Contrast enhancement methods in sodium MR imaging: a new emerging technique

doi:10.4236/jbise.2009.26065

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Vol.2, No.6, 445-457 (2009)

doi:10.4236/jbise.2009.26065

JBiSE

Contrast enhancement methods in sodium MR imaging: a

new emerging technique

Rakesh Sharma1, Avdhesh Sharma2, Soonjo Kwon3, Ross Booth3

1Department of Medicine, Columbia University, New York, USA; 2Image Processing Lab, Department of Electrical Engineering,

Maharana Pratap A&T University, Udaipur Rajasthan, India; 3Biological Engineering, Utah State University, Logan, USA.

Email: rs2010@columbia.edu; soonjo.kwon@usu.edu

Received 23 August 2008; revised 20 July 2009; accepted 25 July 2009.

ABSTRACT

Background: In the last decade, sodium mag-

netic resonance imaging was investigated for its

potential as a functional cardiac imaging tool for

ischemia. Later interest was developed in con-

trast enhancement for intracellular sodium. Lit-

tle success was reported to suppress extracel-

lular sodium resulting in the intracellular so-

dium MRI image acquisition using quantum fil-

ters or sodium transition states as contrast

properties. Now its clinical application is ex-

panding as a new challenge in brain and other

cancer tumors. Contrast enhancement: We

highlight the physical principles of sodium MRI

in three different pulse sequences using filters

(single quantum, multiple quantum, and triple

quantum) meant for sodium contrast enhance-

ment. The optimization of scan parameters, i.e.

times of echo delay (TE), inversion recovery (TI)

periods, and utility of Dysprosium (DyPPP) shift

contrast agents, enhances contrast in sodium

MRI images. Inversion recovery pulse sequence

without any shift reagent measures the intra-

cellular sodium concentration to evaluate ische-

mia, apoptosis and membrane integrity. Mem-

brane integrity loss, apoptosis and malignancy

are results of growth factor loss and poor

epithelial capability related with MRI visible in-

tracellular sodium concentration. Applications

and limitations: The sodium MR imaging tech-

nical advances reduced scan time to distinguish

intracellular and extracellular sodium signals in

malignant tumors by use of quantum filter tech-

niques to generate 3D sodium images without

shift regents. We observed the association of

malignancy with increased TSC, and reduced

apoptosis and epithelial growth factor in breast

cancer cells. The validity is still in question.

Conclusion: Different modified sodium MRI

pulse sequences are research tools of sodium

contrast enhancement in brain, cardiac and

tumor imaging. The optimized MRI scan pa-

rameters in quantum filter techniques generate

contrast in intracellular sodium MR images

without using invasive contrast shift agents.

Still, validity and clinical utility are in question.

Keywords: Sodium MRI; Double Quantum; Inver-

sion Recovery; Contrast Enhancement; Cancer

1. BACKGROUND

Sodium-23 (Na-23) nuclei are in abundance in the body

but exhibit poor magnetic resonance sensitivity and

serve as sodium MRI clinical imaging modalities. The

sodium Na-23 nucleus is detectable by MRI due to its

3/2 spin existing as -3/2, -1/2, 0, ½, 3/2 (5 states).

Physical nature of sodium-23 with spin = 3/2 is suitable

to have a nonvanishing magnetic moment and Larmor

frequency = 11.26 MHz and exhibits four transition

states with five different energy levels in the static mag-

netic field. MR detection sensitivity is very low (9.3 %)

at in vivo concentration (approximately 0.05 %).

Sodium is abundant (100 %) in living body tissue in

3/2 spin state. The sodium ion is the predominent cation

in the extracellular fluid (139 mmol/L), while its intra-

cellular concentration is very low (in the order of 8-10

mmol/L), mainly bound with glycosides and proteins.

The role of increased intracellular sodium due to con-

certed efflux through Na+/K+ ATP-ase intrinsic action,

angiogenesis, and cell proliferation was described in

ischemia, hypoxia, arrhythmia, myocardial edema, and

tumors [1]. The sodium concentration is sensitive to

disease as an indicator of cellular and metabolic integrity.

Na-23 concentration in tissues is present in the order of

tens of millimoles. The low sodium concentration results

in low signal-to-noise ratio of 23Na MR imaging in long

imaging times and/or poor spatial resolution. Therefore,

it needs the use of shift reagents or pulse sequences with

R. Sharma et al. / J. Biomedical Science and Engineering 2 (2009) 445-457

446

filters such as single-, double-, triple-, and multiple

quantum (SQ, DQ, TQ and MQ) filters to enhance the

sodium contrast [2].

Evidence of sodium transfer rates across the blood-

brain barrier dates back to its use in MRI [3] followed by

the use of spin echo pulse sequence for experimental

sodium imaging with first surface coil [4]. Soon after it,

in vivo clinical sodium imaging at clinical 1.5 T scan-

ners was first described using multislice spin echo pulse

sequences [5]. Later developments were made at high

magnetic field MRI scanners using ultrashort echo time

sequences and hardware improvements that permitted

better spatial resolution with shorter imaging times and

better quantitative measurements of tissue sodium con-

centration without use of shift reagents [6].

This paper describes the phases and roles of quantum

filters in pulse sequence design to generate intracellu-

lar/extracellular sodium contrast, high signal-to-noise

ratio, and high resolution in magnetic resonance sodium

images by triple-quantum filter and double inversion

recovery pulse sequence without using paramagnetic

shift reagents. Triple quantum filter was evaluated as a

superior method for high signal-to-noise ratio but needs

a long acquisition time [7]. Other available multiple-

quantum, single-, and double quantum filter tomographic

magnetic resonance pulse sequences generate intracellu-

lar sodium images with high signal-to-noise ratio but

need paramagnetic shift reagents as contrast agents.

1.1. Nature of Sodium MR Signal

At high field magnetic resonance application, the applied

phase-cycled radio frequency pulses at set time intervals

cause an alignment of nuclei populations within the

sample, resulting in a measurable signal in the transverse

plane. Optimized times of echo delay (TE) in single-,

double-, triple quantum filters; optimized times of inver-

sion times (TI) for inversion recovery pulse sequence;

and application of a multiple quantum filter after injec-

tion of paramagnetic shift contrast reagents detected the

distinct intracellular sodium signal to obtain a sodium

weighted image. As a result, two sodium MRI signals

are achieved with distinct MRI signal intensities to

measure sodium intracellular and extracellular concen-

trations in the tissue. The MR signal intensity is related

with tissue sodium content as follows: The MR signal

intensity of Na-23 is:

MR sensitivity = % Na-23/H-1. C (Na-23). A.γ. S2+S,

where % Na-23/H-1 is sodium abundance, C (Na-23) is

tissue concentration, A is MR scanner intrinsic parameter,

γ is Gyromagnetic Ratio and S is nuclear spin.

