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Chapter 5
Yeast Experiments
The goal of these experiments was to characterize the diffusion behavior of water in a
twocompartment system. Yeast cell suspensions have been well characterized by others
and are fairly uncomplicated model systems to understand. The following work contains
multiple sets of experiments using yeast cells as a model system to characterize the
diffusion behavior of water.
5.1. Deconvolution of Restriction Effects on
Compartmental Diffusion Using Combined
Relaxometric and Diffusimetric NMR
5.1.1. Abstract
Diffusion signal attenuation curves in yeastcell suspensions show nonmonoexponential
signal decay which is assumed to arise from separate compartmental contributions to the
overall signal. However, restricted diffusion effects also give rise to non
monoexponential signal decay and are difficult to separate from compartmental signal
contributions. Combined relaxometry and diffusion measurements allows differentiation
COMPARTMENTAL DIFFUSION 99
between compartmental diffusion constants by first separating the compartmental
contributions on the basis of differences in their respective relaxation times. Diffusion
weighted inversionrecovery spinecho experiments were carried out at different b
values. Intra and extracellular compartments in yeastcell suspensions were separated on
the basis of T
1
relaxation by adding an MR contrast agent to the extracellular space.
Once the compartmental signals were distinguished on the basis of T
1
, the relative signal
attenuation for each compartment was used to calculate the separate compartmental
ADCs. With this method, even compartmental diffusion coefficients with similar values
can be distinguished.
5.1.2. Introduction
The cell membrane plays an important role in cell physiology by acting as a protective
barrier between the extra and intracellular compartments. Since the cellular membrane
is a lipid bilayer structure with a highly hydrophobic core (Fox, 1993), it is unclear how
water is transported between the intra and extracellular spaces through a seemingly
impermeable membrane. It has been speculated that transmembrane transport of water
occurs via osmoregulated water channels (Jap and Li, 1995; Skach et al., 1994) or in
conjunction with watersoluble ion transport.
Movement of water between the extra and intracellular spaces, as well as within the
cells, is of particular importance in biomedicine. Studies have shown that changes in the
COMPARTMENTAL DIFFUSION 100
apparent diffusion coefficient (ADC) of water in biological systems are a function of
membrane permeability and changes in the extracellular volume fraction (Latour et al.,
1993b, 1994). Since the water NMR signal is measured simultaneously from the intra
and extracellular space, it is difficult to ascertain whether the changes in the ADC are
caused by drastic changes in one or both of the compartments, or by permeability
changes in the cell membrane.
Nonmonoexponential behavior in the diffusionsignal attenuation curves has been
observed in a number of biological systems and has been attributed to compartmental
effects (Niendorf et al., 1996; Andrasko, 1976b; Vétek et al., 1994; Szafer et al., 1995).
Although it has been reported that changes in the extracellular volume fraction mirror the
changes observed in the rapidly decaying component of the signal decay curve (Trouard
et al., 1997; Niendorf et al., 1994), the volume fraction calculated for the slowly
diffusing component in these experiments differ significantly from the true intracellular
volume fraction (Niendorf et al., 1994). While multicompartment systems in the slow
exchange regime are expected to exhibit nonmonoexponential signal decay curves,
Helmer et al. (1995) have shown that nonmonoexponential diffusionsignal attenuation
curves can be obtained in polystyrene bead packs (a single compartment system) due to
restriction effects alone. Consequently, since the compartmental contributions do not
definitively represent the cellular compartments, it is difficult to ascertain whether the
nonmonoexponential behavior in a highly restricted twocompartment system is due to
compartmental contributions, restrictions, or both of these effects.
COMPARTMENTAL DIFFUSION 101
Since compartmental effects and membrane permeability are closely related, comparable
methods have been used to study both parameters (Andrasko, 1976a; Tanner, 1983;
Pilatus et al., 1997). Several studies have shown the efficacy of using MR contrast
agents to discriminate between extra and intracellular water signals (Labadie et al.,
1994; Stanisz et al., 1998), or to measure membrane permeability (Andrasko, 1987a;
Conlon and Outhred, 1972). MR contrast agents do not cross the cell membrane and thus
selectively affect the extracellular water signal. The addition of MR contrast agents to
the extracellular space drastically alters the relaxation properties of the sample, and, with
sufficient concentrations of contrast agent, the system can be brought into the slow
exchange regime where a biexponential relaxation curve characterizes the separate
contributions from the intra and extracellular compartments (Labadie et al., 1994).
Another method used to discriminate between intra and extracellular water signals is by
adding diffusion gradients to a CarrPurcell MeiboomGill (CPMG) sequence (van
Dusschoten et al., 1996; Herbst and Goldstein, 1989). A major disadvantage of using the
transverse relaxation to distinguish between compartments is that T
2
is sensitive to bulk
magnetic susceptibility effects, diffusion, and exchange. Kleinberg et al. (1994) have
shown that T
2
relaxation times in porous media are reduced by diffusion in the presence
of inhomogeneous magnetic fields arising from magnetic susceptibility differences, while
T
1
is dominated by relaxivity due to hyperfine interactions with the pore surface. While
these studies were performed on porous rocks, the mechanism of relaxation is very
similar for water in the presence of paramagnetic MR contrast agents.
COMPARTMENTAL DIFFUSION 102
In previous experiments (Han et al., 1998a, 1998b), T
1
relaxation was used to separate the
compartmental signal contributions using relaxography. This method effectively
differentiates the compartments on the basis of T
1
relaxation, which then allows other
types of measurements to be performed on the separate compartmental signals (Labadie
et al., 1994; Provencher and Dovi, 1979). These works also show that if the continuous
relaxation spectra display two distinct distributions of relaxation times, comparable
results can be obtained by fitting the data to a biexponential using a constrained nonlinear
leastsquares algorithm (Han et al., 1998a; Silva et al., 1998).
In this study, combined T
1
relaxation and diffusion data were obtained from yeastcell
suspension in the presence of a MR contrast agent. From these data, the effective
compartmental ADCs were calculated and compared to the decay constants calculated
from a biexponential fit of the diffusionsignal attenuation curve. The validity and
reliability of such a model to determine compartmental ADCs is discussed.
5.1.3. Theory
It has been shown that the relationship between a set of relaxationsignal decay data and
the probability distribution of relaxation times is given by the Laplace transform. Thus,
by taking the formal inverse Laplace transform (ILT) of data governed by a distribution
of relaxation times
COMPARTMENTAL DIFFUSION 103
∑


