Its units are therefore millilitres of blood per
100 g of tissue per minute. It is important to distinguish between perfusion and bulk blood
ow (which occurs along major arteries and veins). Perfusion is blood ow at the capillary
level, and is closely related to the delivery of oxygen and other nutrients to the tissue. It is
this quantity which determines whether the energy status of the tissue is likely to become
compromised. Perfusion is therefore an essential parameter, and for this reason much effort
has been put into its measurement. Two main MRI approaches have been developed: bolus
tracking and arterial spin labelling. This section describes the basic principles behind these
techniques.
3.2. Measurement of perfusion using bolus tracking methods
3.2.1. Introduction to MR bolus tracking using dynamic susceptibility contrast agents.
Paramagnetic contrast agents have been used for the past ten years to obtain information
about different physiological parameters related to CBF, cerebral blood volume (CBV) and the
mean transit time (MTT) of blood through a volume of tissue. This technique, usually referred
to as dynamic susceptibility contrast (DSC) MRI, involves the injection of a bolus of contrast
agent and the rapid measurement of the MRI signal loss due to spin dephasing (i.e., decrease
in T
2
and T
2
) during its fast passage through the tissue (Villringer et al 1988). Although the
vascular space is a small fraction of the total tissue volume (5% in the human brain), the
compartmentalization of contrast agent within the intravascular space leads to a signicant
transient drop in signal. This susceptibility effect extends beyond the vascular space (Gillis
and Koenig 1987, Villringer et al 1988) and, in regions with an intact blood:brain barrier
(BBB), dominates over the more local T
1
relaxation enhancement. Since the transit time of the
bolus through the tissue is only a few seconds, a fast imaging technique is required to obtain
sequential images during the wash-in and wash-out of the contrast material (gure 4). The
choice of the imaging technique depends on many factors, such as region of the brain, regional
coverage, time resolution and hardware specications.
3.2.2. Perfusion model for intravascular MR contrast agents. The model used for perfusion
quantication is based on the principles of tracer kinetics for non-diffusable tracers (Zierler
1962, 1965, Axel 1980), and relies on the assumption that in the presence of an intact BBB, the
contrast material remains intravascular. The concentration C
VOI
(t ) of tracer in a given volume
of interest (VOI) can be described in terms of three functions (Axel 1995, Ostergaard et al
1996b):
1. Transport function, h(t): the probability density function of transit time t through the
VOI following an ideal instantaneous unit bolus injection. This reects the distribution of
transit times through the voxel, which is dependent upon the vascular structure and ow.
Figure 4. DSC-MRI in a 4 year old child 12 hours after a stroke in the left basal ganglia (right side
of the images). Sequential SE echo-planar images during the rst passage of a bolus of contrast
agent (TR = 1.5 s), together with the signal intensity time course (in arbitrary units) for three ROIs
(ROI 1: right basal ganglia, ROI 2: left basal ganglia, ROI 3: peripheral branch of the right MCA).
The top left and bottom right images correspond to t = 12 and 25.5 s, respectively. The images
show the signal intensity decrease associated with the passage of the bolus. Three different periods
can be identied in the time course data: the baseline (before the arrival of the bolus), the rst
passage of the bolus and the recirculation period (in this case, a second smaller peak, more clearly
seen in the arterial region (ROI 3)). Note that the stroke region (ROI 2) shows almost no contrast
agent passage due to the very low CBF of that area.
2. Residue function, R(t): the fraction of injected tracer still present in the VOI at time
t following an ideal instantaneous unit bolus injection at time t = 0. By denition,
R(t ) = (1
_
t
0
h() d), where the integral term represents the fraction that has left the
VOI, and R(t = 0) = 1, i.e., all the tracer is present in the VOI at time t = 0.
3. Arterial input function (AIF), C
a
(t): the concentration of contrast agent entering the VOI
at time t .
The concentration C
VOI
(t ), therefore, can be written in terms of a convolution of the
residue function and the AIF (Ostergaard et al 1996b):
C
VOI
(t ) =
_

k
H
_
F
VOI
(C
a
(t ) R(t )) =
_

k
H
_
F
VOI
_
t
0
C
a
()R(t ) d (3.1)
where F
VOI
is the CBF in the VOI, is the density of brain tissue (needed to provide the correct
ow units) and k
H
= (1 H
art
)/(1 H
cap
) accounts for the difference in haematocrit (H)
between capillaries and large vessels, since only the plasma volume is accessible to the tracer.
This expression can be interpreted by considering the AIF as a superposition of consecutive
ideal boluses C
a
() d injected at time . For each ideal bolus, the concentration still present
in the VOI at time t will be proportional to C
a
()R(t ) d, and the total concentration
C
VOI
(t ) will be given by the sum (or integral) of all these contributions. Therefore, in order to
calculate CBF, equation (3.1) must be deconvolved to isolate F
VOI
R(t ), and the ow obtained
from its value at time t = 0.
As mentioned above, CBV can be also obtained from DSC-MRI data. For an intact BBB,
CBV is proportional to the normalized total amount of tracer,
CBV =
_
k
H
_
_
C
VOI
(t ) dt
_
C
a
(t ) dt
. (3.2)
The normalization to the AIF accounts for the fact that, independent of the CBV, if more tracer
is injected, a greater concentration will reach the VOI.
The third physiological parameter that can be calculated, MTT, is the average time required
for any given particle of tracer to pass through the tissue, following an ideal instantaneous bolus
injection. By using the denition of the transport function, MTT can be written as the ratio of
the rst moment of the transport function to its zeroth moment:
MTT =
_
t h(t ) dt
_
h(t ) dt
. (3.3)
In the classical outow experiment (where the concentration in the venous output, C
OUT
(t ),
is measured rather than the concentration at the VOI), the MTT can be calculated from the
rst moment of the tracer concentration (Axel 1995). However, as pointed out by Weisskoff
et al (1993), this MTT is distinct from the rst moment of C
VOI
(t ). Therefore, calculation
of MTT cannot be performed without solving rst equation (3.3), and the rst moment of
C
VOI
(t ) is just an approximation and depends on the topology of the vasculature. CBF,
CBV and MTT are related through the central volume theorem (Stewart 1894, Meier and
Zierler 1954) by MTT = CBV/F
VOI
. Therefore, once CBF and CBV are known, MTT can
also be calculated directly. Conversely, some studies have used the central volume theorem
to estimate a perfusion index (CBF
i
) from the ratio of the CBV to the rst moment of
the concentrationtime curve (used as an approximation to MTT). However, apart from the
previously mentioned dependency on the underlying vascular structure, this perfusion index
is inuenced by the shape of the bolus, since the rst moment contains contributions fromboth
the MTT and the rst moment of the AIF (Axel 1995).
To use this model with MRI data it is necessary to convert the observed MRsignal intensity
variations to changes in contrast agent concentration. It has been shown, both empirically
(Villringer et al 1988, Rosen et al 1990, Hedehus et al 1997) and using Monte Carlo simulations
(Fisel et al 1991, Weisskoff et al 1994, Boxerman et al 1995, Kennan et al 1994), that the
tracer concentration is approximately proportional to the observed change in the relaxation rate
R
2
= 1/T
2
(or R
2
) in normally perfused tissue. By assuming a single exponential relationship,
the change in relaxation rate (R
2
) can be obtained from the change in signal intensity from
the baseline signal before contrast administration (S
0
),
C
VOI
(t ) =
VOI
R
2
=
VOI
TE
ln
_
S
VOI
(t )
S
0
_
(3.4)
where S
VOI
(t ) is the signal intensity measured in the VOI at time t , and TE is the echo time of
the pulse sequence. The proportionality constant
VOI
depends on the tissue, the contrast agent,
the eld strength and the pulse sequence parameters. An equivalent relationship is assumed
for the concentration of the tracer in the arterial input (with a proportionality constant
art
).
