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
5.1.1. Abstract
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
Once the compartmental signals were distinguished on the basis of T1, 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
occurs via osmoregulated water channels (Jap and Li, 1995; Skach et al., 1994) or in
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
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
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
curves can be obtained in polystyrene bead packs (a single compartment system) due to
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.,
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
contributions from the intra and extracellular compartments (Labadie et al., 1994).
Another method used to discriminate between intra and extracellular water signals is by
Dusschoten et al., 1996; Herbst and Goldstein, 1989). A major disadvantage of using the
magnetic susceptibility effects, diffusion, and exchange. Kleinberg et al. (1994) have
shown that T2 relaxation times in porous media are reduced by diffusion in the presence
T1 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
In previous experiments (Han et al., 1998a, 1998b), T1 relaxation was used to separate the
differentiates the compartments on the basis of T1 relaxation, which then allows other
et al., 1994; Provencher and Dovi, 1979). These works also show that if the continuous
results can be obtained by fitting the data to a biexponential using a constrained nonlinear
In this study, combined T1 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
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
T
− I
M xy
= ∑ M 0 i 1 − 2e T1i [5.1.3.1]
i
where Mxy is the NMR signal and TI is the inversion time, the relaxogram generates a
spectrum of relaxation time constants, T1i, and their respective contributions to the overall
signal, M0i (Labadie et al., 1994). It has also been shown that even for relaxograms
different sections of the distribution can represent spatially distinct populations of spins
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
M xy (t ) t
− ' −
t
M norm (t ) =
'
= M 1 − 2e T1 a + M ' 1 − 2e T1'b [5.1.3.2]
M0
0a
0b
where
COMPARTMENTAL DIFFUSION 104
1 1 1 1
(M 0b − M 0 a ) − + +
1 T
1a T1b τ a τ b
M 0' b = 1 − [5.1.3.3]
2 2
1
1 1 1 4
− + − +
T
1b T1a τ b τ a τ aτ b
2
1 1 1 1 1 1 1 1 1 1 4
= + + + − − + − +
T1a 2 T1b T1a τ b τ a
'
T τ τ
1b T1a τ b τ a
a b
[5.1.3.5]
2
1 1 1 1 1 1 1 1 1 1 4
= + + + +
T1'b 2 T1b T1a τ b τ a T − T + τ − τ + τ τ
1b 1a b a a b
[5.1.3.6]
'
where M 0a and T1a' are the fast decaying component, and M 0b
'
and T1b' are the slow
decaying component of the signal; T1a , T1b , M 0 a , and M 0b are the actual compartmental
relaxation times and volume fractions; τ a and τ b are the mean lifetimes (or residence
It is evident that when the system is in slow exchange (i.e., τ a , τ b → ∞ ), the fast and
slow decaying components of relaxation approximate the fast and slow relaxing
compartments ( T1'a → T1a and T1'b → T1b ), and the signalattenuation curve exhibits a
1 1
dominating effect is the exchange rate ( and ) and the signalattenuation curve
τa τb
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
MR contrast agent increases, the width of the distribution will broaden and shift to lower
extracellular T1 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
agent, there will be a complete separation of the relaxographic distributions, where the
fast component of relaxation will be centered around T1'a and the slow component around
T1'b . It is important to note that the T1'a and T1'b are not the actual compartmental
relaxation times, but rather, the effective compartmental relaxation times (the dominating
COMPARTMENTAL DIFFUSION 106
effects on T1'a and T1'b are T1a and T1b , respectively). Similarly, M 0' a and M 0' b are the
It is a nontrivial matter to extract M 0 a and M 0b from M 0' a and M 0' b 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
1 V
τb = [5.1.3.7]
P A
M 0b
τ a = τ b [5.1.3.8]
M 0a
V
where P is the membrane permeability, and is the ratio of the volume to surface area
A
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
(Moffset).
