NMR In Physiology and Biomedicine

University

Preface

This is an exciting time to be associated with the field of in vivo biological magnetic resonance. The past decade has been one of tremendous expansion, both in terms of the numbers of researchers in the field and in terms of the diverse applications of this powerful technique. In previous years, this technology has always been one of promise, wherein most research efforts were expended in proving principles and not in discovering new biological concepts or biomedical applications. The articles in this book prove, without a doubt, that the promise of this technology is being fulfilled.

The purpose of this book is twofold. First, it aims to comprehensively review the entire field of in vivo biological magnetic resonance. Because the field is expanding at a rapid rate, such a book is timely and needed. Therefore, it should serve to update and expand the working knowledge of practicing researchers in in vivo biological magnetic resonance. Second, and equally important, the applications of biological NMR are making inroads into areas that are historically the bailiwick of physiologists: for example, cardiac physiology, neurophysiology, renal physiology, microcirculation, cellular physiology, hepatic physiology, and others. These applications provide new windows on old problems. Thus, a major aim of this book is to introduce nuclear magnetic resonance methods and applications to the working physiologist. The chapters of this book have been written at a level that should be accessible to all researchers, regardless of their area of expertise. To maintain the timeliness of this book, current capabilities and future directions have been emphasized, at the expense of historical review.

Such a book comes into being only through the efforts of many people. First, I must thank Bob Shulman, for readily agreeing to write the foreword and helping me in every way possible during my career. Second, I thank Joe Hoffman. Although he may not know it, this book would never have gotten off the ground without his initial efforts and foresight. Third, I thank my wife, Christine, and my daughters, Julia and Jessica, for those hours I spent with my computer and not with them. Finally, this book is dedicated to my mother, Joyce, and to the memory of my father, Hugh William Gillies, who lived with diabetes for over 50 years.

Robert J. Gillies,     Tucson, Arizona

1

Introduction to Nuclear Magnetic Resonance

E. Raymond Andrew

Publisher Summary

This chapter discusses nuclear magnetic resonance (NMR). NMR is the branch of spectroscopy operating in the radiofrequency region of an electromagnetic spectrum. It arises from the interaction between atomic nuclei and a magnetic field. Compared with other areas of physics, NMR signals are relatively weak and must be sought and managed with care. However, this relative weakness of NMR enables it to probe materials and living systems without disturbing them and yield detailed information in a noninvasive manner. NMR imaging can be applied to systems of any size, large or small. The most intensive effort has been applied to imaging the human body because of the value of NMR imaging in clinical applications. The advantage of NMR microscopy is that it is nondestructive; it is not necessary to cut and mount sections. The biological development of the object studied can be followed continuously without damage.

I INTRODUCTION

Nuclear magnetic resonance (NMR) is a remarkably versatile phenomenon. Discovered in 1946 (Bloch, Hansen, and Packard, 1946, Purcell, Torrey, and Pound, 1946) it was initially important in physics but soon also became an essential analytical and structural technique in chemistry. It swept across the disciplines to biochemistry and physiology and on to medicine where it is now firmly established as a standard modality of diagnostic investigation and research. There have been applications in fields as diverse as archeology, oil prospecting, and veterinary science; few areas of scientific endeavor remain untouched by NMR. In this chapter we provide an introduction to NMR, in the context of physiology and medicine, particularly for those readers with little previous acquaintance with the subject. For further discussion of basic principles of NMR the reader is referred to Harris (1983), Slichter (1990), Abragam (1961), and Ernst, Bodenhausen, and Wokaun (1987) in order of increasing difficulty.

NMR may be regarded as a branch of spectroscopy, though not all its applications are strictly spectroscopic, operating in the radiofrequency region of the electromagnetic spectrum broadly from 0 to 10⁹ Hz. It arises from the interaction between atomic nuclei, as found in all forms of matter, and a magnetic field. This interaction is not a strong one; compared with many other areas of physics NMR signals are relatively weak and must be sought delicately and husbanded with care. However, this relative weakness of NMR is also a source of its strength, enabling NMR to probe materials and living systems without significantly disturbing them and thus yielding detailed information in a noninvasive manner.

Our starting point therefore is the atomic nucleus.

II MAGNETIC PROPERTIES OF ATOMIC NUCLEI

Many atomic nuclei possess an intrinsic angular momentum or spin. The largest measurable component of this angular momentum is I , where I is called the nuclear spin is the natural unit of angular momentum, given by h/2π, where h is Planck’s constant.