Due to the Gyromagnetic Ratio of Na-23 = 11.25

MHz/T relative MR sensitivity is lesser. Moreover, sen-

sitivity is further decreased due to less sodium content in

the tissue: about 1/1000th MR sensitivity of proton, so it

needs use of high magnetic field imaging scanner and

data acquisition techniques with contrast enhancement

mechanisms, such as MQ, SQ, DQ and TQ. Sodium

concentration is higher in blood than that in myocardium

and other tissues. This high sodium density in blood

contributes to tissue contrast in sodium MR imaging.

However, a very small amount of Na-23 is MR visible in

free ionic extracellular form due to poor sensitivity.

Other intracellular forms of invisible sodium are mainly

bound to proteins and phospholipids in the tissue, so

intracellular sodium MRI visualization needs quantum

filters.

1.2. Quadruple Interactions and Sodium MR

Signal

Sodium signal is conventionally represented as quadru-

ple interactions between Na-23 and its molecular envi-

ronment. In sodium nuclei, magnetic resonance excita-

tion and subsequent transition of electrons to different

energy levels of nuclear spins produce single-, double-,

triple-, and multiple quantum sodium transition MR sig-

nals as said earlier. These different quantum transitions

can be filtered by use of specific SQ, DQ, TQ and MQ

pulse sequence techniques by use of selective phases in

pulse sequence design as described later. These tech-

niques also produce spectral patterns reflecting the per-

centage of invisible intracellular sodium. These sodium

spectra are iterated superimposed positions of different

spectra and represent simulated signals from intracellular

sodium in the cytoplasm including interstitium, mito-

chondria, intravascular space, and so on. However, this

invisible “intracellular” Na-23 results from the presence

of a broad frequency peak (T2, very short component). It

masks the corresponding frequency peak. Another con-

tributory factor to intracellular Na-23 MR invisibility is

long correlation times for short and long T2 components.

For the heart, the correlation time is in the range of 10-12

to 10-9 seconds. As a result, intracellular sodium is in-

visible. The double quantum pulse technique (instead of

single quantum pulse technique) selects only double

quantum transitions of sodium. These double quantum

transitions allow sodium to be more ordered with long

correlation time. These pulse sequence techniques visu-

alize the intracellular sodium.

1.3. Nature of Sodium Nuclei and MRI

Properties

The Larmor frequencies of sodium in extracellular and

intracellular sodium populations are the same because

they have the same resonant frequencies. Therefore,

three different approaches are used in distinguishing

these two types of sodium nuclei. These include different

relaxation times between intracellular and extracellular

sodium, the use of paramagnetic shift reagents, and the

use of special multi-quantum pulse sequences.

R. Sharma et al. / J. Biomedical Science and Engineering 2 (2009) 445-457

447

1.4. Sodium Relaxation Times

Based on the observation that tissue transverse relaxa-

tion time for the intracellular space is smaller than that

of extracellular space, intracellular space can be imaged

by shortening the spin-echo time. Intracellular sodium

signal intensity proportionally increases due to intracel-

lular space. Sodium transverse relaxation time (T2) is

biexponential in nature, while sodium is bound with

negatively charged macromolecules – mainly proteins or

metabolites. Perhaps the longitudinal relaxation time (T1)

also behaves as biexponential.

1.4.1. Paramagnetic Shift Reagents

Paramagnetic shift reagents are chelates of paramagnetic

lanthanide ions with anionic complexes. These sub-

stances don’t cross the intact cell membranes. These

reagents interact with extracellular sodium and introduce

a frequency shift in the resonance frequency of extracel-

lular sodium. This shift allows the advantage of produc-

ing two separate spectral peaks arising from intracellular

and extracellular sodium. Common lanthanide ions used

as paramagnetic shift reagents are Dy3+ (Dysprosium),

Tm3+ (Thulium), Gd3+ (Gadolinium), PPP5- (Tripoly-

phosphate), and TTHA6- (Tetraethylene Triaminehexa

acetic acid). These reagents bind with intracellular cal-

cium and cause physiological disturbances and, in some

cases, toxicity, so their use is limited. To overcome such

problems, several approaches were recently developed

based on the modification of pulse sequences and hard-

ware improvements as described in the following sec-

tion.

1.5. Quantum Filters

These are MR pulse sequences that allow direct observa-

tion of multiple quantum transitions. These filters usu-

ally are constructed by phase cycling manipulations. The

general pulse sequence scheme is 90°-t/2-180°-t/2-90°

-π-90°-acquire, where t is creation time and π is evolu-

tion time by use of pulsed field gradients. Characteristi-

cally, these filters are sensitive to variations in phase

cycles up to 5° and magnetic field inhomogeneity. The

filter can be single-, double-, triple-, or multiple quan-

tum and allow single, double-, triple-, and multiple

quantum transitions. In the following sections, we intro-

duce readers to our strategy of quantum filters used in

sodium MRI pulse sequence design.

1.5.1. Single Quantum Pulse Sequence

Due to its multiple spin state transitions, an alternative

MR single quantum approach is used to measure intra-

cellular Na content based on the interaction of Na poly-

anions and their resultant effects on nuclear spin transi-

tions. Spin 3/2 nuclei (such as Na-23) have a nonvan-

ishing quadrupole moment, allowing interaction with

electrostatic field gradients. The pulse sequence is a spin

echo sequence.

1.6. Multiple Quantum Pulse Sequence for

Origin of Double- and Triple Quantum

For intracellular sodium, using only multiple-quantum

(MQ) NMR requires paramagnetic shift reagents (SRs)

that have distinct disadvantages including: toxicity, pos-

sible drug interaction, expanded space, and imperme-

ability to the blood brain barrier. It is known that the

correlation time, tc of the time variations of the electro-

static field gradients in spin 3/2 nuclei satisfies the rela-

tion if wLtc >> 1, where wL is the Larmor frequency.