¹

\

− =
−
i
T
T
i xy
i
I
e M M
1
2 1
0
[5.1.3.1]
where M
xy
is the NMR signal and TI is the inversion time, the relaxogram generates a
spectrum of relaxation time constants, T
1i
, and their respective contributions to the overall
signal, M
0i
(Labadie et al., 1994). It has also been shown that even for relaxograms
displaying only a single distribution, the corresponding relaxographic images from
different sections of the distribution can represent spatially distinct populations of spins
(Pályka et al., 1994; Dardzinski et al., 1994).
The twocompartment exchange equation has been analytically solved by Kärger et al.
(1988), where the normalized signal for an NMR inversionrecovery experiment is given
by the equation
( )
( )


¹

\

− +


¹

\

− = =
− −
'
1
'
1
2 1 2 1
'
0
'
0
0
b a
T
t
b
T
t
a
xy
norm
e M e M
M
t M
t M [5.1.3.2]
where
COMPARTMENTAL DIFFUSION 104
( )
¦
¦
¦
)
¦
¦
¦
`
¹
¦
¦
¦
¹
¦
¦
¦
´
¦
+


¹

\



¹

\

− +


¹

\

−
+ +


¹

\

− −
− =
b a a b a b
b a b a
a b
b
T T
T T
M M
M
τ τ τ τ
τ τ
4 1 1 1 1
1 1 1 1
1
2
1
2
1 1
1 1
0 0
'
0
[5.1.3.3]
1
'
0
'
0
= +
b a
M M [5.1.3.4]
+


¹

\



¹

\

− +


¹

\

− −


¹

\

+ + + =
b a a b a b a b a b a
T T T T T τ τ τ τ τ τ
4 1 1 1 1 1 1 1 1
2
1 1
2
1 1 1 1
'
1
[5.1.3.5]
+