The principal constituents of atomic nuclei are protons and neutrons, collectively nucleons. Protons and neutrons have almost the same mass, so the number of nucleons in the nucleus determines the mass of the nucleus and this integer is the mass number A of the nucleus. Neutrons carry no electric charge but protons carry a positive electric charge e, the same in magnitude as that of the electron, but opposite in sign. Consequently the number of protons in the nucleus determines the electric charge on the nucleus and this integer is the atomic number Z of the element.

. Consequently if A is even, the nuclear spin number I is an integer, whereas if A is odd the nuclear spin number is a half integer. Within a nucleus the protons tend to pair off oppositely and the neutrons similarly tend to pair off oppositely. Consequently a nucleus which has an even number of protons and an even number of neutrons has zero resultant angular momentum, I = 0. Such nuclei, with both Z and A even, always have I = 0 and are of no interest in NMR. Examples of such even–even nuclei are ¹²C and ¹⁶O, the principal isotopes of carbon and oxygen.

has a magnetic moment very close to e /2m, the Bohr magneton (m is the electron mass). Indeed a simple classical calculation shows that the ratio of the magnetic moment of a charged spinning object to its angular momentum should be of order e/m. It is therefore to be expected that a magnetic moment should accompany the spin of an atomic nucleus and that the value of this moment should be of the order of a quantity called the nuclear magneton e /2M, where M is the mass of the proton. This expectation is realized and measured nuclear magnetic moments range from −2.13 nuclear magnetons for ³He to +6.14 nuclear magnetons for ⁹³Nb. We should note that since the mass of the proton is 1836 times larger than that of the electron, nuclear magnetic moments are very small compared with the magnetic moment of the electron. Even–even nuclei not only have zero spin but also have zero magnetic moment. However, all elements have at least one isotope with nonzero spin and magnetic moment.

. are spherical and have no electric quadrupole moment. Examples of nuclei with an electric quadrupole moment are ²H, ¹⁴N, and ²³Na. The electric quadrupole moment can sometimes yield additional useful information, but more often it represents a complicating factor one would rather avoid. Nuclei with spin greater than 1 have higher moments (octupole, hexadecapole, and so on) but these are of no practical importance to biomedical NMR and can be ignored.

III NMR NUCLEI OF INTEREST IN PHYSIOLOGY AND MEDICINE

Some nuclei of potential interest in physiology and medicine are shown in Table I, together with their values of I, nuclear magnetic moment μ, and natural abundance. The most common elements in the human body and other living systems are hydrogen, carbon, and oxygen. For hydrogen the principal isotope is ¹H, a single proton. Its isotopic abundance is almost 100%, its magnetic moment is high, and its physiological concentration is also high. It is therefore an excellent nucleus for NMR. The second hydrogen isotope is ²H, the deuteron. Its magnetic moment is low and its natural abundance is weak. Moreover having spin 1 it has a quadrupole moment. ²H is therefore not a favorite NMR nucleus, but it has occasional specialized NMR uses (See Chap. 13, Evelhoch, this volume). ³H, the triton, has a good magnetic moment but is radioactive with a half-life of 12 years. It therefore has only very restricted specialized NMR uses.

TABLE I

Properties of Some Nuclei of NMR Interest

Although the principal isotope of carbon, ¹²C, has I and can be used in NMR. However μ is low and the natural abundance is only 1.1%. Despite the consequent weakness of its NMR response, ¹³C is nevertheless a useful NMR nucleus in physiology and medicine. Moreover the ¹³C NMR signals may be substantially enhanced from particular molecular species by isotopic enrichment (see Chap. 26, Sherry and Malloy; Chap. 22, Canioni and Quistorff, this volume).

The principal isotope of oxygen, ¹⁶O, also has I and can be used in NMR. The isotopic abundance is low, 0.04%, but is partly compensated by the high physiological concentration of oxygen in vivo, ¹⁷O has an electric quadrupole moment. Thus ¹⁷O has several disadvantages and is not a popular NMR nucleus. Nevertheless some useful biomedical NMR work has been done with it.

has a very low isotopic abundance, 0.4%. Consequently neither are greatly used.

, 100% isotopic abundance, and a magnetic moment almost as large as that of ¹H. Regretfully there is little fluorine occurring naturally in the human body, most of it in fluoroapatite in teeth. However, the normal absence of ¹⁹F in the body makes fluorine compounds ideal as NMR tracers and enables the pathways of fluorine-containing drugs to be followed (see Chap. 16, London; Chap. 18, Bental and Deutch, this volume).