These nuclei can display biexponential relaxation and

MQ spin transitions do occur in the nuclei and these

transitions are detected by specific pulse sequences

called multiple- quantum filters.

A phase-cycled RF pulse sequence applied over a

slice-select axis selects both echo and anti-echo signals

of spin undergoing multiple-quantum spin transitions at

resonating Larmor frequency. The RF pulse has both a

flip angle and a phase angle. The phase angles are cycled

using multiple-quantum transition filter and simultane-

ously produce an output signal proportional to the sum

of echo and anti-echo signals of MQ coherence. It in-

cludes the evolution period with a flip angle of 180° to

refocus the RF pulse to avoid inhomogeneity-induced

amplitude deterioration. This output signal is measured

by induction current in the RF coil during the realign-

ment process and is used in tomographic MQ transition

23Na MRI images.

The pulse sequence comprises: a preparation period of

length tP extending from time t0, an evolution period of

length te and a detection period of length td. The RF

pulse sequence manipulates the selected nuclei to exhibit

them with single quantum coherence. During the evolu-

tion period, the pulse sequence implements a MQ filter

which isolates the coherence (e.g. double- or triple

quantum) and simultaneously selects both echo and anti-

echo signals corresponding to the coherence by; a) an

evolution period first flip angle RF pulse timewise cen-

tered at tP which converts the selected nuclei from sin-

gle-quantum coherence to the selected multiple- quan-

tum coherence; b) an evolution period of second 90° flip

angle RF timewise centered at tP+te, converts the se-

lected nuclei from multi-quantum to single-quantum

coherence. Therefore, during the detection period, se-

lected nuclei exhibit single quantum coherence to gener-

ate a single quantum signal.

The resultant collective phase angle of evolution pe-

riod RF pulse= ΦE=Φ3-2Φ4+Φ5, wheree ΦE is collective

radio frequency phase during evolution time, and Φ4 &

Φ5 are phases of 90° RF pulses. A further preparation

period of 90° flip angle radio frequency slice-selecting

pulse, a preparation 180° flip angle refocusing pulse, and

a detection period 180° flip angle radio frequency are

applied – centered at t0, ½ tP and tP+te+1/2 td, respec-

tively. The preparatory 90° flip angle RF pulse selects a

R. Sharma et al. / J. Biomedical Science and Engineering 2 (2009) 445-457

448

slab of sample perpendicular to a slice-selection axis as

shown in Figure 1. The preparatory 90° flip angle

slice-selection RF pulse has phase angle Φ1, the pre-

paratory 180° Rf refocusing pulse has Φ2, and detection

period 180° flip angle RF pulse has phase angle ΦP of

the RF pulses during preparation time (evolution period

90° flip angle RF phase Φ3) as described earlier(8). The

phase angle is ΦP= Φ1-2Φ2+Φ3 and the output signal will

be Φ= ΦP-m ΦE+ Φ5+2 Φ6+ ΦR, where m is the integer of

magnetization, Φ is the phase of detected signal, and ΦR

is the phase of receiver.

For double quantum filtered images, m=+2, and for

triple quantum filtered images, m=+3. The phases will

be Φ1= (n/2)π, Φ2=[n/2+int(n/4)] π, Φ3=(n/2)π,

Φ4=Φ5=Φ6=0 and ΦR =nπ for double quantum filter; and

phases will be Φ1= (n/3)π, Φ2=[n/3+int(n/6)] π,

Φ3=(n/3+1/2)π, Φ4=0, Φ5=(1/6) π, Φ6=0 and ΦR

=[n+int(n/6)]π for triple quantum filter, where n/6 is

integer part of the variable n/6. The variable n is positive

integer of 0…N-1. N is a multiple of 8 and 12 for dou-

ble- and triple quantum filters respectively.

The magnetic gradient pulse sequence is also applied

along with the RF pulse sequence for positional infor-

mation in the data. A slice-select gradient pulse, first and

second phase encoding gradient pulse, and read-out gra-

dient pulse are applied along read-axis after detection

period 180° RF refocusing pulse during output signal

observed as shown in Figure 1. The read-out pulse area

before time tP+te+td is equal to the area of read-out gra-

dient pulse. These gradient pulses exhibit simultaneous

synchrony with RF pulses.

1.6.1. Inversion Recovery Pulse Sequence

The inversion recovery technique can suppress the ex-

tracellular sodium nuclei with specific ranges of the lon-

Figure 1. The figure illustrates a series of radiofrequency

pulses and a synchronized series of magnetic gradient pulses

for phase cycled evolution period 180° flip angle refocusing

pulse.

Figure 2. A scheme of inversion recovery pulse sequence is

presented. On top, one cycle of IR with two recovery curves is

shown for a single slice. First, 180˚ pulse is flipping the

magnetization Mz for first recovery followed by 90˚ pulse for

FID and second recovery within TR period. At bottom, the

application of two inversion pulses πA and πB is shown for

slices A and B with simultaneous application of RF and gradi-

ent pulses.

gitudinal relaxation time, T1 using fast spin-echo pulse

sequence. The sequence has two sets of slice selective

inversion pulse trains (long recovery period TI1 for ex-

tracellular sodium and short recovery period TI2 for in-

tracellular sodium nuclei). The first inversion pulse is

played during a long recovery period TI1, during which

extracellular sodium magnetization recovers just past its

null point while longitudinal magnetizations of intracel-

lular sodium nuclei are almost fully restored. The second

set of inversion pulse is played during short recovery

period TI2, when intracellular Na nuclei magnetizations

reach their null points following TI2 interval and become

180° out-of-phase. After inversion pulses, a fast spin-

echo acquisition sequence is applied to achieve mag-

netization in z-direction (Mz) as following:

Mz=M0 [(1-2e-TIe/T1e + e-TR/T1e)(1-2e-TIi/T1i) (1)

where ‘e’ represents extracellular sodium nuclei and ‘i’

represents intracellular sodium nuclei. M0 is initial lon-

gitudinal magnetization, TI1 & TI2 are long and short

inversion times, and T1e & T1i are longitudinal relaxation

times of extracellular and intracellular sodium nuclei,

respectively.

The relaxation time for intracellular Na nuclei varies

from 5-10 milliseconds (for Na bound to large molecules

or dispersed in highly order environment) to 51 milli-

seconds (for extracellular sodium nuclei in free solution).