Figure 5. T
2
-weighted turbo spin-echo (TSE) (top left), diffusion-weighted image (top middle),
average ADC map and DSC-MRI data from the same 4 year old child as in gure 4, 12 hours after
a stroke in the left basal ganglia (right side of the images). The images in the bottom row are the
CBF, MTT and CBV maps obtained from the dynamic data shown in gure 4. A subtle region of
hyperintensity can be seen in the TSE image. This ischaemic region is much more clearly seen on
the diffusion and perfusion maps.
There are a number of assumptions for the model described above apart from the already
mentioned intact BBB: rst, that the owis stable during the measurement, and that the contrast
agent is really a tracer (it has no effect on the CBF and has negligible volume itself); second,
that the change in T
1
relaxation is negligible; third, that the recirculation of the tracer (see
gure 4) is negligible or eliminated. This can be achieved, either by truncating the curve, or
by tting a portion of the curve to an assumed bolus shape function, typically a gamma-variate
function (Starmer and Clark 1970, Berninger et al 1981). Finally, since the AIF is estimated
from a major vessel (such as the MCA), the dispersal and delay of the bolus as it reaches the
VOI must not be signicant. This last assumption is likely to be invalid during ischaemia and
can introduce an underestimate of the calculated perfusion.
3.2.3. Quantitative parameters available from DSC-MRI. Although deconvolution methods
allow regional CBF, CBV and MTT to be calculated (as shown in gure 5), quantication
using more simplistic approaches has commonly been used. These different approaches to the
quantication of data obtained using DSC-MRI can be divided into three main categories:
(a) quantication of absolute CBF;
(b) quantication of relative CBF (relCBF) and
(c) quantication using summary parameters, such as time to peak (TTP), bolus arrival time
(BAT), maximum peak concentration (MPC), full width at half maximum (FWHM), peak
area (PA) and rst moment of the peak (C
(1)
VOI
), as well as parameters dened from the
coefcients of the gamma-variate function.
The rst two approaches are very technically demanding since they require an accurate
characterization of the AIF and its deconvolution from the residue function. The AIF depends
not only on the shape of the injected bolus, but also on the cardiac output, the vascular geometry
and the cerebral vascular resistance. The AIF can be estimated by measuring the signal loss
in a region of interest positioned on a feeding cerebral artery, such as the carotid artery or the
middle cerebral artery (MCA) (Porkka et al 1991, Rosen et al 1991, Perman et al 1992), and
it has been shown to be proportional to measurements obtained invasively by arterial blood
sampling in animals (Porkka et al 1991, Rosen et al 1991). Several methods to deconvolve
equation (3.1) have been proposed. Ostergaard et al (1996b) have recently compared the
performance of some of them, both using Monte Carlo simulations and experimental data.
From the methods studied, they concluded the model-independent approach using the singular
value decomposition (SVD) technique was the most accurate, independent of the underlying
vascular structure (R(t )) and volume (CBV).
The use of summary parameters, on the other hand, does not require the deconvolution
of the measured signal and it has been widely used, both in animal and human studies
due to its much simpler and less time-consuming processing. However, there is no simple
relationship between the summary parameters and CBF. They also depend on other factors,
such as CBV, MTT, bolus volume and shape, injection rate and cardiac output. This makes
their interpretation less straightforward, and in general it will depend on assumptions about
the underlying vascular structure (Weisskoff et al 1993, Gobbel et al 1991). Furthermore,
accurate comparisons between subjects, or repeated measurements in follow-up studies, are
not possible. However, when no information about the AIF is accessible, summary parameters
are the only quantitative option and, in many cases, they can be useful in helping to distinguish
between various pathological and physiological situations.
3.3. Measurement of perfusion using arterial spin-labelling methods
3.3.1. Introduction to arterial spin labelling (ASL). The arterial spin-labelling (ASL)
methods for perfusion measurement are based on the fact that the magnetization and relaxation
characteristics of tissue water are affected by the inow of blood water. An MR image can be
made sensitive to CBF if the magnetic state of blood water spins is different to that of the tissue
water spins. In general, two images are acquired in an ASL experiment: one in which blood
and tissue water magnetizations are different (the spin-labelled image) and one in which the
two magnetic states are the same (the control image). Subtraction of the spin-labelled from
the control image results in an image whose intensity is directly related to perfusion. This
section outlines the model used to quantify perfusion and describes the most common pulse
sequences used in ASL techniques.
3.3.2. Tissue model for quantication of perfusion with ASL. Longitudinal relaxation of
tissue water magnetization is classically described by the Bloch equation (Abragam 1961):
dM
z
(t )
dt
=
M
0
z
M
z
(t )
T
1
(3.5)
where M
z
(t ) is the longitudinal magnetization per gram of tissue at time t , M
0
z
is the fully
relaxed value of M
z
(t ) and T
1
is the longitudinal relaxation time. This equation can be extended
to include the effects of ow by the addition of two extra terms:
dM
z
(t )
dt
=
M
0
z
M
z
(t )
T
1
+ f M
a
(t ) f M
v
(t ). (3.6)
The rst term (f M
a
(t )) represents the gain of magnetization by the tissue caused by inow,
and the second term (f M
v
(t )) accounts for the loss of magnetization due to outow (M
a
(t )
is the magnetization of the inowing (arterial) blood per ml, M
v
(t ) is the magnetization of
the outowing (venous) blood per ml and f is blood ow (in ml g
1
s
1
). In a well mixed
compartment, such as brain tissue and microvasculature, the magnetization of the venous
blood is related to that of the brain tissue via the blood:brain partition coefcient of water
(Herscovitch and Raichle 1985), by:
M
v
(t ) =
M
b
(t )
(3.7)
where M
b
(t ) is the longitudinal magnetization per gram of brain tissue water, and so
equation (3.6) can be written as:
dM
b
(t )
dt
=
M
0
b
M
b
(t )
T
1
+ f M
a
(t )
f
M
b
(t ). (3.8)
Therefore, it can be seen that by altering the state of the arterial magnetization M
a
, one can
modify the apparent relaxation and magnetization of the brain tissue water. If the state of
the inowing blood water is known and the change of magnetization of the tissue water is
measured, then solving equation (3.8) for f will enable the calculation of blood ow. In
general, arterial magnetization is labelled in ASL by spin inversion. The different ways in
which this can be achieved constitute the various ASLtechniques, as described in the following
sections.
3.3.3. Continuous arterial spin labelling. The original ASL technique, proposed by Detre
et al (1992, Williams et al 1992), is now known generally as continuous arterial spin labelling
(CASL). In this approach, blood water is continuously inverted as it ows into the brain for a
period of several seconds, allowing a ow-dependent steady state of brain tissue magnetization
to develop (see gure 6(a)). An image is then acquired using a rapid imaging technique such
as echo-planar imaging (EPI). A control image is acquired in which spin labelling of arterial
water is not performed. The difference between these two images can be calculated from
equation (3.8), and leads to the following equation for blood ow (Williams et al 1992):
f =

T
1app
(M
cont
b
M
label
b
)
2M
cont
b
(3.9)
where is the efciency of spin inversion (see next paragraph), M
cont
b
and M
label
b
are the steady
state control and spin-labelled tissue magnetizations respectively, and T
1app
is the longitudinal
relaxation time of brain tissue including the effect of ow i.e.
1
T
1app
=
1
T
1
+
f
. (3.10)
Equation (3.10) is a fundamental equation of ASL, showing how the apparent longitudinal
relaxation time is directly related to perfusion.
Continuous arterial spin inversion is achieved using adiabatic fast passage (AFP). An off-
resonance radio-frequency (RF) pulse is applied in the presence of a magnetic eld gradient in
the slice select direction. As blood ows into the brain, the effective magnetic eld experienced
by the blood water sweeps from being parallel to the main eld to being anti-parallel. As long
as the adiabatic condition (Abragam 1961) is satised, i.e.