, has a good magnetic moment, and is 100% abundant. It is present in many aspects of the body’s chemistry, for example, in metabolites, phospholipids, and nucleic acids. It has widespread use in the NMR investigation of living systems.

. Other nuclei which have some potential in biomedical NMR are ²H, ¹⁴N, ¹⁵N, ¹⁷O, ²³Na, ²⁵Mg, ³⁵C1, ³⁹K, ⁴³Ca.

IV NUCLEAR MAGNETIC RESONANCE

Let us consider the behavior of a magnetic nucleus in a magnetic field. From the standpoint of classical mechanics when a nuclear magnetic moment μ is placed in a magnetic field B (Fig. 1) it experiences a torque tending to turn it parallel to the field direction.¹ Since the nucleus is spinning it does not respond to this couple by turning parallel to the field, but precesses around it like a gyroscope under the influence of gravity. The angular frequency of precession ω is proportional to the couple, and therefore to B, and was first calculated almost a century ago by Sir Joseph Larmor (1900):

FIGURE 1 Classical precession of a magnetic nucleus in a magnetic field.

(1)

This is known as Larmor’s theorem and ω/2π is the Larmor frequency. The proportionality constant γ is the nuclear gyromagnetic ratio, the ratio of the nuclear magnetic moment μ to its angular momentum I :

(2)

For a proton in a field of 1 T the precession frequency ω/2π given by (1) is 42.6 MHz, a radiofrequency. We are all sitting in the earth’s magnetic field of about 0.5 × 10−4 T. So inside each of us some 10²⁸ protons are busy precessing at about 2 kHz. We are not, I think, very much aware of this induced activity which is continuously going on inside us!

. When placed in a magnetic field the nucleus may be found in one of two states (eigenstates) having energy

(3)

as indicated in Fig. 2. The two states and eigenvalues of energy are labeled with magnetic quantum numbers m . Suppose now we irradiate the proton with electromagnetic radiation of just such a frequency v that the photons or quanta have energy hv exactly equal to the separation of the two levels of energy. Then if the proton is in the lower energy eigenstate it can be exactly promoted to the upper level. If on the other hand it is initially in the upper state it can be resonantly stimulated to emit a photon and fall to the lower state. We therefore have a resonant exchange of energy between the proton and the electromagnetic field. This is nuclear magnetic resonance.

FIGURE 2 Eigenvalues of energy of a nucleus of magnetic moment μ in a magnetic field B.

This condition requires

(4)

and this frequency v, which satisfies (4), is called the nuclear magnetic resonance frequency. We may rewrite Eq. (4), using (2) with I for the proton, as

(5)

We see that the NMR frequency derived from this elementary quantum calculation is exactly the same as the Larmor frequency of Eq. (1). This happy agreement of quantum mechanics with classical mechanics makes it possible to use classical mechanics to describe many aspects of NMR, which is usually easier than using the more rigorous quantum mechanics.

It is worth noting that we are using the word resonance in NMR in the same sense as in other areas of physics, for example in mechanics, acoustics, and resonant electrical circuits. Whenever an applied vibration coincides in frequency with a natural internal mode of a system we have resonance. In our case the natural classical mode with which the radiofrequency is in resonance is the Larmor frequency of precession of the nucleus in the applied magnetic field.

, which covers the important nuclei lH, ¹³C, ¹⁵N, and ³¹P. However, the result is also true for general spin I. When placed in a magnetic field B the nucleus has (2I + 1) eigenvalues of energy with equal spacing μB/I running from –μB to +μB. Transitions between adjacent states take place when the quanta of the applied electromagnetic radiation exactly match the separation between adjacent states,

(6)

which reduces to (4) when I . Using (2) we again see that (6) leads to

(7)

which is the Larmor frequency.

A full listing of nuclei with I ≥ is given in the Appendix together with their magnetic moments, natural abundances, and NMR frequencies.

Since the nuclear magnetic moments precess at the Larmor frequency when in a magnetic field, it is often convenient to view their behavior in a frame of reference which is rotating relative to the laboratory at the Larmor frequency. This is shown in Fig. 3. Figure 3a shows the nuclear magnetic moment μ precessing with the Larmor frequency ω relative to the laboratory frame. Figure 3b shows the nuclear magnetic moment vector in the frame of reference rotating with the Larmor frequency around the magnetic field direction. In this rotating frame of reference the vector μ is at rest and we see later that this frame is very useful for visualizing the behavior of nuclear spins. It is usual to use coordinates x,y,z in the laboratory frame and coordinates x′,y′,z′ in the rotating frame.