Based on 3D projection reconstruction imaging pulse

sequence, echo time TE as short as 0.1 ms can be

R. Sharma et al. / J. Biomedical Science and Engineering 2 (2009) 445-457

449

achieved. This captures the more rapid decay of short T2

sodium components in the NMR signal so as to further

improve the signal to noise (SNR).

As shown in Figure 2, radio frequency pulse at 180˚

is followed by a 90˚ pulse for slice excitation utilizing

echo-train length with any option of selective or nonse-

lective, single or composite, sinc, sech or tyco pulse as

needed. So, inversion pulses are played out during in-

version TI1 and TI2 periods for both types of sodium

nuclei. Data acquired includes projection reconstruction

with slice selection or without slice selection using

spin-echo or gradient echo. Thus, inversion/acquisition

periods are played out for several slices per repetition of

fast spin echo (FSE) sequence exhibiting two distinct

null points for both sodium nuclei populations.

2. MRI PHYSICS OF CONTRAST

ENHANCEMENT BY INVERSION

RECOVERY AND TWO NULL POINTS

After first 180° RF pulse flips the magnetization from

M-z  Mz’, the two different inversion times (TI1 and TI2)

for two sodium populations exhibit two null points and

growth of longitudinal magnetizations. In this sequence,

soon after TI, a 90˚ RF pulse is applied to flip the longi-

tudinal magnetization into the x-y plane for the T1

growth curve within repetition time TR. At this point,

frequency induction decay (FID) is measured when the

longitudinal magnetization is flipped into the x-y plane.

Thereafter 180˚ RF pulses are repeatedly applied. The

magnetization signal ‘S’ may be measured as: [S] α

M0(1-2e-TI/T1)(1-eTR/T1), as T1 recovery curves for both

180˚ and 90˚ degree pulses applied.

The intracellular and extracellular populations of nu-

clei have different longitudinal relaxation times, so they

generate intracellular/extracellular sodium contrast. To

set the IR pulse sequence to suppress the contribution of

sodium nuclei with specific ranges of T1, the inversion

time TI1 or TI2 is set as (ln 2)(T1ex), where T1ex is the

composite longitudinal relaxation time. This inversion

time then determines the time between the 180o and 90o

pulses in the IR pulse sequence.

2.1. Projection Reconstruction Methods

First, projection reconstruction was proposed based upon

Fourier transformation of the FID (free induction decay)

signals [9].

Now, these FID signals are obtained by rotating a

magnetic field gradient with back-projection to deter-

mine the spin-density function or enhanced contrast. So

far, this technique has been used only for animal hearts.

These signals can be gated to heart rate to reduce the

motion artifact. Signals are reconstructed from 12 pro-

jections. Each projection is obtained from an average of

320 FID signals. For image matrix 64 x 64 total data

acquisition, time is approximately 15 minutes.

2.2. Three-Dimensional Fourier Techniques

In this technique, phase-encoding is made in two or-

thogonal directions, and a fixed gradient is applied in a

third direction of read-out gradient [10]. The pulse- en-

coding steps are applied, typically 40 steps along each

phase-encoding direction. As a result two echo signals

with different spin-echo times are generated and added

in a coherent manner to enhance contrast. This phe-

nomenon also improves the signal-to-noise ratio. This

technique is used for the human brain. In cardiac sodium

imaging the utility of this technique is limited to animal

heart experiments. Typically short TR = 100 ms is used

to get data in 3 hours, including different spin echoes

summation. The application of greater sodium signal in

the left anterior descending artery was observed in oc-

clusion, followed by re-perfusion of circumflex in the

coronary artery after ischemia.

2.3. Hybrid Spin Echo Techniques

The spin-spin T2 relaxation time of intracellular sodium

is shorter than that of the extracellular sodium compart-

ment, so it is difficult to observe it to create contrast, and

it needs a hybrid spin-echo pulse sequence. In the hybrid

approach, both projection-reconstruction and Fourier

encoding schemes are used at the same time. Fourier

analysis does slice selection in one direction while an-

other projection-reconstruction is performed in two other

orthogonal directions. This technique is good for clinical

MR scanners with gradient refocused sequence with

modified head coil to generate echo time as short as 2.8

msec [11].

2.4. Surface Coil Techniques and Coil

Sensitivity

Sodium imaging at short T2 relaxation times can be per-

formed by use of a customized radio-frequency coil fo-

cused on the region-of-interest to improve the signal-to-

noise ratio. The pulse sequence is applied with optimiz-

ing the minimum saturation time of preamplifier and

duration of 90-degree RF pulse. This effect minimizes

the magnetic homogeneity influence over the free in-

duction image and the short T2 can be imaged. This

technique is relatively better than others for human

cardiac sodium imaging on clinical scanners. First,

using different transmitter gains optimizes the gain for

90° pulse, and different transmitter gains at increased

and decreased gain values yields 23Na images at different

flip angles of 45°, 90°, and 135° with matrix sizes 32 x

32 x 32 points. Nonlinear least squares fit of the pixels

intensities in all three images determines the B1 field

strength (for 5% maximum intensity on image) as a

function of the related [Na]i image intensities in re-

sponse to the local RF coils for receive and transmit sen-

sitivities [13].

R. Sharma et al. / J. Biomedical Science and Engineering 2 (2009) 445-457

450

The image signal intensity depends on coil transmit

and coil receive sensitivity as:

|)sin(|

0kkkk MRI  (2)

where Ik is the signal intensity of the kth pixel, Rk is a

sensitivity at the coil center, and M0k is the equilibrium

magnetization. The flip angle Φk is a function of the

transmitter gain setting (TG) as:

20/

)10)( TG

kk CR (3)

(C · Rk) and M0k were each fitted with a single parameter

for each pixel. The constant ‘C’ is independent of spatial

position and receives sensitivity. It is also directly pro-

portional to the transmit field distribution for the coil

[13]. After normalization, the product (C · Rk) yields the

relative sensitivity Rk for the kth pixel. The B1 (expressed

as B1/2 ) depends on reference transmit power, TGref:

))2(/(10)(

20/





ktCR

Bref

 (4)

where tp is the pulse duration.