1/T
1a
, 1/T
2a
_ (1/B
1
)Gv _ B
1
(3.11)
(where T
1a
, T
2a
are the longitudinal and transverse relaxation times of arterial blood water,
B
1
is the amplitude of the RF pulse, G is the amplitude of the eld gradient, v is the blood
Figure 6. Spin-labelling strategies for the three most common ASL techniques: (a) continuous
ASL, (b) EPISTAR and (c) FAIR. In (a), the dashed line indicates the plane of inversion of the
adiabatic fast passage RF pulse. In (b) and (c), the shaded areas indicate the regions which are
inverted for pulsed spin labelling. The imaging slice is represented by diagonal lines.
velocity and is the magnetogyric ratio), then the magnetization of the owing spins will
remain aligned with the effective magnetic eld, and so will switch from being fully relaxed to
being inverted. In practice, this means that inversion must take place in a major artery where
the blood velocity is sufcient to satisfy the above condition. However, a range of velocities
may be present, some of which may meet the adiabatic condition and others of which may not.
This results in imperfect inversion of arterial blood water magnetization. To account for this
effect, a term known as the inversion efciency () is introduced into equation (3.9) above.
is dened as follows: if all owing water is inverted using an AFP pulse, has a value of 1;
if no inversion occurs, = 0. Values of using AFP in vivo are typically in the region of
0.70.8.
Several additional factors affect the accuracy of a perfusion measurement made using
CASL. The most important of these are:
Magnetization transfer (MT). The application of a long off-resonance RF pulse which
is used to perform the AFP inversion of arterial blood water has a secondary effect on
the tissue magnetization in the slice of interest. Although no direct saturation of MRI-
observable water in the imaging slice occurs due to the pulse being applied several kHz
off-resonance, the magnetization of water bound to macromolecules can be affected, since
the resonance peak of this group of spins is very wide. Magnetization transfer between
free and bound tissue water then causes the observed MRI signal to decrease. For
a more complete discussion of MT see Wolff and Balaban (1989), but sufce it to say
that these effects will cause the expected signal difference between the spin-labelled and
control CASL images to decrease. Proper quantication of perfusion then requires MT
effects to be accounted for (Zhang et al 1992, McLaughlin et al 1997). The most important
consequence of this relates to the acquisition of the control CASL image. In order for the
control image to experience exactly the same MT effects as the spin-labelled image, an
off-resonance RF pulse must be applied prior to its acquisition. This is usually done with
the magnetic eld gradient reversed in polarity, so that the plane of inversion for the
control image is positioned above the head of the subject, and no blood water inversion
results (see gure 6).
Arterial transit times. The equation for the calculation of blood ow (equation (3.9)) was
derived assuming that blood water arrives at the tissue of interest in a state dened by ,
the inversion efciency. However, during the time it takes for the blood to travel from
the point where it is inverted to where it reaches the capillary bed and exchanges into the
tissue, relaxation causes the degree of inversion to decrease. To account for this effect,
one could measure the transit time and incorporate it into the Bloch equation analysis
described above (Zhang et al 1992). However, in most situations, a range of transit times
will exist across an image, and signal-to-noise and time restrictions prevent determination
of transit time on a pixel-by-pixel basis in practical applications of the technique (Ye
et al 1997). In 1996, Alsop and Detre introduced a modication of the standard CASL
technique in which they put a delay between the end of the labelling period and the image
acquisition (Alsop and Detre 1996b). They showed this approach allows quantication
of perfusion without sensitivity to differences in transit times, as long as the delay used
is longer than the longest transit time present and the T
1
-values of blood and the tissue
of interest are approximately the same. Although there is a loss of signal to noise in the
difference images (control labelled) using this technique, the resulting CBF maps are
of higher quality (see gure 7) due to their insensitivity to transit time effects and the
absence of vascular artefacts (see next paragraph).
Vascular signal contamination. The Bloch equation model described above predicts the
behaviour of tissue water magnetization in the presence of ow. In addition to tissue, a
typical MRI voxel also contains blood signal from capillaries and possibly from larger
vessels. The signal in the larger vessels is labelled by the AFP inversion pulse, and so
will potentially contribute to a signal difference between the spin-labelled and control
images. However, if the blood in these vessels does not perfuse tissue in the same voxel,
its contribution to the difference signal is not desired. The blood is merely passing through
the voxel, possibly en route to perfusing tissue further downstream. In order to eliminate
the signal from these larger vessels, ow-sensitive crusher gradients can be used (Detre
et al 1992, Ye et al 1997). Alternatively, the technique of Alsop and Detre described above
(Alsop and Detre 1996b), in which a delay is inserted between the end of the labelling
period and the image acquisition, allows most of the faster moving blood to wash out of
the imaging slice prior to image acquisition. In this way, vascular artefacts are eliminated,
except in regions where extended transit times cause spin-labelled blood to still be present
in the vasculature at the time of image acquisition (such as regions with collateral ow).
Multi-slice CASL imaging. Due to the need for equivalent MT effects in both the
spin-labelled and control CASL images, multi-slice acquisition using this technique is
complicated. Two methods have been proposed to tackle this problem. In one (Silva et al
1995), an RF surface coil with a small spatial range is placed on the neck and used to
Figure 7. Multi-slice perfusion images acquired using continuous ASL with various post-labelling
delays. With short delays, the majority of spin-labelled blood resides in the vasculature (top row).
As the delay increases, spin-labelled blood distributes throughout the brain tissue in a manner
directly related to CBF. Vascular artefacts are greatly reduced at long delay times (lower rows).
(Image kindly provided by Dr David Alsop.)
perform spin labelling. The B
1
eld generated by this coil does not reach the imaging
slices and thus causes no MT effect. In the second approach (Alsop and Detre 1996a,
1998), the control RF pulse is designed to achieve identical MT effects to the inversion
pulse throughout the whole brain. For the exact details of these sequences, the reader is
referred to the relevant reference. While not yet in wide use, these methods hold great
promise for the future if CASL is to become a practical clinical and research tool.
An example of a CASL image acquired using various post-labelling delays is shown in
gure 7. The images show that with a short delay, the difference signal is higher but has a
very non-uniform distribution throughout the brain, consistent with the signal deriving mostly
from arteries and arterioles. As the delay increases, the vascular contamination and transit
time sensitivity are reduced, resulting in more uniform and accurate CBF maps.
3.3.4. Pulsed arterial spin labelling. The difference between CASL and pulsed arterial
spin-labelling (PASL) techniques is the way in which spin labelling is performed. In PASL, a
short (i.e. several millisecond) RF pulse is used to invert the magnetization of all the water in a
region adjacent to the imaging slice(s). Blood in this region then ows into the imaging slice
during an inowtime TI, at the end of which the image is acquired. Asecond image is required
in which owweighting is not applied, and subtraction of the two images results in a difference
image whose signal intensity is proportional to ow. The advantages of PASL over CASL are
that the inversion region can be placed very close to the imaging slice (since the inversion
process does not rely on blood velocity), thus minimizing transit time effects, and that the
magnetization transfer effects are less for short RF pulses compared to long continuous pulses.
The main disadvantage of PASL is a reduced sensitivity to ow, since the inverted blood water
relaxes during the inow time. Several distinct pulse sequences have been developed which
use PASL to measure perfusion:
1. Echo-planar imaging and signal targeting with alternating radio-frequency (EPISTAR)
was the rst PASLsequence to be proposed (Edelman et al 1994) and is shown schematically in
gure 6(b). The general principle of the technique is similar to the CASL approach. Following
saturation of the imaging slice (to reduce unwanted signal from static tissue water), a slab of
water proximal to the imaging slice is inverted and blood fromthis region is allowed to owinto
the imaging slice during TI. As with CASL, the inowof inverted blood water reduces the tissue
magnetization in the imaging slice. A control image with equivalent MT effects is acquired
by either applying the inversion distal to the imaging slice (Edelman et al 1994) or by using a
double inversion pulse to remove the spin label (Edelman and Chen 1998). The difference in
signal between the labelled and control images can be calculated from equation (3.8), and is
given by (Calamante et al 1996, Kwong et al 1995):
M = 2M
0
b
f
_
exp(TI/T
1app
) exp(TI/T
1a
)
1/T
1a
1/T
1app
_
(3.12)
where all parameters are as dened previously. If one assumes the T
1
of blood and tissue to
be the same, = 1 and f/ _ 1, equation (3.12) can be simplied to:
M = 2M
0
b
TI
f
exp
_
TI
T
1
_
. (3.13)
This equation is commonly used to calculate blood ow from PASL measurements. It should
be noted, however, that under certain circumstances (e.g. measuring blood owin white matter,
where the T
1
of tissue and blood are signicantly different) the assumptions made to derive
this equation are invalid and the full equation (3.12) must be used for accurate quantication of
perfusion. It is also interesting to compare equation (3.13) with equation (3.9), which shows
that the expected signal difference for PASL is a factor of approximately [exp(TI/T
1
)] less
than CASL under ideal conditions.