¹

\



¹

\

− +


¹

\

− +


¹

\

+ + + =
b a a b a b a b a b b
T T T T T τ τ τ τ τ τ
4 1 1 1 1 1 1 1 1
2
1 1
2
1 1 1 1
'
1
[5.1.3.6]
where
'
0a
M and
'
1a
T are the fast decaying component, and
'
0b
M and
'
1b
T are the slow
decaying component of the signal;
a
T
1
,
b
T
1
,
a
M
0
, and
b
M
0
are the actual compartmental
relaxation times and volume fractions;
a
τ and
b
τ are the mean lifetimes (or residence
times) of the spins in their respective environments.
COMPARTMENTAL DIFFUSION 105
It is evident that when the system is in slow exchange (i.e., ∞ →
b a
τ τ , ), the fast and
slow decaying components of relaxation approximate the fast and slow relaxing
compartments (
a a
T T
1
'
1
→ and
b b
T T
1
'
1
→ ), and the signalattenuation curve exhibits a
biexponential decay. Conversely, when the system is in fast exchange ( 0 , →
b a
τ τ ), the
dominating effect is the exchange rate (
a
τ
1
and
b
τ
1
) and the signalattenuation curve
exhibits a monoexponential decay.
For a twocompartment system displaying a single relaxographic distribution, the system
is in fast exchange and the single distribution represents the weighted average of both the
intra and extracellular water signals. Adding MR contrast agent to the extracellular
space effectively acts as a shift reagent in relaxographic space. As the concentration of
MR contrast agent increases, the width of the distribution will broaden and shift to lower
values of relaxation times. With increasing concentrations of contrast agent, the
extracellular T
1
will become the dominating term in Eqs. [5.1.3.5] and [5.1.3.6] and the
system will shift from the fast (residencetime dominated) to the slowexchange
(relaxationtime dominated) regime. At some concentration of extracellular MR contrast
agent, there will be a complete separation of the relaxographic distributions, where the
fast component of relaxation will be centered around
'
1a
T and the slow component around
'
1b
T . It is important to note that the
'
1a
T and
'
1b
T are not the actual compartmental
relaxation times, but rather, the effective compartmental relaxation times (the dominating
COMPARTMENTAL DIFFUSION 106
effects on
'
1a
T and
'
1b
T are
a
T
1
and
b
T
1
, respectively). Similarly,
'
0a
M and
'
0b
M are the
effective compartmental volume fractions.
It is a nontrivial matter to extract
a
M
0
and
b
M
0
from
'
0a
M and
'
0b
M since a priori
knowledge of the residence times as well as one of the relaxation time constants is
necessary. A reliable estimate of the residence times can be obtained from (Conlon and
Outhred, 1972; Andrasko, 1976b; Kärger et al., 1988)

¹

\

=
A
V
P
b
1
τ [5.1.3.7]


¹

\

=
a
b
b a
M
M
0
0
τ τ [5.1.3.8]
where P is the membrane permeability, and
A
V
is the ratio of the volume to surface area
of the cells. Since the residence times are a function of the compartmental ratios, at least
one of these quantities must be measured independently (Herbst and Goldstein, 1989).
It can be shown that for a system in slow exchange, a constrained nonlinear leastsquares
fit to Eq. [5.1.3.2] is comparable to results obtained using CONTIN (Han et al., 1998b).
COMPARTMENTAL DIFFUSION 107
Taking this one step further, Eq. [5.1.3.2] can be modified (Eqs [5.1.3.9] and [5.1.3.10])
to include echo time (TE) effects, gradient (b) effects, and the inversion efficiency
(M
offset
).
( )
( )
0
, ,
, ,
M
b TE T M
b TE T M
I xy
I norm
= [5.1.3.9]
( )
offset
bD T
TE
T
T
b
bD T
TE
T
T
a I norm
M e e e M e e e M b TE T M
b b b
I
a a a
I
+