Local field B1 and longitudinal relaxation time T1

determine the correction factors for saturation for each

pixel k, SFk as follows:

)]

cos(1[

)

sin(][









TTR



 (5)

where B1k is the B1 field in the respective ROI and is the

gyromagnetic ratio for [Na]i using the pulse time tp=0.4

msec, and the TR=120 msec [13].

2.5. Rotating Frame Techniques

Conventionally, sodium imaging can be done using the

rotating frame technique based on superimposing a

magnetic field gradient on the excitation field B1. As a

result, during this excitation period, spatial localization

and spatial encoding may be performed very fast to gen-

erate sodium contrast. In this process, loss of MR signal

intensities is also minimized. In an isolated perfused

rabbit heart, sodium-rotating frame imaging experiment

was completed in 12 msecs in a 128 x 128-image matrix

with a clinical 1.5 T scanner [12].

2.5.1. Use of Contrast Agents

Different shift agents such as dysprosium or Tm (DOTP)

contrast agents increase the sensitivity and specificity of

sodium MRI by shifting intracellular sodium magnetiza-

tion away from extracellular sodium magnetization (ex-

hibited well as shifted peaks). Dextran-magnetite in-

creases the contrast between tumor and surrounding soft

tissue sodium images. Normal reticulo-endothelial cells

take up a greater amount of magnetic particles than tu-

mor cells do, which supports the potential for tumor so-

dium imaging by contrast agents. However, a major role

of sodium imaging seems to distinguish myocardial

ischemia.

Techniques used for sodium in vivo MRI: Several

approaches have been used. Despite little success, these

techniques are gaining interest in physiological MR im-

aging using intracellular sodium. Some landmark tech-

niques have been described for image generation.

2.5.2. Spectrally Weighted Twisted Projection

Imaging

The 3D sodium MRI imaging can be made faster by

reducing the T2 signal attenuation effects. This is im-

plemented by 3D twisted projection imaging. At high

spatial frequencies, the sample density is reduced, and as

a result the reduced readout time is achieved. This leads

to decreased T2 signal attenuation, which translates into

improved signal-to-noise ratio (SNR) without a loss of

resolution [14].

2.5.3. Biexponential Relaxation Effect and

Sodium Concentration Mapping

Dual-frequency RF coils with identical B1 field distribu-

tions at the two different observation frequencies pro-

vide sodium and proton mapping. These optimize both

dual frequencies in dual quadrature RF coils. Sodium

and proton channels are decoupled to make dual-quad-

rature birdcage configurations. The fourth harmonic of

sodium frequency happens to be very close to proton

frequency. This is suitable for echo-planar imaging and

metabolite quantification [15].

2.5.4. 3-D Triple Quantum-Filtered Twisted

Projection Na-23 Imaging

A new approach was developed based on a three pulse,

six-step, coherence transfer filter (with fast twisted pro-

jection imaging sequence) to generate spatial maps of

the TQ signal. In principle, three pulse coherence filter

leads to TQ sodium images. In these images, the image

intensity depends on the spatial variation of the flip an-

gle. This image intensity is lesser than the flip angle

chosen in the four pulse TQ filter. This technique allows

the generation of RF inhomogeneity corrected TQ so-

dium images after TQ signal variation. The pulse se-

quence of TQ filtering is based on spherical tensor op-

erators or a density matrix. It consists of three non-se-

lective RF pulses, and sometimes a fourth RF pulse be-

tween the three first and the second pulses, to refocus the

main magnetic field inhomogeneities [16]. In this pulse

sequence, RF induces different flip angles as θ30, 2θ30,

θ150, θ0…. flip angles where the phase stepped up through

the values 30°, 90°, 150°, and so on. The second pulse is

intended for the B inhomogeneities refocusing and is

absent in the case referred to as “three pulse”. The

preparation time is the period between two phases and

evolution time is the period between θ150 and θ0.

R. Sharma et al. / J. Biomedical Science and Engineering 2 (2009) 445-457

451

2.6. Simultaneous Quadruple and Double

Quantum Na-23 MR Imaging

Na-23 is a quadruple nucleus with biexponential relaxa-

tion or non-vanishing quadruple coupling. Both proper-

ties contribute to generate Na-23 multiple-quantum (MQ)

coherence [17]. The Multiple Quantum Filter pulse se-

quence can be represented as Eq.6:

(θ1,φ1)-tP/2-(θ2,φ2)-tP/2(θ3,φ3)-tE-(θ4,φ4)-Acq(T,φR), (6)

where refocused preparation time, φP and non-refocused

evolution time, φE may be represented as Eq.7:

43321 ;2



 EP (7)

where φ1-4 denote the phase of each RF pulse with flip

angle θ1-4, φR is receiver phase, and T is acquisition time,

each θ1=π/2 and θ2=π.

This sequence is good for detecting well-defined and

ordered structures in biological tissues. Both quadru-

ple-order and double-quantum signals can be generated

simultaneously by the same RF sequence without any

loss in signal amplitude. In addition, the acquired MQ

signals can be readily decomposed into their second –

and third – rank components, i.e. into quadruple order

and biexponential relaxation components. This allows

reduction of the scan time to acquire MQ signals of dif-

ferent coherence orders and ranks, which should prove

useful for in vivo studies.

2.7. Double Spin-Echo Imaging and

Transverse Relaxation Time

This approach of imaging is good for myocardial water

content analysis based upon the calculation of transverse

relaxation time from dual-quantum spin-echo MRI [18].

Right and left ventricles can be assessed for quantifica-

tion of myocardial edema. The transverse relaxation

times (T2) of ventricles can be calculated from the signal

intensities within multiple regions of interest over myo-

cardium as shown in Eq.8:

T2 =[(TE2-TE1)/ln(I1/I2)], (8)

where TE1 is first echo time, TE2 is second echo time,

and I1 & I2 are image amplitudes of first & second spin

echoes.

2.8. Double-Quantum-Filtered Na-23 MR

Spectroscopy

The extracellular EC-Na and intracellular IC-Na con-

tents are better visualized by multiple-quantum-filter

spectra acquired in the absence of chemical shift or re-

laxation reagents. The intracellular IC-Na sensitivity can

be enhanced by double-quantum-filter (DQF) Na-23

NMR to measure the detection of Na+ motion anisotropy

with the presence of residual quadruple splitting (19).