FIGURE 3 (a) Magnetic nucleus processing in laboratory frame of reference. (b) Magnetic nucleus at rest in frame of reference rotating relative to the laboratory frame at the Larmor frequency.

V NMR IN BULK MATTER

So far we have considered a single proton. We now go on to consider matter in bulk. Let us take a vial enclosing 1 ml water, which therefore contains about 10²³ hydrogen nuclei. We place this vial in a magnetic field B (Fig. 4) and let the nuclei come to equilibrium. Some will align with the field corresponding to the eigenstate with m and some against the field corresponding to the eigenstate with m . In equilibrium there will be a small preponderance aligned with the field. From statistical mechanics we know that the ratio of these populations is given by the Boltzmann factor

FIGURE 4 A 1-ml vial of water in a magnetic field B. The protons line up with a small preponderance along the field direction constituting a resultant nuclear magnetization M.

(8)

since in practice 2μB/kT 1. At room temperature in a field of 1 T the excess protons pointing with the field are 8 × 10−6. It is on this small but finite excess of population pointing with the field that we depend for our observation of nuclear magnetic resonance from matter in bulk. At extremely low temperatures, below 1 mK, the Boltzmann factor can be quite sizeable and the approximation in (8) is no longer valid. However, for applications in physiology and medicine the approximation is generally a very good one. The small preponderance of nuclear magnetic moments pointing with the field, shown in Fig. 4, constitutes a macroscopic magnetic moment, which is called the nuclear magnetization M.

Let us now apply an electromagnetic field at exactly the Larmor frequency to the vial of water. As we see in a moment this has the effect of turning the magnetization M away from the direction of B, all the time precessing around B at the Larmor frequency. When M has turned through 90° (Fig. 5), let us shut off the electromagnetic field and leave M precessing about B in a plane perpendicular to B. This short pulse of radiofrequency field is called a 90° pulse.

FIGURE 5 A 90° pulse of resonant radiation turns the nuclear magnetization through 90° and leaves it precessing in a plane perpendicular to the magnetic field B.

Suppose we place a small coil of a few turns of copper wire around the vial (Fig. 6). Then by Faraday’s Law of Electromagnetic Induction the rotating magnetization will generate an electromotive force (a voltage) in the coil at the Larmor frequency. We can attach the coil to the input of a radio receiver tuned to the Larmor frequency; we can amplify the signal, detect it, and display it on an oscillograph. This is the NMR signal.

FIGURE 6 The precessing nuclear magnetization induces an oscillatory voltage in the coil surrounding the vial. This is the NMR signal.

Because the NMR signal is induced in the coil by the precessing nuclear magnetization it is sometimes called a nuclear induction signal (Bloch et al., 1946). As indicated in Fig. 6 the signal decays; the NMR signal is therefore often referred to as a free induction decay or FID for short.

NMR signals can be obtained from all magnetic nuclei, namely, from all nuclei with nonzero spin. NMR signals have been observed from matter in all its manifestations: from solids, liquids, and gases; from metals, semiconductors, and insulators; from polymers and liquid crystals; and from living organisms, animals, and humans.

VI BASIC EQUIPMENT

We now consider what equipment is needed for detecting and displaying the proton NMR signal from our vial of water. A basic equipment diagram is shown schematically in Fig. 7. First the vial must be placed in the magnetic field of a magnet. Most NMR magnets nowadays are superconducting magnets and Fig. 7 illustrates a superconducting magnet which provides a magnetic field B in its vertical bore. The magnet is the most important and usually the most expensive component of the whole NMR system and we discuss magnets in more detail in the next section. The vial, placed inside the radiofrequency coil of wire, is mounted centrally in the magnet. A radiofrequency generator, operating at the NMR frequency, supplies pulses of radiofrequency current in the radiofrequency coil, thus generating pulses of radiofrequency (rf) field experienced by the protons in the sample within the coil. This rf coil in its housing is called the rf probe.

FIGURE 7 Schematic diagram of equipment required to detect nuclear magnetic resonance.

The coil is also connected to a radio receiver tuned to the NMR frequency, which picks up the NMR signal, amplifies and detects it, and passes it to the oscillograph for display. A dedicated computer is programmed to instruct the entire system; it also receives the NMR signals in digital form, records them in its memory, and performs any calculations required on them.