¹

\

− +


¹

\

− =
−
− −
−
− −
' '
2
'
1
' '
2
'
1
2 1 2 1 , ,
'
0
'
0
[5.1.3.10]
where the variables represented denote the effective time constants and fractional
contributions (Fig. 5.1.4.1). It should be noted that while the T
1
and T
2
terms are
sensitive to the presence of MR contrast agent, the diffusion coefficient values are
relatively unaffected by such paramagnetic substances. Hence, the compartmental
differentiation on the basis of relaxometry in the presence of MR contrast agents will
have little effect on the subsequent measurements of the compartmental diffusion
coefficients.
Although exchange effects are present, the spin densities associated with each of the
relaxographic peaks represent the medium effectively sampled by those spins during the
COMPARTMENTAL DIFFUSION 108
echo time. Hence, for systems in slow exchange,
'
0a
M represents the components of
relaxation dominated by spins in the fastdecaying compartment (
a
M
0
), while
'
0b
M
represents the components of relaxation dominated by spins in the slowdecaying
compartment (
b
M
0
). Furthermore, despite the pulse sequence used, spins will effectively
experience the same medium as long as the evolution times (time interval between
excitation and acquisition) are identical.
5.1.4. Methods
5.1.4.1. Yeast Preparation
Baker’s dry yeast (Fleishmann’s Yeast, Inc., Oakland, CA), 1.5 g, was rehydrated at
room temperature in a 50 cc centrifuge tube by adding 40 cc of distilled H
2
O. The
suspension was bubbled with medicalgrade air and, after a starving period of
approximately 3 hrs, the suspension was centrifuged for 8 min at 3500 rpm (IECCentra8
Centrifuge, International Equipment Company, USA). Yeast cells were washed four
times with 5 mM gadopentate dimeglumine (GdDTPA, Magnevist®, Berlex, Wayne,
NJ). After the final wash, the wet/dry mass ratio was adjusted to 3.25:1 and NMR
experiments were performed. The elapsed time between the end of the sample
preparation and the beginning of NMR data acquisition was about ten minutes.
COMPARTMENTAL DIFFUSION 109
5.1.4.2. NMR Experiments
Combined Relaxometry and Diffusion (CRD)
All data were obtained on a GECSIII 2.0T/45 cm imaging spectrometer with ±20 G/cm
selfshielded gradients operating at 85.5 MHz for
1
H. The studies were performed using
a 5mmdiameter, 9turn solenoid coil. The magnetbore temperature was 19°C. NMR
data were acquired using an inversionrecovery pulse sequence with diffusionweighted
spinecho detection (Fig. 5.1.4.1). Thirtytwo inversion times (TI), with logarithmic
temporal spacing (TI = TI
0
*a
n1
, TI
0
= 2.0 ms, a = 1.250) and two signal averages, were
used to measure each T
1
relaxation curve. Measurements were repeated for eighteen
different bvalues (1410, 5639, 12688, 22557, 35245, 50754, 69082, 90229, 114196,
140983, 170590, 203016, 238262, 276327, 317212, 360917, 407442, and 456786 s/cm
2
).
Other acquisition parameters were ∆ = 41.5 ms, δ = 6.0 ms, predelay = 2000 ms. Half
sineshaped gradient pulses with amplitudes of 1.7, 3.5, 5.2, 6.9, 8.7, 10.4, 12.1, 13.9,
15.6, 17.3, 19.1, 20.8, 22.5, 24.2, 26.0, 27.7, 29.4, and 31.2 G/cm were used for the
diffusion weighting. Since the maximum gradient strength was ±20 G/cm for each axis,
gradients were applied simultaneously in three orthogonal directions to achieve the
necessary gradient values. Halfechoes were acquired with a spectral width of ±1250 Hz
and 2K data points. Separate measurements were performed at TE = 55 and 65 ms to
characterize echotime effects.
COMPARTMENTAL DIFFUSION 110
PulsedFieldGradient SpinEcho (PFGSE) Experiment
To examine the behavior of the signal as a function of bvalue, a standard pulsedfield
gradient spinecho (PFGSE) sequence (Fig. 5.1.4.2; Stejskal and Tanner, 1965) with 32
bvalues, TE = 55 ms, ∆ = 41.5 ms, δ = 6.0 ms, and two signal averages was used. The b
values were varied by keeping the diffusion time constant and increasing the gradient
strength from 0.97 G/cm to 31.04 G/cm, in increments of 0.97 G/cm. Gradients were
applied simultaneously in three orthogonal directions in order to achieve these gradient
values.
Fig. 5.1.4.1. Inversionrecovery spin echo pulse sequence with diffusion gradients. TI
ranged from 2 to 2019 ms in logarithmic increments where TI = TI
0
*a
n1
, TI
0
= 2.0 ms,
a = 1.250, and n = 0…31, TE = 55 ms, g = 1.73 to 31.2 G/cm in 1.73 G/cm increments,
∆ = 41.5 ms, δ = 6.0 ms, and bvalues of 1410, 5639, 12688, 22557, 35245, 50754,
69082, 90229, 114196, 140983, 170590, 203016, 238262, 276327, 317212, 360917,
407442, and 456786 s/cm
2
. Gradients were applied in all three orthogonal directions
simultaneously to achieve these bvalues.
COMPARTMENTAL DIFFUSION 111
5.1.4.3. Data Analysis
InversionRecovery Data
Data were evaluated using CONTIN (Provencher, 1982a; 1982b; Provencher and Dovi,
1979) and also by constrainedbiexponential fitting to the equation
Fig. 5.1.4.2. Pulsedfieldgradient spinecho (PFGSE) pulse sequence. The diffusion
time was kept constant and the bvalue varied by incrementing the total gradient
strength from 0.97 G/cm to 31.04 G/cm. Gradients were applied simultaneously in all
three orthogonal directions. Other acquisition parameters were TR = 2000 ms, TE =
55 ms, ∆ = 41.5 ms, and δ = 6.0 ms.
COMPARTMENTAL DIFFUSION 112
Offset
T
T