However, the application of this technique is limited to

the experimental heart studies using the pulse sequence:

90°-τ/2- 180° -τ/2 -θ-t1-θ-t(Acq), where τ denotes the crea-

tion time and t1 is evolution time, θ is RF flip angle.

The magnetization M (τ, t, θ) for the MQF Na-23

NMR signal, when the quadruple splitting factor (ωQ) is

zero, is given by Eq.9:

M(τ, t, θ)=αM0[exp(-τ/T2s) - exp ( -τ/T2f )]

x [exp(-t/T*2s )-exp(-t/T*2f)] (9)

x sin2θ(1 - 3 cos2θ)

where α is a factor which depends on the degree of

quantum coherence , M0 is equilibrium magnetization,

T2f & T2s are fast & slow transverse relaxation times, and

T*2f & T*2s are the corresponding inhomogeneity-

broadened times. An important implication for this tech-

nique is the possibility of measuring intracellular sodium

concentration in animal hearts with the DQF spectrum in

the absence of shift or relaxation reagents. Significant

attenuation of the DQF spectrum derived from extracel-

lular Na+ allows to differentiate the NMR spectrum from

intracellular Na+.

2.9. Double Quantum Filtering and

Spin-Quantum Coherence

Conventionally, the double-quantum filtering method for

sodium imaging is completed in a four-step phase-cy-

cling scheme. In this process, the radio-frequency pulses

generate single-quantum coherence. Its phase appears

similar to the phase of a double-quantum coherence sig-

nal in the pulse sequence. Consequently, the interse-

quence stimulated echo passes through the double-

quantum filtration in the phase cycling scheme and gra-

dient pulses in the DQ pulse sequence, so the sin-

gle-quantum coherence is an unwanted component in

this pulse sequence. It is eliminated by the use of spoil-

ing RF pulses followed by dephasing gradient pulses

incorporated into the DQ filtering pulse sequence. These

spoiling pulses disperse the pulse transition and elimi-

nate the magnetization components of the intersequence

stimulated echo in the DQ pulse sequence [20]. Another

good way to eliminate single quantum coherence is to

increase the repitition time TR. Moreover, a low signal

level of DQ coherence also demands many signal aver-

aging at short TR to keep short scan time and not a

longer TR.

2.10. Chemical Shift Selective Acquisition

of Multiple Quantum-Filtered Na-23

In cardiac imaging, MQ signals over a wide off-reso-

nance bandwidth is a problem which causes interference

between echo and anti-echo. It is due to a resonance off-

set insensitive to the flip angle of the creation RF pulse;

usually the second π/2 pulse. Off-resonance effects are

applied to eliminate the MQ signal in the presence of a

chemical shift. It suppresses the MQ signal over a wide

range of off-resonance bandwidth [21]. Existing tech-

R. Sharma et al. / J. Biomedical Science and Engineering 2 (2009) 445-457

452

niques for chemical-shift-selective acquisition of SQ

signals can be combined with MQ off-resonance effects

to enhance the selectivity of chemical shift.

2.11. Double Quantum Filtering and

Elimination of Intersequence

Stimulated Echo

In cardiac sodium imaging the ‘intersequence stimulated

echo’ is an unwanted component because the phase of

this echo has the same properties as that of the dou-

ble-quantum (DQ) signal. Using a partition method and

computer simulation can eliminate the magnetization

components of the intersequence stimulated echo [22].

The DQ filter pulse sequence with non-refocused prepa-

ration time (τP) and evolution time (τE) may be ex-

pressed as: (θ1,φ1)-τP- (θ2, φ2)-τE-(θ3φ3)-Acq (t2 ,φR),

where φn denotes the phase angle of each RF pulse

with the flip angle φn; t2 is acquisition or detection time

and φR is receiver phase.

2.12. Multiple Quantum Filters of Arbitrary

Phases of Na-23 Nuclei

These Triple Quantum filtered data acquisition, uses the

pulse sequence with arbitrary phase values: 90°(θ1)- t/2

-180°(θ2) - t/2 -90°(θ3) -δ - 90°(θ4) - acq(θ5) (T), where δ is

evolution time, t is the acquisition time, θ1, θ2, θ3, θ4,

and θ5 are the phase values. Multiple-quantum-filtered

(MQF) Na-23 NMR spectroscopy may provide increased

sensitivity in detecting IC Na and thus offers the possi-

bility of monitoring changes in IC Na content without

SR. The amplitude of the MQF spectrum is determined

by several factors such as transverse relaxation times,

creation times, and the amount of Na that exhibits MQ

coherence. By the behavior of relaxation and greater

IC-Na MQ coherence, it is possible to have MQF spec-

trum with intracellular sodium to measure IC-Na content

[23].

2.13. Spiral Cardiac Respiratory Gate

Multi-Shot Functional MRI as

Possibility

Physiological fluctuations (possibly by sodium) are

known to be a major contribution to noise in fMRI data.

Cardiac noise adds noise to both the magnitude and

phase of the image in regions localized near vessels and

ventricles. Another factor, respiratory motion, has both

global changes in the phase of image and localized

variations in the image magnitude, particularly near ven-

tricles. Recently, an empirical model was used to capture

the observed features of physiological noise in fMRI

data to reduce intracellular ionic noise in fMRI data us-

ing a 3-D cylindrical-stack spiral pulse sequence [24].

Briefly, spiral waveforms of sequence were applied on

the x and y gradients. The waveforms were designed get

low slew rate limited except for the first few points, and

their maximum amplitude was 0.75 G/cm to give a 3.8 x

3.8 mm nominal in-plane resolution. Six interleaves

were measured by adjusting the spiral waveforms (mul-

tiplying the x and y gradients by coefficients of a rota-

tion matrix) to effectively rotate the k-space trajectory in

the kx - ky plane. At the end of each TR interval, crusher

gradients were used to minimize spillover of the FID

signal into subsequent intervals. Their amplitudes were

set to generate about 3π dephasing over each voxel di-

mension of covered volume of interest. It results in a

“stacked spirals” k-space acquisition scheme. With the

spiral acquisition, all gradient moments, Mn(t), can be

shown as Eq.10:

Mn(t)

∫G(t)tndt (10)

with t = 0 at the center of the RF pulse, are zero at the

center of k-space (t = tc).