2. Flow-sensitive alternating inversion recovery (FAIR) (Kwong et al 1995, Kim 1995)
is the second of the two main PASL techniques. It is based on the acquisition of a pair of
inversion recovery images (see gure 6(c)). The rst image is acquired following a slice-
selective inversion pulse, and the second image is acquired following a global inversion. In
both cases the inowtime TI is the same, and so the signal fromstatic tissue in the imaging slice
is the same. The difference between the images comes from the difference in magnetization
of inowing blood in the two acquisitions: in the slice-selective inversion recovery (ssIR)
image, inowing blood is fully relaxed and so increases the apparent relaxation rate of the
tissue water; in the global (non-selective) inversion recovery (nsIR) image, inowing blood
is initially inverted and subsequently relaxing during TI and so affects the relaxation rate of
the tissue water less. In fact, the signal difference due to perfusion in FAIR follows exactly
the same behaviour as that of EPISTAR (equations (3.12) and (3.13)), despite the raw image
contrast being very different (since EPISTAR raw images are acquired after a saturation and
FAIR raw images are acquired following an inversion). The reason for this is that the signal
difference derives from exactly the same source: inowing blood being fully inverted in one
case and fully relaxed in the other. This point is illustrated in gure 8, which shows the raw
spin-labelled images for EPISTAR and FAIR at several inow times and the corresponding
subtraction images. The subtraction images are very similar for the two techniques.
Figure 8. Raw images and subtraction images (control labelled) for EPISTAR and FAIR as
a function of inversion time TI. (a) EPISTAR raw images, (b) FAIR raw images, (c) EPISTAR
subtraction images and (d) FAIR subtraction images. TI values from left to right: 200, 400, 600,
800, 1000 and 1200 ms. While the static tissue contrast is very different, the subtraction images
are dependent only on the inow of spin-labelled blood, and so are very similar. (Image kindly
provided by Dr Eric C Wong, reprinted with permission.)
In order to properly quantify blood ow using FAIR, it is necessary to acquire images
at a range of inow times and t the signal differences to the biexponential curve dened by
equation (3.12). There are a total of ve unknowns in this equation (, M
0
b
, f , T
1app
and
T
1a
). Three of these (, M
0
b
and T
1app
) can be obtained by tting the ssIR data to a standard
inversion recovery monoexponential curve (M
ssIR
= M
0
b
(1 2 exp(TI/T
1app
))]). Fitting
the biexponential curve of equation (3.12) for f and T
1a
(or preferably just f if T
1a
is known
or measured separately) will then yield a value for CBF. Using this method, it is also possible
to estimate transit time effects (Yang et al 1998); however, the total time for a single CBF
measurement is rather long (approximately 30 minutes).
The main limitations of PASL techniques are similar to those of CASL, though the extent
of their effect may be different:
Transit time effects are reduced in PASL compared to CASL since the edge of the labelling
region is generally closer to the imaging slice in PASL. This means that the physical
distance that spins have to travel is less, and ideally is very short indeed. The main
limiting factor for the proximity of the labelled region and imaging slice is the shape of
the respective pulse proles. Usually, adiabatic hyperbolic secant inversion pulses (Silver
et al 1984) are used in PASL, which produce inversions with quite sharply dened edges.
However, the distance between the edge of the labelled region and the imaging slice is
typically of the order of the imaging slice thickness itself (several millimetres). This
compares typically to several centimetres in CASL. Nevertheless, transit times can cause
problems for CBF quantication in PASL, and methods have been proposed to reduce the
sensitivity to these effects, either by pulse sequence manipulation (Wong et al 1998) or
by the use of sharper inversion pulses (FOCI pulses; see Ordidge et al 1996 and Yongbi
et al 1999).
Vascular contribution to the perfusion signal is not accounted for in the PASLmodel (in the
same way that it is not included in the CASL model). If an inow time of sufcient length
is used, it is reasonable to assume that all spin-labelled blood will either have exchanged
with tissue or washed through the imaging slice. However, to properly quantify CBF,
PASL images are needed at a range of inversion times. Motion-sensitive crusher gradients
can be used to destroy signal from water which is moving rapidly, though the choice of
amplitude of these gradient is rather arbitrary. This is a problem which has yet to be
properly resolved.
Multi-slice PASL can be performed fairly easily for both EPISTAR (in which several
images are acquired progressively further from the inversion region) and FAIR (in which
the slice-selective inversion is widened to accommodate extra imaging slices). However,
this causes problems by introducing a range of transit times for the different slices. Transit-
time-insensitive techniques are therefore preferable for multi-slice imaging, though these
are still under development and subject to certain limitations (see Wong et al 1997).
3.4. Advantages and disadvantages of MR perfusion techniques
Although neither ASL nor DSC-MRI require ionizing radiation, ASL has the extra benet
of using an endogenous contrast agent. This allows multiple measurements to be performed
without any restriction. However, a drawback of ASL is that the intrinsic perfusion signal (i.e.
the difference between the spin-labelled and control images) is generally only a few per cent
of the control image intensity, requiring good signal to noise for an accurate measurement.
In DSC, signal intensity changes of the order of tens of per cent are observed at normal CBF
levels.
Both techniques behave reasonably well in the range of normal CBF values, although they
have difculties when dealing with very high or very lowow. In the limit of very lowow, the
rst obvious limitation is the low SNR of the measurement. This can be improved in the ASL
technique by increasing the number of averages, which is not an option in the typical single
bolus DSCstudies, due to its dynamic character. Afurther problemwhen measuring lowows
is related to the presence of long arrival delays for the arterial blood to the VOI, such as those
resulting from collateral circulation in stroke regions. Although the latest ASL sequences
proposed (Alsop and Detre 1996b, Wong et al 1998) are less sensitive to differences in transit
time (provided the appropriate sequence parameters are chosen), very long transit times may
make use of the required parameters impractical. In PASL, the transit time insensitivity can
be further improved by slice prole optimization (Pell et al 1998). Another possibility is to
take account of the different transit times, by measuring them (Zhang et al 1992, Ye et al
1997), or by using the bolus arrival time information from a DSC experiment. However, this
problem is one of the fundamental limitations of the ASL technique for measuring CBF in the
presence of very long transit time delays. In the case of a very long delay, it may be impossible
to have tagged signal arriving at the tissue and, therefore, no effect will be observed. The
presence of very long bolus arrival times is also a problem in DSC studies. The transit of
the bolus from the site of injection (typically a vein) to the VOI can introduce a delay and
dispersion (spread of the bolus shape). If these are not accounted for, they may cause an
overestimation of MTT and underestimation of CBF (Ostergaard et al 1996a, 1998, 1999,
Schreiber et al 1998). While one can account for the dispersion from the site of injection
(vein) to the artery where the AIF is measured, the extra dispersion from the artery to the input
of the VOI (where the true AIF should be obtained) cannot be easily ascertained. This extra
dispersion is particularly signicant in cases of collateral ow to ischaemic areas. To address
this problem, Ostergaard et al (1999) have recently incorporated a mathematical vascular
model into the DSC analysis. They show that the vascular model approach is less sensitive
to vascular delay and dispersion than the conventional deconvolution approach. Their results
are very promising, but further validation on a large number of patients with cerebrovascular
diseases is needed.