b
T
T

a xy
M e  M e  M M
b
I
a
I
+


¹

\

+


¹

\

=
1 1
2 1 2 1
0 0
[5.1.4.1]
where M
xy
represents the measured signal intensity, M
0a
and T
1a
are the M
0
and T
1
of the
fastdecaying component of the signal, M
0b
and T
1b
are M
0
and T
1
of the slowdecaying
component of the signal, and finally M
0Offset
represents the inversion efficiency. A
standard nonlinear leastsquares algorithm available in IDL (Research Systems, Inc.,
Boulder, Colorado) was used for the constrainedbiexponential fitting. The T
1
and M
0
values obtained from the constrainedbiexponential fit were compared for consistency to
results obtained using CONTIN. The resulting M
0a
and M
0b
at each bvalue were then
used to calculate the respective ADCs of both the fast and slowrelaxing component of
the overall signal.
In a similar fashion, the echotime dependence of the signal was studied for the two sets
of inversionrecovery curves generated at TE = 55 and 65 ms. The inversionrecovery
data were fitted to Eq. [5.1.4.1] and the resulting M
0a
and M
0b
at each echo time were
fitted to a twopoint T
2
measurement according to Eq. [5.1.4.2].
( )


¹

\

−
=
65
55
ln
55 65
) (
2
xy
xy
M
M
ms T [5.1.4.2]
COMPARTMENTAL DIFFUSION 113
This yielded an approximation for the T
2
relaxation times of the fast and slow decaying
signals.
PFGSE Data
The diffusion attenuation curves generated by the PFGSE sequence were fitted to a
biexponential diffusionattenuation curve using
b a
bD
b
bD
a xy
e M e M M
− −
+ =
0 0
[5.1.4.3]
where M
0a
and M
0b
are the calculated fast and slow components of M
0
for the
biexponential decay, and D
a
and D
b
are their respective decay constants.
5.1.5. Results
5.1.5.1. [GdDTPA] and Relaxographic Peak Separation
Relaxography data for yeastcell suspensions in 3.0 mM GdDTPA shows that there are
clearly two distinct components, and is consistent with the results obtained by Labadie et
al. (1994). At 2.0T and 19°C, the T
1
of 3.0mM GdDTPA in the bulk solution was
measured to be 60 ms.
COMPARTMENTAL DIFFUSION 114
5.1.5.2. InversionRecovery Experiments with Varying Gradient Strengths
and Echo Times
The bvaluedependent inversionrecovery curves and the fit to the curves (Fig. 5.1.5.1)
show an excellent fit of the data to Eq. [5.1.4.1]. The calculated T
1
and M
0
values for the
8000
3750
500
4750
9000
0 700 1400 2100
TI (ms)
S
i
g
n
a
l
I
n
t
e
n
s
i
t
y
Fig. 5.1.5.1. Inversionrecovery curves at different gradient values. Inversion
recovery data acquired with the following NMR parameters: TI ranged from 2 to
2019 ms in logarithmic increments where TI = TI
0
*a
n1
, TI
0
= 2.0 ms, a = 1.250, and
n = 0…31, TE = 55 ms, g = 1.73 to 31.2 G/cm in 1.73 G/cm increments, ∆ = 41.5
ms, δ = 6.0 ms, and bvalues of 1410, 5639, 12688, 22557, 35245, 50754, 69082,
90229, 114196, 140983, 170590, 203016, 238262, 276327, 317212, 360917,
407442, and 456786 s/cm
2
. All of these curves display biexponential behavior and
were fitted to
Offset
T
TI