The zeroth [M0(t)] and first [M1(t)] order moments of

the slab select null at gradient at t = tc by using two addi-

tional gradient phases after the slice selection lobe. Fur-

thermore, the magnetization were nulled at t =TR i.e.,

M0(t) for the phase encode gradient and M0(t) and M1(t)

for the spiral waveforms. These spiral waveforms are

made of trapezoid waveforms at amplitude with mini-

mum gradient moments. In addition, a phase encoding

scheme is inverted and quadratic RF phase modulation is

performed to minimize stimulated echoes. Additional

gradient pulses can minimize stimulated echoes after

acquisition. Another good practice to decrease the scan

time is inserting extra ‘dummy views’ to avoid extra

acquisitions when the physiological phase corresponds

to the view far away from the actual view to acquire,

although these ‘dummy views’ reduce fMRI temporal

resolution and lead to improper assessment of functional

activity & physiological variance. Cardiac ordering

shows greater utility due to shorter cardiac periods. It

can reduce inter-image variance by using k-space post-

processing techniques and may enhance sodium contrast.

This technique is very promising but longer scan times

are required.

3. CLINICAL SODIUM MRI TRIALS

Sodium MRI can be used in anatomical imaging appli-

cations in brain, tumors and cardiac cycle (25-29). Spe-

cial attention was focused on achieving intracellular so-

dium images. Triple Quantum filter in spin echo acquisi-

tion mode and inversion recovery methods are consid-

ered useful for it without using shift contrast agents.

However, the triple-quantum filtered technique requires

a very long repetition time. Inversion recovery technique

suffers from poor nulling and poor suppression of ex-

tracellular or intracellular sodium population (30-31).

Moreover, for imaging applications with the resolutions

of 64 or 128 pixels, physiological noise corrupt even

better regions without variation. These are referred to as

‘ghosts’, and may be shifted away from the area of in-

R. Sharma et al. / J. Biomedical Science and Engineering 2 (2009) 445-457

453

terest or avoided by using the ‘view ordering’ technique.

It was done by sorting the views for which physiological

period T = T0 (α =1) ‘low frequency sort mode’ or T = T0

x N/2 ‘high frequency sort mode’, where α =N/2,

N=total number of views). For cardiac sodium MRI data

acquisition in real time, the physiological phase and

corresponding view are obtained again and again till all

views are expended. Physiological ghosts, phase varia-

tion is cause of further inter-image fluctuations in car-

diac MRI time course and affect activation related

changes in the acquired signal.

The sodium concentration may be measured by plac-

ing a phantom next to tumor or animal during MR im-

aging. The mean sodium concentration per kilogram wet

body weight in the region of interest will be:

SFR

Na kk



][ (13)

The coefficients a and b were calculated from the so-

dium concentrations C1 and C2 and signal intensities I1

and I2 of the two phantoms as

Intracellular sodium concentration measurement: Sev-

eral attempts to measure the intracellular sodium con-

centration including sodium flame photometry and ion

electrodes in serum; ratiometric, electrom beam CT,

atomic force (AFM) spectroscopy in tumors; and sodium

MRS in myocardium suggested the role of ATP energy

and Na+/K+ pump in ischemia & tumor apoptosis.

Therapies that alter tumor ion homeostasis or affect/

destroy tumor cell membrane integrity are likely to gen-

erate changes that are observable with 23Na MR imaging

and sodium concentration measurements [29,31]. For

different methods, the concentration of intracellular so-

dium can be measured based on NMR peak areas by

subtraction or magnetization ratio and intracellular/ex-

tracellular volumes as following:

SFR



 (14)

and

2aC

SFR

b (15)

where R1 and R2 are sensitivity factors.

In the following section, we describe our experiments

in support of increased sodium in malignancy and asso-

ciation with apoptosis in breast tumors and epithelial

growth factor in breast isolated cancer cells. For simplic-

ity, we introduce readers to the biochemical basis of in-

creased sodium in tumor cells. The low sodium-hydro-

For MQ method, gen exchange kinetics by slow sodium-hydrogen trans-

porter is center point. In malignant tumor cells, acidic

pH enhances the intracellular sodium that impairs both

the sodium-hydrogen transporter ability and Na+/K +

pump resulting in reduced [Na+/K+] ATPase enzyme to

release sodium (high intracellular sodium concentration

inside cells) from mitochondrial oxidation which also

triggers cells to slow down apoptosis or other associated

epithelial growth factors [high intracellular Na with low

apoptosis and EGF]. The low concentration of intracel-

lular sodium is sensitive to any malignancy change and

serves as a diagnostic rapid MRI imaging assay to test

drugs.



)(,0

)(0

(~][/

][

1][ xvMRinR

yvM

MQi MNaVV

Na 





















(11)

where [x] and [y] denote the corresponding tissue and

standard phantom intracellular sodium concentrations. K

is a constant equal to MM0,v (y) /[y]. VR/Vin is the ratio of

reference and intracellular volumes in tissue.

For DQ method,

[Nai+]DQ= {(Ain/Aref)-(intercept)}/(slope) (12)

where Ain and Aref represent areas under tissue and

phantom intracellular sodium peaks. 3.1. Chemosensitivity Rapid Assays Using

Sodium MRI

For SQ method,

IC[Na] = {Ain/Aout}{Vout/Vin}{EC[Na]} (13) Very limited studies reported the value of intracellular

sodium in a chemosensitivity assessment by cell prolif-

eration and apoptosis in prostate and breast tumors.

However, its applications are expanding to glioma, car-

tilage, and liver [32,33]. A technical advance was re-

ported using an inversion recovery pulse sequence and

optimization of inversion times to achieve intracellular

sodium images in less time. Its application was applied

in texotere chemosensitivity response to PC 3-induced

mouse prostate and MCF 7-induced rat breast tumors as

shown in Figure 3. The enhanced contrast of increased

intracellular sodium MRI signal was correlated with

histopathology characterization of tumor tissues [34,35,

Ain and Aout are areas of the SQ 23Na NMR peaks for IC

and EC spaces; EC [Na] is extracellular Na concentration.

With these measurements, changes can be observed

much earlier than with the effects of anatomic remodel-

ing. 1H MR imaging FLAIR sequence, use of MR con-

trast agents, and T2-weighted imaging along with 23Na

MR imaging may improve tumor visualization of a ne-

crotic core or proliferating zones by multiparametric

analysis methods. Other approaches of enhancement of

3D 23Na images signal-to noise ratio depend on receivers

and coils and the twisted-projection imaging pulse se-

quence [32].