The case of very high ows can also be problematic for both MRI perfusion techniques.
For ASL, the assumption of complete exchange may become invalid (Silva et al 1997a,b), and
if this is not taken into account the perfusion will be underestimated (since only a fraction
of the arterial magnetization is exchanged). In the DSC method, very high ow will cause
narrowing of the R
2
response curve, making it potentially difcult to accurately characterize
for a given time resolution. However, in practice this does not seem to be a source of difculty,
since dispersion fromthe site of injection causes the bolus to be quite widely spread even when
CBF is high.
Both techniques can be used in a multi-slice protocol, although the maximum number of
slices is limited in each case. In DSC-MRI, the coverage is given by a compromise between
the number of slices and the time resolution (for typical repetition time values, a coverage of
1015 slices can be achieved using EPI). For the case of ASL, transit times limit the maximum
number of slices (typically between ve and ten slices can be obtained, depending on the
spatial resolution). Another possibility for whole brain coverage is the use of 3D acquisition
methods (Petrella et al 1997, Alsop and Detre 1999), though this tends to reduce the spatial
resolution.
3.5. Other techniques for measuring perfusion-related parameters
In addition to the techniques which have been designed specically to obtain quantitative
measures of CBF, several other approaches have been developed in parallel which derive
their contrast from parameters closely related to perfusion. These approaches will be briey
summarized here, though for a full description of these methods, the reader is referred to the
relevant literature.
3.5.1. Blood-oxygen-level-dependent (BOLD) imaging. Blood-oxygenation-level-
dependent (BOLD) MRI is based on the magnetic properties of blood, which are dependent
on the oxygenation state of haemoglobin (Thulborn et al 1982, Ogawa et al 1990). Because
deoxygenated haemoglobin is more paramagnetic than oxygenated blood and normal tissue,
it can act as an endogenous intravascular paramagnetic contrast agent. Deoxygenation
results in an increased magnetic susceptibility difference (due to an increase of paramagnetic
deoxyhaemoglobin) in and around the vascular compartment, and thereby is expected to cause
signal loss in both T
2
- and T
2
-weighted images. This phenomenon is now widely used for
MR functional neuroimaging (fMRI), in which local increases of image intensity have been
observed in association with specic brain activation tasks (see section 4.2.2). However,
the most elementary analysis of the biophysical and haemodynamic events relating to brain
activation lead to the conclusion that many factors can affect the MRsignal in such studies. The
list includes blood volume, blood ow, arterial and venous haemoglobin saturation, oxygen
extraction rate, blood viscosity and haematocrit. This makes direct interpretation of BOLD
images very difcult. For an extensive review of the BOLD technique, see Ogawa et al
(1998).
3.5.2. Steady-state contrast agents for measurement of blood volume. The duration of the
effect of the contrast agents used in DSC-MRI is limited to the time taken for the bolus
to pass through the vasculature of the organ of interest; recirculation of the tracer after the
rst pass is minimal. However, certain intravascular contrast agents, such as ultrasmall
superparamagnetic iron oxide (USPIO), have longer blood half-lives, since they are only slowly
eliminated from the blood pool (Weissleder et al 1990). As a result, it is possible to estimate
dynamic changes in blood volume by measuring changes in R
2
and R
2
of the tissue, which
are directly related to the blood volume containing the contrast agent. The paramagnetism
of USPIO completely overwhelms any effects due to changes in the levels or concentration
of deoxyhaemoglobin, thus simplifying the analysis of the signal intensity changes. Under
certain circumstances CBV and CBF are very closely correlated, and so using this method it is
possible to estimate CBF behaviour. However, this is not generally the case, and measurement
of CBV alone can usually only provide a limited amount of information regarding perfusion
status.
3.5.3. Intra-voxel incoherent motion (IVIM). The structure of the microvasculature of the
brain is such that, on the scale of a typical imaging voxel, the ow of blood through capillaries
can be viewed as incoherent, pseudo-random motion. Each voxel contains typically several
thousand capillaries, all of which are made up of multiple straight segments oriented at many
different angles (Le Bihan 1995b). Blood owing through the capillaries can thus be modelled
as random, diffusion-like motion with an effective diffusion coefcient D
. The value of D
will dependonfactors suchas the vascular geometryandthe rate of perfusion, andwill generally
be much higher than the effective diffusion coefcient of tissue water. The application of
diffusion-sensitizing gradients (as described in section 2.2) will cause the signal intensity from
a voxel to decay with biexponential characteristics: a rapid initial decrease as the microvascular
signal is eliminated at relatively low b-values, followed by a more gradual decline which is
determined by the tissue ADC. Quantication of perfusion based on measurement of the low
b-value behaviour of signal intensity has been attempted, but dynamic range problems (due to
low CBV compared to tissue volume) and uncertainty of a correct vascular model have limited
the usefulness of this method (see King et al 1992 and Pekar et al 1992).
4. Applications of diffusion and perfusion imaging
4.1. Diffusion-weighted imaging in biological systems
Diffusion-weighted imaging (DWI) (see section 2) and measurements of the apparent diffusion
coefcient (ADC) (see section 2.3.2) of water have been applied to various organs in the body,
including brain, liver, pancreas, uterus, breast, bone and muscle. This holistic applicability
has meant that a diversity of conditions and systems may be studied in both research and
clinical environments. Examples of such conditions include neurological disease (see below);
brain myelination; liver cirrhosis or carcinomas; pancreatic cysts or tumours; bone marrow
imaging for compression fractures; osteogenic sarcomas; musculoskeletal neoplasms; breast
pathologies; uterus anisotropy and hyaline cartilage deterioration.
Due to the sensitivity of the diffusion-weighted images to motion, it is essential that the
organ or systemof interest remains stationary, or that the imaging technique used is insensitive
to any gross movement. Because the brain is situated within the connes of the skull, and
therefore suffers less motion than organs of the chest or abdomen, it is well suited to the DWI
and has therefore been the focus of attention for many years (Hoehn-Berlage 1995). There
are reports of DWI changes in numerous neurological conditions such as stroke, subarachnoid
haemorrhage, Alzheimers disease, AIDS, epilepsy, head injury and brain tumours. Despite
the applicability of DWI to a range of neurological conditions the main area of interest has
been, and continues to be, in the eld of stroke research (Moseley et al 1990, Baird and Warach
1998).
In the following section we discuss why DWI has become so predominant in the
investigation of stroke. We will also demonstrate that DWI provides not only the opportunity
to identify the very early changes following a stroke, but also the opportunity to investigate the
underlying pathophysiology and treatment of this condition (Moseley et al 1990b, Van Bruggen
et al 1994). Further we examine DWI in the context of the mechanisms of change during
cerebral ischaemia; CBF thresholds; metabolic correlations; temporal evolution of diffusion
change in experimental and human stroke and reperfusion.
4.1.1. Diffusion-weighted imaging: why the focus on stroke? Ischaemic stroke, which is
commonly caused by thrombotic or embolic occlusion of a cerebral artery resulting in tissue
damage, is the most common neurological disorder causing death or disability among adults
living in industrialized nations. Stroke is a devastating condition and ranks third as a cause of
death, surpassed only by heart disease and cancer (Mas and Zuber 1991). In any given year in
Britain, 250 000 people suffer from some form of stroke, and in 1990, the Ofce of Population
Census and Surveys for England and Wales reported a mortality of approximately 15 000 men
and 26 000 women (Warlow1998). In the United States, it is estimated that annually, that there
are as many as 550 000 hospitalizations and 150 000 deaths, and the average cost of treatment
per person for the rst stroke is estimated to be $103 000. The total cost of stroke for 1990 in
the USAwas $40.6 billion (Taylor et al 1998). Despite substantial declines in incidents during
the last two decades (Mas and Zuber 1991), stroke is still a leading neurological reason for
hospitalization and one of the most common causes of long term disability. From the above
statistics we can see that stroke is a signicant problem, and for the clinician investigating and
treating a stroke patient, an accurate diagnosis together with a technique to assess the effect of
therapy is essential. Before the advent of DWI, stroke patients would undergo a computerized
tomography (CT) scan or standard MRI investigation. However, ischaemic changes in the CT
scan or standard magnetic resonance images were not observed until approximately 6 hours
following the initial stroke event. In 1991, introduction of DWI allowed the identication of
a stroke minutes following the initial event, with the opportunity to monitor therapy and aid
in a prognosis for the patient (Baird and Warach 1998); the reasons and interpretation for the
change in water diffusion are discussed below.