b
T
TI

a xy
M e  M e  M M
b a
+


¹

\

+


¹

\

=
1 1
2 1 2 1
0 0
The solid lines on the plot represent the fit to the data while the points represent the
actual data.
COMPARTMENTAL DIFFUSION 115
fast and slow components of decay at different bvalues are summarized in Table
5.1.5.2.1. The bvaluedependent behavior of M
0a
exhibits a larger attenuation than M
0b
.
This indicates that M
0a
is associated with the fasterdiffusing extracellular spins while
M
0b
is associated with slowerdiffusing intracellular spins.
8000
3750
500
4750
9000
0 700 1400 2100
TI (ms)
S
i
g
n
a
l
I
n
t
e
n
s
i
t
y
Fig. 5.1.5.2. Inversionrecovery curves at different echo times. Inversionrecovery
data were acquired with the following NMR parameters: TI = TI
0
*a
n1
, TI
0
= 2.0 ms, a
= 1.128, TE = 55 and 65 ms. Both curves display biexponential behavior and
were fitted to
Offset
T
T

b
T
T

a xy
M e  M e  M M
b
I
a
I
+


¹

\

+


¹

\

=
1 1
2 1 2 1
0 0
The solid lines on the plot represent the fit to the data while the points represent the
actual data
COMPARTMENTAL DIFFUSION 116
The TEdependent inversionrecovery curves also exhibit biexponential behavior. The
calculated M
0
and T
1
values associated with the fast and slow components of relaxation
are summarized in Table 5.1.5.2.2. The estimates of T
2
relaxation times for M
0a
and M
0b
were 16 ms and 34 ms for the fast and slow components, respectively.
Table 5.1.5.2.1. Calculated T
1
and M
0
values for the fast and slow components of
relaxation at different bvalues. T
1a
and M
0a
represent the effective relaxation time and
spin density for the fast decaying extracellular compartment while T
1b
and M
0b
represent
the effective relaxation time and spin density for the slow decaying intracellular
compartment at a 55 ms echo time. No T
1
value was available for the fit of the inversion
recovery curve to the highest bvalue. This indicates a quenching of the extracellular
compartment at this bvalue.
bvalue (s/cm
2
) M
0a
T
1a
(ms) M
0b
T
1b
(ms)
1410 4855 59 3182 328
5639 4310 54 3171 296
12688 3780 54 3188 284
22557 3587 57 2697 333
35245 2986 55 2720 320
50754 2519 55 2663 310
69082 2127 57 2580 324
90229 1668 56 2531 319
114196 1363 57 2430 331
140983 1007 56 2462 325
170590 561 43 2709 305
203016 405 24 2642 294
238262 317 52 2478 318
276327 205 37 2424 313
317212 185 55 2309 326
360917 118 52 2302 321
407442 233 83 2173 368
456786 134 *** 2275 319
COMPARTMENTAL DIFFUSION 117
5.1.5.3. Calculation of the Biexponential DiffusionDecay Constants from the
PFGSE Data
The nonmonoexponential diffusionsignal attenuation curve obtained using the PFGSE
sequence is shown in Fig. 5.1.5.3. The curve exhibits biexponential behavior and was
fitted to Eq. [5.1.4.3] to yield
b b
xy
e e M
6 5
10 75 . 1 10 64 . 1
564 . 0 436 . 0
− −
× − × −
+ = [5.1.5.1]
There was excellent correspondence between the data and the fit as can be seen in Fig.
5.1.5.3.
5.1.5.4. Calculation of ADCs Associated with the Fast and Slow Relaxing
Components of the Signal
The dependence of M
0a
and M
0b
on bvalue is shown in Fig. 5.1.5.4. The behavior of
both M
0a
and M
0b
exhibit nonmonoexponential attenuation. When fitted to a single
exponential, M
0a
(the component associated with the extracellular space) showed a
Table 5.1.5.2.2. Calculated T
1
and M
0
values for the fast and slow components of
relaxation at different echo times. T
1a
and M
0a
represent the effective relaxation
time and spin density for the fast decaying extracellular compartment while T
1b
and
M
0b
represent the effective relaxation time and spin density for the slow decaying
intracellular compartment at 55 ms and 65 ms echo times.
TE55 TE65
M
0a
4855 2600
T
1a
59 ms 53 ms
M
0b
3182 2367
T
1b
328 ms 257 ms
COMPARTMENTAL DIFFUSION 118
reasonably good fit while M
0b