Openly accessible at

R. Sharma et al. / J. Biomedical Science and Engineering 2 (2009) 445-457

454

Figure 3. Inversion recovery pulse sequence using optimized inversion

time generated high resolution intracellular sodium images of tumor (right

on the phantom image) in each panel. On right, a tumor image is enlarged

to show regional heterogeneity of MCF 7 induced breast tumor in rat. P

represents image of agarose-sodium phantom and alphabets at different

points indicate different stages of tumor growing cells to correlate with

histology as shown in table to highlight the accuracy of sodium MR signal

intensity and tumor pathology staging [36].

Figure 4. A Comparison between normal and malignant hu-

man breast epithelial cells (grown in a 2D monolayer and 3D

culture matrix) shows a role of EGF in luminal structure for-

mation. Normal human breast epithelial (NHBE) cells (grown

in the 3D culture matrix) form a luminal structure of normal

phenotype. (a) Normal human breast epithelial cells in

monolayer without rBME (2D monolayer) with normal EGF,

(b) NHBE cells in rBME (3D matrix) with reduced EGF, (c)

NHBE cells in rBME (3D) with normal EGF, (d) Malignant

human breast epithelial cells (HTB-132 obtained from ATCC)

in rBME (3D) with reduced EGF. Notice the role of FGF and

the rBME matrix used in association with malignant character-

istic of cells.

36]. However, the exact cause of increased TSC is not

known. In another set of breast cancer culture cells, we

observed the reduced EGF in tissues sensitive to sodium

ion homeostasis and metabolic integrity.

3.2. Correlation between Intracellular

Sodium Signal and Malignancy

Elevated TSC in breast lesions measured by non-inva-

sive 23Na MRI appears to be an indicator at the cellular

level associated with malignancy. This Na-23 MRI

method may have potential to improve the specificity of

breast MRI with only a modest increase in scan time per

patient [37]. Several physiological and biochemical

changes associated with proliferating malignant tumors

may cause an increase in total tissue sodium concentra-

tion (TSC) as a result of impaired sodium-hydrogen

transporter and [Na/K] pump activity resulting in re-

duced EGF (Figure 5).

Normal and malignant epithelial ductal cells were

grown on a reconstituted basement membrane extract

(rBME). In the event of the correct signaling from

growth factor and extra-cellular matrix proteins, isolated

human breast epithelial (HBE) cells formed their origi-

nal phenotype in vivo. HBE cells in the absence of re-

constituted basement membrane extract (rBME) failed to

assemble organized structures, and arrested growth when

R. Sharma et al. / J. Biomedical Science and Engineering 2 (2009) 445-457

455

EGF+ECMproteins  

GlucoseNa

transporter

IntracellularboundNa‐Proteins‐OH

‐

NullPoints (A)and(B)(TQ,DQandMQfiltersshowdifference)

MagneticMomentNa

‐P

Freeextr a ce l l u l a r Na

‐

(A) LongT

(B) LongT

ATPase

Cell

Membrane

(outer) (inner)

Sodium

ETS

Figure 5. This schematic description represents the origin of in-

tracellular sodium across the membrane and different null points

of sodium inside and outside generate contrast due to different

longitudinal relaxation constants as basis of sodium MRI. The in-

creased intracellular sodium leakage out of cells is associated with

hypoxia and apoptosis in cancer. EGF: Epidermal Growth Factor,

ECM: Extracellular matrix, and ETS: Electron Transfort System.

they reached confluence. HBE cells on-top of rBME

could display an acinar structure with a dead luminal

space in the absence of epidermal growth factors (EGF)

(Figure 4(b)). This simulated the ductal structures of

breast epithelial cells found in vivo. Under the same

condition, carcinoma cells exhibited colony overgrowth,

luminal filling, and loss of intracellular sodium (Figure

4(d)). These changes eventually reduce the resistance

against apoptosis and enhance the cell proliferation re-

sulting in severe morphological deformities visible by

microscopy and imaging as earlier reported elsewhere

[37]. It was observed that EGF disrupted the formation

of luminal structures of HBE either in monolayer or in 3

dimensional cell cultures with rBME. It may be attrib-

uted with the possibility if luminal structures bound with

sodium get free by the disruption after adding EGF.

When EGF was reduced or removed from the cultures,

normal phenotypes (e.g. luminal structure and uniformed

size) of human breast epithelial cell were obtained. With

the variation of the time to reduce EGF, we could gener-

ate different sizes of luminal structure of HBE

3.3. Limitations of Sodium Contrast and

Sensitivity

Due to high concentrations of extracellular sodium, its

suppression by using inversion recovery pulses makes it

difficult to get absolute intracellular sodium images.

However, intracellular sodium images may be indicators

of edema, interstitial space, cell proliferation, malig-

nancy, and hypoxia as criteria to divide tumor dormancy,

slow-growing, and fast growing regions. Newer tech-

niques without the use of shift reagents using high reso-

lution RF coils generate 3D23Na images with high sig-

nal-to noise ratio in less than 15 minutes, permitting use

of combined 23Na and 1H MR imaging protocols with

total examination times of about 45 minutes. Moreover,

sodium imaging in myocardium and cardiac ischemia is

well investigated as a clinical tool.

4. CONCLUSIONS

A present state-of-art for sodium MR imaging is pre-

sented with a focus on quantum filters in pulse se-

quences and their variants in order to acquire intracellu-

lar sodium images with hands-on software pulse se-

quence design and MRI physics principles. Triple quan-

tum filtering scheme and non-invasive inversion recov-

ery fast spin-echo pulse sequences are described for dis-

tinct intracellular sodium MR images. Our experiments

on breast tumors and isolated culture cells indicated the

association of malignancy with increased intracellular

sodium and reduced apoptosis and reduced EGF in cells

with possibility of other growth factors involved.

5. ACKNOWLEDGEMENTS

The present work was supported by the grant GIA 0086 funded by

Aventis Pharmaceutical Company Inc. for design and development of

sodium MRI technique. Partly the work was presented at workshop

ISMRM: Minimization of data acquisition with maximum outcome in

2002. The study for the normal and malignant breast epithelial cell

culture was supported by NIH (1 R21 CA 131798-01A1).

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