4.1.2. Mechanisms of DWI changes following a stroke. Moseley et al observed the rst DWI
changes in cerebral ischaemia and attributed this ADC reduction of tissue water diffusion to
an osmotically obliged shift of extracellular water to intracellular compartments, as a result
of a disruption of ion homeostasis and formation of cytotoxic oedema (Moseley et al 1990b).
This occurs due to the following chain of events: a decreased blood supply following a stroke
reduces the amount of oxygen and glucose delivered to the tissue, and this is accompanied by
a decrease in the available adenosine triphosphate (ATP), the main source of cellular energy.
ATP normally provides energy to pump Na
+
out of and K
+
into the cell to maintain ionic
homeostasis. When the supply of ATP ceases, there is an accumulation of intracellular Na
+
;
this causes an inux of osmotically obliged water, which leads to cell swelling (cytotoxic
oedema) and, in time, cell death.
Later work by Benveniste et al supported Moseleys early hypothesis, with studies in which
the ADC decreased during induction of cytotoxic oedema by ouabain (a specic inhibitor of
Na
+
, K
+
-ATPase) administration (Benveniste et al 1992). It was suggested that the reduction
of ADC is indeed caused by an inux of extracellular water, which has a high intrinsic
diffusion, to intracellular compartments, where the water diffusion would be retarded due
to various intracellular obstacles and the relatively high cytoplasmic viscosity. Consequently,
it is now accepted that an ischaemia-induced drop of the tissue water ADC is associated with
the development of cytotoxic oedema.
Despite this work, the exact biophysical mechanisms of water ADC reduction remain
unclear. Several further hypotheses have been suggested to account for the mechanisms
underlying the ADCchanges in ischaemia. Latour et al have suggested, for example, that DWI
changes are caused by a reduction in extracellular diffusion due to an increase in the tortuosity
of the extracellular space that occurs after cell swelling (see section 2.3.3) (Latour et al 1994,
Hasegawa et al 1996). Alternatively, Helpern et al suggested that a reduction in cell membrane
permeability is a possible cause (Helpern et al 1992), whereas DWI of intracellular metabolites
and compartment specic markers indicate that a decrease in intracellular diffusivity might
be involved (van der Toorn et al 1994, Duong et al 1998). This reduction in intracellular
diffusivity may be due to a decrease in the intracellular circulation or an increase in viscosity
(Duong et al 1998).
Afurther parameter that may be calculated fromdiffusion-weighted images is that of tissue
anisotropy (see section 2.3.4). Anisotropy gives an indication of the directional dependence
of water movement and therefore the connes of the biophysical environment, which may be
expected to change under such conditions as stroke. This indeed is the case, and following
a stroke it is not only the ADC that changes but also the tissue anisotropy. Although both
grey and white matter are anisotropic (Lythgoe et al 1997), it is a change in the anisotropy
of the cerebral white matter that is notable during the rst 24 hours following an ischaemic
event (Sorensen et al 1999b), with changes in grey matter at later times (Zelaya et al 1999).
This information provides a novel technique to probe the changes in cellular structure due to
a reduction in cerebral blood ow.
4.1.3. Relation of diffusion-weighted imaging to cerebral blood ow. In 1992, using a gerbil
model of forebrain ischaemia, Busza et al found that DWI signal enhancement only occurred
when the CBF fell below 20 ml/100 g min
1
(Busza et al 1992). This is similar to the ow
threshold for the maintenance of tissue high-energy metabolites necessary for normal cellular
ion homeostasis (Crockard et al 1987, Allen et al 1988). These results differ from those
achieved for the CBF threshold in a rat model of focal ischaemia, which is approximately
35 ml/100 g min
1
(Perez Trepichio et al 1995, Kohno et al 1995). This discrepancy may be
due to the differences in species, properties of global and focal ischaemic lesions or technique
for the measurement of CBF. Further work, using a middle cerebral artery occlusion (MCAO)
rat model, has shown a time-dependent increase in CBF threshold from 34 ml/100 g min
1
(30 minutes) to 41 ml/100 g min
1
(120 minutes) over a 4 hour period, indicating the growing
sensitivity of the tissue to perfusion decits (Kohno et al 1995). This work was conrmed by
Mancuso et al who demonstrated that after 30 minutes the area occupied by more than 15%
reduction in ADCcorresponded to CBF levels below25 ml/100 g min
1
, and after 90 minutes
this threshold had increased to 3035 ml/100 g min
1
(Mancuso et al 1995). In summary the
CBF must be reduced to a certain level to induce an ADC decrease, although this threshold is
dependent upon species and duration of the reduced CBF.
4.1.4. Relationship of DWI to metabolic alterations. Mintorovitch et al correlated DWI signal
change during ischaemia, with tissue water content, ATPase activity, and electrolyte content
(Mintorovitch et al 1994). Within 30 minutes of ischaemia, DWI changes had occurred with
a concomitant decrease of 30% to 40% in ATPase activity, while tissue water content and
electrolyte concentrations were normal. By 60 minutes the ATPase activity had decreased
further, the water and sodium content had increased and the potassium concentration was
lowered. These data indicate that DWI monitors very early disturbances of cellular ion
pumps, at times when such changes are thought to be potentially reversible. Autoradiographic
techniques for measuring CBF, ATP, glucose, lactate and pHhave been used to study the spatial
relationship between DWI changes and metabolism following MCAO in the rat (Kohno et al
1995). In contrast to changes observed in global ischaemia, in which the CBF thresholds for
changes in DWI and high-energy metabolites are similar (Busza et al 1992, Allen et al 1988),
Kohno et al reported that during the early phase of focal cerebral ischaemia (30 minutes) the
area of hyperintensity seen on DWI was signicantly larger than the region of ATP depletion,
although it matched the area exhibiting tissue acidosis (Kohno et al 1995). This difference
became progressively smaller with the evolution of the lesion, such that by 7 hours the area
of tissue damage, as indicated by DWI, was identical to the region of ATP depletion and
histological infarction. If DWI changes result from an alteration in compartmentation of
tissue water, then the latter observations indicate that water redistribution occurs prior to the
loss of high energy metabolites. These observations do not necessarily contradict the former
statements, since the anaerobic production of metabolites, including lactate, can also give rise
to redistribution of water, which might be expected to result in DWI changes. Consistent
with this is a good agreement between lactate production and DWI change (Decanniere et al
1995), and further that DWI changes precede anoxic depolarization following cardiac arrest
in experimental studies (de Crespigny et al 1999). The understanding that DWI indicates
acute changes in the cellular dynamics is further emphasized in recent work using a piglet
model of secondary energy failure, in which a gradual ADC decline is observed following
a hypoxicischaemic insult. Here a close relationship between a decrease in ADC and high
energy phosphates was observed, while a nonlinear relationship to decrease in the nuclide
triphosphate pool (Thornton et al 1998).
As mentioned previously, several studies have shown that cytotoxic-oedema-related
changes in brain tissue water ADC are paralleled by lactate accumulation, decrease in high
energy phosphates and intracellular pH, and an increase in inorganic phosphate. However,
a recent study showed that administration of the neurotoxin N-methyl-D-aspartate (NMDA)
results in a ADC reduction, which is interesting as the NMDA-mediated excitotoxicity neither
leads to profound energy failure nor to a signicant lactate accumulation, but does promote
depolarization, ion movements and cell swelling. Therefore, the metabolic basis underlying
the acute reduction in brain water ADCis different in an excitotoxic insult compared to cerebral
ischaemia (Dijkhuizen et al 1996).