(component associated with the intracellular space)
produced a poorer fit. The fits to a monoexponential attenuation function were
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 200000 400000 600000
b value
l
n
(
S
/
S
0
)
Fig. 5.1.5.3. Diffusion attenuation curve for yeastcell suspension acquired using the
PFGSE pulse sequence in Fig. 5.1.4.2. Nonmonoexponential behavior in diffusion
attenuation can be seen in yeast samples. The diffusion attenuation curves were
acquired with the following NMR parameters: TE = 55 ms, ∆ = 41.5 ms, δ = 6.0 ms,
g = 0.48*n G/cm where n = 1…32. The data were fitted to a biexponential decay
curve
b b
xy
e e M
6 5
10 75 . 1 10 64 . 1
564 . 0 436 . 0
− −
× − × −
+ =
The solid line on the plot represents the fit to the data while the points themselves
are the actual data
COMPARTMENTAL DIFFUSION 119
b
xy
e M
fast
6
10 59 . 8
559 . 0
−
× −
= ; R
2
= 0.928 [5.1.5.2]
and
b
xy
e M
slow
7
10 44 . 6
441 . 0
−
× −
= ; R
2
= 0.687 [5.1.5.3]
y = 2.88E+03e
6.44E07x
R
2
= 6.87E01
y = 3.65E+03e
8.59E06x
R
2
= 9.28E01
100
1000
10000
0 250000 500000
b value
C
a
l
c
u
l
a
t
e
d
M
0
Fig. 5.1.5.4. Calculated M
0a
and M
0b
from water in yeastcell suspensions plotted as
a function of bvalue. Both curves display nonmonoexponential diffusion
attenuation. The M
0
values were fitted to
b
xy
e M
fast
6
10 59 . 8
559 . 0
−
× −
= ; R
2
= 0.928
for the fast component and
b
xy
e M
slow
7
10 44 . 6
441 . 0
−
× −
= ; R
2
= 0.687
for the slow component.
COMPARTMENTAL DIFFUSION 120
There was a significant discrepancy between this method and the PFGSE sequence when
the ADC value was calculated for the fastdecaying compartment.
1.6
1.2
0.8
0.4
0
0 200000 400000 600000
b value
l
n
(
S
/
S
0
)
Normalized Data
Normalized Fit (PFGSE)
Normalized Fit (CRD)
Fig. 5.1.5.5. Normalized plot of the calculated fits using the two different methods.
PFGSE fit to the biexponential is displayed along with the two fits obtained from the
combined relaxography and diffusion (CRD) fits. Both the monoexponential and
biexponential fit to the calculated values of M
0
are shown. It is evident that all three
curves exhibit distinct behavior.
COMPARTMENTAL DIFFUSION 121
5.1.6. Discussion
Differentiation of compartmental signals using MR contrast agents has been shown to be
effective for yeastcell suspensions. For this particular system, where a complete
separation of relaxographic peaks was observed, it is believed that the measured M
0
values are dominated by compartmental rather than exchange effects. It was also
observed that fits of M
0fast
versus bvalue to a monoexponential produced good results
while the corresponding fit to M
0slow
produced a much poorer result. Since the
compartmental effects are thought to be deconvolved using relaxometry, there is no
theoretical rationale for fitting the calculated M
0
values to a biexponential. The larger
errors associated with the fits for the intracellular ADC could partly be due to the reduced
dynamic range for the attenuation of the intracellular signal as compared to the
extracellular signal. Another possibility is that the intracellular water experiences an
environment with greater restrictions, hence, the diffusion attenuation exhibits non
monoexponential behavior. In either case, it becomes necessary to be able to measure
M
0fast
, M
0slow
, T
1fast
, and T
1slow
to a high degree of accuracy. Furthermore, if indeed the
intracellular water displays a nonmonoexponential decay due to restriction, more b
values would be necessary to observe this effect.
The calculated T
2
values from the echotime dependent inversionrecovery curves show
that there is significant signal loss when a 55 ms echo time is used. In both the extra and
intracellular compartments, there is a greater than 80% signal attenuation due to the echo