M xy (TI , TE , b )
M norm (T I , TE , b ) = [5.1.3.9]
M0
T
− I' − TE' T
− I − TE'
M norm (TI , TE , b ) = M 1 − 2e
' T1 a e T2 a e −bDa' + M ' 1 − 2e T1'b e T2 b e − bDb' + M
0a
0b
offset
[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 T1 and T2 terms are
sensitive to the presence of MR contrast agent, the diffusion coefficient values are
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, M 0a represents the components of
'
relaxation dominated by spins in the fastdecaying compartment ( M 0 a ), while M 0b
compartment ( M 0b ). Furthermore, despite the pulse sequence used, spins will effectively
experience the same medium as long as the evolution times (time interval between
5.1.4. Methods
Baker’s dry yeast (Fleishmann’s Yeast, Inc., Oakland, CA), 1.5 g, was rehydrated at
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
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
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 1H. The studies were performed using
a 5mmdiameter, 9turn solenoid coil. The magnetbore temperature was 19°C. NMR
spinecho detection (Fig. 5.1.4.1). Thirtytwo inversion times (TI), with logarithmic
temporal spacing (TI = TI0*an1, TI0 = 2.0 ms, a = 1.250) and two signal averages, were
used to measure each T1relaxation 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/cm2).
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,
necessary gradient values. Halfechoes were acquired with a spectral width of ±1250 Hz
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 = TI0*an1, TI0 = 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/cm2. Gradients were applied in all three orthogonal directions
simultaneously to achieve these bvalues.
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
values.
COMPARTMENTAL DIFFUSION 111
InversionRecovery Data
Data were evaluated using CONTIN (Provencher, 1982a; 1982b; Provencher and Dovi,
T
 I T
 I
M xy
= M 0 a 12e T1a + M 12e T1b +M [5.1.4.1]
0b
Offset
where Mxy represents the measured signal intensity, M0a and T1a are the M0 and T1 of the
fastdecaying component of the signal, M0b and T1b are M0 and T1 of the slowdecaying
component of the signal, and finally M0Offset represents the inversion efficiency. A
Boulder, Colorado) was used for the constrainedbiexponential fitting. The T1 and M0
values obtained from the constrainedbiexponential fit were compared for consistency to
results obtained using CONTIN. The resulting M0a and M0b at each bvalue were then
used to calculate the respective ADCs of both the fast and slowrelaxing component of
In a similar fashion, the echotime dependence of the signal was studied for the two sets
data were fitted to Eq. [5.1.4.1] and the resulting M0a and M0b at each echo time were
T2 (ms ) =
(65 − 55) [5.1.4.2]
M xy55
ln
M xy
65
COMPARTMENTAL DIFFUSION 113
This yielded an approximation for the T2 relaxation times of the fast and slow decaying
signals.
PFGSE Data
The diffusion attenuation curves generated by the PFGSE sequence were fitted to a
where M0a and M0b are the calculated fast and slow components of M0 for the
5.1.5. Results
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 T1 of 3.0mM GdDTPA in the bulk solution was
measured to be 60 ms.
COMPARTMENTAL DIFFUSION 114
9000
Signal Intensity
4750
500
3750
8000
0 700 1400 2100
TI (ms)
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 = TI0*an1, TI0 = 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/cm2. All of these curves display biexponential behavior and
were fitted to

TI

TI
M xy = M 0 a 12e T1a
+ M 0b 12e T1b
+ M Offset
The solid lines on the plot represent the fit to the data while the points represent the
actual data.
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 T1 and M0 values for the
COMPARTMENTAL DIFFUSION 115
4750
500
3750
8000
0 700 1400 2100
TI (ms)
Fig. 5.1.5.2. Inversionrecovery curves at different echo times. Inversionrecovery
data were acquired with the following NMR parameters: TI = TI0*an1, TI0 = 2.0 ms, a
= 1.128, TE = 55 and 65 ms. Both curves display biexponential behavior and
were fitted to
 I
T
 I
T
M xy = M 0 a 12e T1a
+ M 0b 12e T1b
+ M Offset
The solid lines on the plot represent the fit to the data while the points represent the
actual data
fast and slow components of decay at different bvalues are summarized in Table
5.1.5.2.1. The bvaluedependent behavior of M0a exhibits a larger attenuation than M0b.
This indicates that M0a is associated with the fasterdiffusing extracellular spins while
Table 5.1.5.2.1. Calculated T1 and M0 values for the fast and slow components of
relaxation at different bvalues. T1a and M0a represent the effective relaxation time and
spin density for the fast decaying extracellular compartment while T1b and M0b represent
the effective relaxation time and spin density for the slow decaying intracellular
compartment at a 55 ms echo time. No T1 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.