4.1.5. Monitoring ischaemic lesion development in animals and humans. It was rst
demonstrated, by the use of DWI, that following occlusion of a cerebral artery, the resultant
ischaemic lesion in both rat and cat expands primarily during the rst 2 hours after MCAO
(Roussel et al 1994, Hoehn-Berlage et al 1995b). Although the lesion size has nearly fully
evolved at 2 hours, the ADC value continues to decrease for a period of up to 46 hours
following MCAO (40% of control) (Knight et al 1994), with some studies indicating lowest
ADC values at 24 to 48 hours (4050% of control) (Hoehn-Berlage et al 1995a; Kohno et al
1995). Inthe chronic stages of cerebral ischaemia, the ADCof water exhibits a different pattern.
At approximately 2448 hours after a vessel occlusion, the ADC rises and slowly returns to
normal at 3 days (Knight et al 1991, Helpern et al 1993). Following this, a subsequent
increase in the diffusion of water above the ischaemic control can be observed after 1 week
(Verheul et al 1992, Knight et al 1994). The elevated ADC of tissue water, above control
values, is associated with cellular lysis, the loss of cellular barriers, combined with excessive
accumulation of oedematous water (Pierpaoli et al 1993).
Following a stroke in man, a reduced ADChas been observed as early as 105 minutes after
the initial onset (Warach et al 1992) and as with the animal studies, a pseudo-normalization
occurs at between 4 and 10 days (Baird and Warach 1998). The large variation in human results
may depend on the both the site and size of the lesion and depth of ischaemia, together with
method for the calculation of ADCwith respect to diffusion anisotropy (see section 2.3.4). For
diagnostic purposes, temporal evolution of ADC following a stroke provides information to
discriminate between acute and chronic lesion, based on the low or high ADC value, provided
the insult is imaged at the appropriate time (Baird and Warach 1998).
4.1.6. Recirculation following cerebral ischaemia. There are four ADC patterns that occur
on CBF recirculation subsequent to an ischaemic period:
the ADC remains decreased at the ischaemic level (Busza et al 1992)
the ADC normalizes and remains normal (Mintorovitch et al 1991)
the ADCnormalizes followed by a secondary decline at approximately 24 hours (Thornton
et al 1997)
the ADC normalizes for a short period, then gradually declines (Pell et al 1999a, Lythgoe
et al 1999).
In 1991 Mintorovitch et al rst demonstrated that the hyperintense regions of the ischaemic
brain seen on DWI could be reversed if the occlusion was removed 33 minutes after the MCAO
in the rat (Mintorovitch et al 1991). More recently it was shown that the size of the lesion based
on the diffusion-weighted images declined if the owwas restored 1 hour following occlusion,
but not after 2 hours (Minematsu et al 1992). Even a 15 minute period of cardiac arrest in cats,
which resulted in a decline of water ADC values, subsequently normalized within 30 minutes
of successful cardiac resuscitation (Fisher et al 1995). At this point in time, DWI furnished
the observer with information about regions of reversibility within the area of DWI change,
provided that time of ischaemia was considered. In the quest for better prognostic information,
the DWI data were further rened using quantitative measures of the ADC of water. ADC
thresholds for reversibility of the lesion in a rat MCAO model have been estimated to be
either less than 0.55 10
3
mm
2
s
1
(Dardzinski et al 1993) or a reduction of greater than
0.25 10
3
mm
2
s
1
relative to the contralateral hemisphere (Hasegawa et al 1994). During
MCAO in cats, lesions were reversible if reperfusion occurred before the ADC decreased to
less than 70% of the control. These data may point to a prognostic role for the measurement
of ADC values, although the temporal component cannot be ignored.
After reperfusion of a previously ischaemic region, the low ADC values can return
to normal levels. However some studies are now reporting a secondary decline in
ADC, which, as yet, is little understood. The secondary decline had been termed
secondary or delayed energy failure and initial reports using MRS in infants suffering
an hypoxic/ischaemic insult during birth (birth asphyxia) demonstrated a delayed fall in
[PCr]/[Pi] (phosphocreatine/inorganic phosphate) at 2448 hours (Azzopardi et al 1989). This
hypoxicischaemic condition has been recently modelled in piglets and a secondary decline
has also been observed in ADC values (Thornton et al 1997). This effect is not limited
to a hypoxic/ischaemic insult, since a delayed ADC decrease, following normalization, has
been observed at 24 hours post-reperfusion in a rat MCAO model (Van Bruggen et al 1998).
In another rat model, Dijkhuizen et al using 20 minutes of hypoxic/ischaemic insult in adult
rats, demonstrated secondary reductions of ADC at 24 hours (Dijkhuizen et al 1998). This
delayed effect has also been conrmed in the immature rat and may be suggestive of delayed
neuronal death (Tuor et al 1998). The underlying reasons for this process are still unclear.
However, hypoperfusion following reperfusion produces gradual changes in ADCwhich could
result froma mismatch between CBF and metabolic rate in the hypermetabolic post-ischaemic
brain, giving rise to a delayed ADC decrease (Pell et al 1999a). The nal pattern is somewhat
unusual, that is, following reperfusion the ADC normalizes for a short period then gradually
declines over a prolonged period of several hours (Pell et al 1999a, Lythgoe et al 1999).
Interestingly it is these regions which were previously thought to undergo delayed cell damage
(tissue damage that occurs days after the initial event) (Crain et al 1988, Pulsinelli et al
1982). As yet there are no clear explanations for the CBF changes and suggestions for the
increased vascular tone include abnormality of the synthesis or degradation of nitric oxide
within the endothelium, or adhesion of platelets or leukocytes to the vascular wall (Hossmann
1997).
4.2. Perfusion imaging
Over recent years, the methods for measuring perfusion using MRI (see section 3) have been
used in an increasing number of applications. This section outlines the use of perfusion imaging
in both the research and clinical environments for the detection of blood ow changes during
ischaemia and subsequent reperfusion, together with a comparison of the information gained
from DWI.
4.2.1. MRI perfusion measurements in ischaemia. While DWI may provide unique
information about the effect of an ischaemic insult as early as a few minutes post-ictus, it is
clearly desirable to obtain information regarding the integrity of the vascular bed. Pioneering
studies in a primate model, showed that a reduction of CBF below 12 ml/100 g min
1
for
>2 hours produces focal infarction or non-recoverable tissue, demonstrating a link between
degree and duration of ischaemia and eventual tissue injury (Morawetz et al 1974). Prompted
by this work, MRI techniques have been developed to measure CBF and its related parameters
(see section 3), which are nowintegrated into both the experimental and clinical environments.
Early DSC-MRI experiments in a rat model of focal cerebral ischaemia, demonstrated an
absence of contrast agent in the ischaemic core of the lesion (Quast et al 1993). Interestingly,
in the periphery of the lesion, a diffusionperfusion mismatch was observed, that is, a decrease
in CBF without a change in diffusion. This effect was also noted in a similar study using
DWI and DSC-MRI in a unilateral MCAO cat model, in which perfusion decits not great
enough to cause energy failure may go unnoticed on DWI (Roberts et al 1993). The sensitivity
of perfusion measurements to minor alterations in blood ow is further illustrated in a cat
model of hypoperfusion (Derugin and Roberts 1994). Here, the diffusion-weighted images
showno evidence of abnormality in the territory of the occluded MCA, whereas the DSC-MRI
blood ow measurements show decreased CBF throughout the MCA region. This early work
using DSC-MRI has been followed by studies using the non-invasive ASL techniques for
the quantitation of CBF in animal models. The combined data of perfusion and diffusion,
allows three tissue signatures to be assigned following an ischaemic insult: normal perfusion
and diffusion, denoting a region of normal tissue; tissue that has a CBF decrease without
diffusion changes corresponding to a region of hypoperfusion; and a region in which there
is a concomitant diffusion and perfusion decrease, corresponding to a severely compromised
region (Calamante et al 1999) (see gure 9).