COMPARTMENTAL DIFFUSION 122
time used for this experiment. More importantly, since the measured T
2
values were not
dissimilar enough, it would have been difficult to differentiate the compartments on the
basis of T
2
alone. One of the advantages of using T
1
is that the underlying mechanism for
spinlattice relaxation is the hyperfine interaction of the
1
H
2
O protons with the
paramagnetic center of the MR contrast agent (Kleinberg et al., 1994). This avoids
complications arising from throughspace bulksusceptibility effects that can affect the T
2
relaxation time of water in compartments that have no MR contrast agent present.
The PFGSE data exhibit nonmonoexponential diffusionsignal attenuation behavior.
Although an excellent fit to this data can be obtained using a biexponential, the
interpretation of these data becomes problematic since part of the curvature most likely
arises from restriction effects. Since the intracellular compartment is highly restricted
(≈4µmdiameter yeast cells; Tanner, 1983), the restriction effects are probably non
negligible at the diffusion time and gradient strengths used for this experiment. For this
reason, the discrepancy between the PFGSE biexponential fit and the CRD fit for the data
was not surprising. Due to the relatively low packing density of the yeastcell
suspension, restriction effects in the extracellular space are probably minimal. This
hypothesis is substantiated by the monoexponential signal decay of the deconvolved
extracellular component. However, there is a significant discrepancy between the fits for
the intracellular ADC between the PFGSE and CRD methods. We believe this disparity
is due to restriction effects. Since the M
xy
and ADC values measured from the PFGSE
experiment are a representation of the environment sampled by the water molecules
COMPARTMENTAL DIFFUSION 123
during the evolution time, it is possible that the space sampled during this echo time is a
combined function of exchange and restrictions. While the sampled space would be the
same for the CRD experiment, the compartmental contributions are deconvolved from the
inversionrecovery data before the ADC is calculated. This allows for independent
calculations of ADC from each compartment without the worry of contamination due to
exchange effects.
One of the effects observed in this study was the eventual quenching of the extracellular
signal at high bvalues. This can account for the missing value of T
1
measured for the
inversionrecovery curve at the highest bvalue. This idea of quenching the signal could
prove to be useful in studies where only a single compartment is of interest.
In order to understand the restriction effects on the overall signal, it would be useful to
study the timedependent behavior of water in a restricted twocompartment system.
Investigation of the timedependent behavior of compartmental water would provide
valuable information on the microstructure of the environment that the
1
H
2
O samples
(Helmer et al., 1995; Latour et al., 1993a; Norris and Niendorf, 1995).
A major advantage of using combined relaxometry with diffusion (CRD) gradients is that
two different compartments with similar ADC values can be distinguished on the basis of
differences in relaxation times of the respective compartments.
COMPARTMENTAL DIFFUSION 124
In conclusion, we have shown that compartmental contributions in the measurement of
ADC can be deconvolved by combined relaxometry and diffusimetry. This method
unequivocally differentiates between the intra and extracellular signals before fitting for
the ADC, thus removing the confounding effects of restriction on the measured ADC
values and also allowing for ADC measurements of two compartments with similar ADC
values.
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