calculated M0 and T1 values associated with the fast and slow components of relaxation
are summarized in Table 5.1.5.2.2. The estimates of T2 relaxation times for M0a and M0b
Table 5.1.5.2.2. Calculated T1 and M0 values for the fast and slow components of
relaxation at different echo times. T1a and M0a represent the effective relaxation
time and spin density for the fast decaying extracellular compartment while T1b and
M0b represent the effective relaxation time and spin density for the slow decaying
intracellular compartment at 55 ms and 65 ms echo times.
TE55 TE65
M0a 4855 2600
T1a 59 ms 53 ms
M0b 3182 2367
T1b 328 ms 257 ms
sequence is shown in Fig. 5.1.5.3. The curve exhibits biexponential behavior and was
−5 −6
M xy = 0.436e −1.64×10 b
+ 0.564e −1.75×10 b
[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 M0a and M0b on bvalue is shown in Fig. 5.1.5.4. The behavior of
both M0a and M0b exhibit nonmonoexponential attenuation. When fitted to a single
exponential, M0a (the component associated with the extracellular space) showed a
COMPARTMENTAL DIFFUSION 118
0
0 200000 400000 600000
0.2
0.4
0.6
ln (S/S0)
0.8
1
1.2
1.4
1.6
b value
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
−5 −6
M xy = 0.436e −1.64×10 b + 0.564e −1.75×10 b
The solid line on the plot represents the fit to the data while the points themselves
are the actual data
reasonably good fit while M0b (component associated with the intracellular space)
10000
y = 2.88E+03e6.44E07x
R2 = 6.87E01
Calculated M0
1000
y = 3.65E+03e8.59E06x
R2 = 9.28E01
100
0 250000 500000
b value
Fig. 5.1.5.4. Calculated M0a and M0b from water in yeastcell suspensions plotted as
a function of bvalue. Both curves display nonmonoexponential diffusion
attenuation. The M0 values were fitted to
−6
M xy fast = 0.559e −8.59×10 b
; R2 = 0.928
for the fast component and
−7
M xy slow = 0.441e −6.44×10 b
; R2 = 0.687
for the slow component.
−6
M xy fast = 0.559e −8.59×10 b
; R2 = 0.928 [5.1.5.2]
and
−7
M xy slow = 0.441e −6.44×10 b
; R2 = 0.687 [5.1.5.3]
COMPARTMENTAL DIFFUSION 120
0
0 200000 400000 600000
0.4
ln (S/S0)
0.8
1.2
1.6
b value
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 M0 are shown. It is evident that all three
curves exhibit distinct behavior.
There was a significant discrepancy between this method and the PFGSE sequence when
5.1.6. Discussion
effective for yeastcell suspensions. For this particular system, where a complete
values are dominated by compartmental rather than exchange effects. It was also
observed that fits of M0fast versus bvalue to a monoexponential produced good results
while the corresponding fit to M0slow produced a much poorer result. Since the
theoretical rationale for fitting the calculated M0 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
environment with greater restrictions, hence, the diffusion attenuation exhibits non
M0fast, M0slow, T1fast, and T1slow to a high degree of accuracy. Furthermore, if indeed the
The calculated T2 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 T2 values were not
dissimilar enough, it would have been difficult to differentiate the compartments on the
basis of T2 alone. One of the advantages of using T1 is that the underlying mechanism for
1
spinlattice relaxation is the hyperfine interaction of the H2O 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 T2
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
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 Mxy and ADC values measured from the PFGSE
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 T1 measured for the
inversionrecovery curve at the highest bvalue. This idea of quenching the signal could
In order to understand the restriction effects on the overall signal, it would be useful to
valuable information on the microstructure of the environment that the 1H2O 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
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.