Not only has DSC-MRI been useful to monitor CBF changes during ischaemia, but may
also be applied to monitor the effects of reperfusion following the initial ischaemic insult.
Figure 9. Typical maps obtained from the rat brain following middle cerebral artery occlusion
(MCAO). (a) Continuous ASL perfusion map showing reduced ow in the occluded (left)
hemisphere, acquired 4 hours post-occlusion. (b) ADC map, also acquired 4 hours post-
occlusion, showing the region of reduced diffusion which corresponds to the area of cytotoxic
oedema. (c) Schematic representation of the rat brain after MCAO, highlighting three main areas:
unaffected area; moderately affected area, with reduced CBF but normal ADC, and severely
affected area, in which both CBF and ADC are signicantly reduced.
Three patterns of reperfusion have been noted in a cat model following MCAO: complete
reperfusion (normal TTP, see section 3.2.3); initial hyperaemia (shortened TTP) and persistent
hypoperfusion (prolonged TTP). These three CBF patterns have now been quantied using
the non-invasive MRI technique of pulsed ASL (Pell et al 1999b) (see gure 10). In addition,
in studies of transient ischaemia or hypoxic ischaemia, DSC-MRI has been used to detect the
change in CBV during and after the insult (DArceuil et al 1998). These data illustrate that
CBF measured with either contrast agent or arterial spin labelling techniques, can explicitly
measure responses of various haemodynamic parameters under conditions of ischaemia and
reperfusion, and provided additional information to that of DWI alone.
In addition to animal models of cerebral ischaemia, DSC-MRI and ASL techniques have
now been applied to the study of cerebral perfusion in humans. In patients studied with
acute stroke, enlargement of the ischaemic lesion occurred when there was a larger perfusion
decit (measured with relative MTT) but not when the perfusion abnormality was equivalent
to or smaller than the DWI lesion (Baird and Warach 1998). In addition to the MTT and
DWI volumes, Sorensen et al have studied the CBF and CBV perfusion parameters in acute
stroke lesions. In 19 cases studied within 10 hours of onset, the volume of MTT abnormality
(mean = 119 cm
3
) and CBF abnormality (mean = 112 cm
3
) were larger than the mean CBV
(47 cm
3
) and the mean DWI volume (35 cm
3
). The nal infarct volume (mean 67 cm
3
) was
between the DWI/CBV and the MTT/CBF volumes (Sorensen et al 1997). This indicates that
the CBF images may reveal abnormalities not shown on the blood volume maps, although
the lesion areas from the CBV maps correlate better with nal infarct regions (Sorensen et al
1999a). These data indicated the importance of measuring multiple parameters, which enhance
predictive models and also raise the possibility of using time-dependent therapies to reduce
infarction during the investigation of stroke.
Figure 10. Non-invasive MRmeasurement of CBFusinga pulsedASLtechnique (FAIR), following
ve minutes of forebrain ischaemia in the gerbil brain. The time courses are taken from regions
of interest in the left and right cerebral cortex. Quantitative CBF measurements demonstrate an
initial rise following reperfusion, with subsequent prolonged hypoperfusion. MRI offers the only
methods to obtain non-invasive longitudinal time course data in individual subjects.
4.2.2. Blood-oxygenation-level-dependent MRI: T
2
and T
2
in ischaemia. Since BOLD MRI
is sensitive to changes in regional tissue oxygenation status (see section 3.5.1) (Ogawa et al
1998, van Zijl et al 1998), it can be used to monitor acute deoxygenation following induction
of ischaemia as well as reoxygenation after reperfusion (de Crespigny et al 1992, Roussel
et al 1995). BOLD MR signal intensity, measured by T
2
-weighted MRI, drops immediately
upon the onset of ischaemia, and rises when reow occurs. A transient overshoot in signal
intensity during reperfusion has been described and may reect post-reperfusion hyperaemia
(de Crespigny et al 1992). These haemodynamic responses indirectly report on local changes
in CBF, CBV and oxygen extraction fraction and their individual contributions cannot easily
be distinguished. More recently, early changes in T
2
-values have been reported in conditions
of ischaemia (Busza et al 1994, Grohn et al 1998, Calamante et al 1999) and oligaemia (Grohn
et al 1998, Calamante et al 1999). In the latter studies of cerebral ischaemia, two patterns
were observed following an initial T
2
decrease:
1. T
2
-values remained depressed throughout the study without an ADC changeindicating
a mild hypoperfusion condition (Grohn et al 1998, Calamante et al 1999).
2. T
2
- and ADCvalues are decreased throughout the study, indicating a severe hypoperfusion
condition (Roussel et al 1995, Calamante et al 1999).
The combination of T
2
or T
2
and ADC measurements may provide MRI tissue signatures
for the status of the tissue. These data highlight that changes in T
2
are not always
related to vasogenic oedema and early changes in T
2
may provide information as to the
pathophysiological nature of ischaemia or oligaemia.
4.2.3. Functional magnetic resonance imaging (fMRI). Since the introduction of functional
magnetic resonance imaging (fMRI), this technique, initially employing exogenous contrast
agents (Belliveau et al 1991) and subsequently using BOLD (Ogawa et al 1992) (see
section 3.5.1), has become a widespread method for investigating brain function. Many
excellent reviewarticles have been written on this subject, and the reader is referred to these for
further information (e.g. Turner et al 1998, Le Bihan et al 1995, Bandettini and Wong 1997,
Frackowiak et al 1997). However, BOLD fMRI has been criticized for its inability to make
quantitative measurements of physiologically relevant parameters. It has become apparent
that perfusion, and to a lesser degree diffusion MRI, may have an important role to play in the
quantication of haemodynamic parameters associated with neuronal activation. Recent work
that has performed simultaneous BOLD and perfusion MRI has shown that the information
may be combined to estimate the change in cerebral metabolic rate of oxygen consumption
(CMRO
2
) caused by cerebral activation (Davis et al 1998, Kim et al 1999, Hoge et al 1999).
Also, it has been shown that diffusion imaging is sensitive to the magnetic eld gradients set up
by intravascular susceptibilty contrast agents, and that the measured ADCis proportional to the
intravascular susceptibility shift (Does et al 1999). These developments, though still at a very
early stage, show that there is good potential for MRI to perform quantitative brain mapping.
5. Conclusion
Although MRI scientists have sought to quantify the physically meaningful parameters of tissue
water diffusionandcerebral perfusion, the intricacies of biological tissue ona cellular scale, and
the numerous experimental difculties encountered, have complicated the simple measurement
and interpretation of these quantities. Nevertheless, it is clear that diffusion measurements
provide a powerful tool for investigating tissue microstructure, and perfusion measurements
provide a readily understood assessment of tissue function. As techniques develop further, and
our understanding of the underlying relationships between these measurements and the tissue
structure and function improves, both methods offer great promise in extending our knowledge
of both normal tissues and tissue that has, or is suffering, damage. Since MRI is non-invasive,
the measurements may be repeated to follow tissue changes over the time-scales of seconds,
minutes, days or even years. The value of this information is enhanced by the additional ability
of MRI to provide high resolution structural images using standard MRcontrast during the same
examination. In addition, MR spectroscopy can provide important biochemical information
related to tissue health and function. These capabilities have ensured that both methods have
found extensive application in animal models of disease and human studies. With currently
available hardware, diffusion imaging can be made accessible to the clinician (and radiologist)
who is prepared to provide the acute scanning service necessary to make it a useful tool, and
perfusion should follow closely. Future developments in MR hardware (i.e. improvements in
system stability and signal-to-noise performance) should make both these methods available
for routine diagnostic studies.
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radio-frequency (RF) pulse creates coherent transverse magnetization and the

RF pulse. If the spins

pulse (Hong and Dixon 1992) or the alternation

) RF pulses with diffusion gradients placed after the rst and

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