When NMR is used in applications in physics and chemistry the samples examined are usually about 1 cm³ of solid or liquid material and a superconducting magnet with a bore diameter of 5 cm is adequate to accommodate them. However, when NMR is used in physiology and in medicine the object of interest is often a live animal or a live human being. In this situation a very much larger magnet is needed in order to accommodate the living system in the required magnetic field. Nevertheless, whatever the application, the basic equipment is essentially the same, comprising five basic components: a magnet, a radiofrequency generator or transmitter, a radio receiver, a display system, and a computer. We see later that in order to generate an NMR image, or to produce an NMR spectrum from a particular region of anatomy, certain additional components are needed.

VII MAGNETS

Early NMR experiments on small samples were carried out using iron electromagnets or permanent magnets. Some NMR experiments were also carried out in the earth’s magnetic field and NMR has been used for measuring geomagnetic fields from aircraft and from oceanographic vessels and for detecting archeological artifacts at historic sites. However, almost all NMR measurements in physics, chemistry, and biochemistry are now carried out using superconducting magnets with a vertical bore 50 or 89 mm diameter. An example is seen in Fig. 8. There has been a steady upward trend in the magnetic field of such magnets to take advantage of the increased NMR sensitivity and the increased NMR spectral resolution which higher magnetic field strengths afford. Currently standard magnetic field strengths are 9.4, 11.7, and 14.1 T for proton high-resolution NMR operation at 400, 500, and 600 MHz, respectively. Magnets operating at 17.6 T are now becoming available for operation at 750 MHz and prototypes are under design for 900 MHz and 1 GHz and, when realized, will be very expensive. All these magnets have a highly stable magnetic field, constant to at least 1 part in 10⁹, and a very uniform magnetic field varying by no more than a few parts in 10⁹ over 1 cm³. Adjustments to perfect the uniformity of the magnetic field are often made by supplying adjustable small currents to a set of orthogonal coils. Such a set of coils are called shim coils and the process of optimizing the uniformity is called shimming the magnet.

FIGURE 8 A 14.1-T superconducting magnet for high-resolution NMR spectroscopy. In this magnet the proton NMR frequency is 600 MHz. (Photograph courtesy of Oxford Instruments PLC.)

For NMR investigations of the human body much larger magnets are required. Large superconducting magnets are available with a horizontal bore typically 1 m in diameter. As illustrated in Fig. 9 such magnets are about 2 m long and stand 2 m tall. The human subject lies on a couch and is conveyed into the interior of the magnet bore. Standard magnets for medical magnetic resonance have fields from 0.5 to 2 T. Experimental whole-body magnets have been made at 3, 4, and 5 T. Horizontal magnets of smaller bore diameter are available for NMR investigations on infants and on animals. Large electromagnets and permanent magnets are also sometimes used for clinical purposes.

FIGURE 9 A 1.5-T superconducting magnet for clinical magnetic resonance imaging (University of Florida Hospital).

VIII CIRCULAR POLARIZATION OF THE ELECTROMAGNETIC RADIATION

We return now to our 1-ml vial of water immersed in a magnetic field B and take a more detailed look at the resonance process. In equilibrium the nuclear magnetization M is aligned along B (Fig. 10a). If M were somehow turned away from B it would precess around B at the Larmor frequency. Viewed in the rotating frame (Fig. 10b) M would lie along the Z axis in equilibrium, but if turned away from B it would not be seen to precess in this frame. The field B is effectively extinguished in this frame, though its direction does define the axis Z about which the rotating frame rotates. Suppose now that we apply a small magnetic field B1, rotating with the Larmor frequency in the XY plane as shown in Fig. 10c; what effect will this have? The effect is actually somewhat complicated but is made much easier by looking at it in the rotating frame. In the rotating frame (Fig. 10d) B1 is at rest, directed along X′. Indeed in this frame B1 along X′ is the only field M experiences because in this frame B is extinguished. So M precesses around X′ with angular frequency ω1 = γB1. Let us turn B1 off when M has turned through 90° and leave it pointing along Y′. Now let us see how this manipulation looks in the laboratory frame. M is precessing in the XY plane, perpendicular to B, and we have achieved the consequences of a 90° pulse discussed in section V. In the laboratory frame the motion of the vector M is a slow motion through 90° at angular frequency ω1 superposed on a rapid precession at the Larmor frequency ω, the tip of the vector M tracing a spiral pattern on a hemispherical surface (Fig. 10c).

FIGURE 10 Effect of 90° pulse viewed in laboratory frame (a) and (c) and in rotating frame (b) and (d).

Suppose the frequency of rotation of B1 is not quite equal to the Larmor frequency. In the rotating frame B1 is not now at rest. The magnetization M will not move steadily through 90° but instead will exhibit only minor perturbations according to the relative phases of the two motions with no net effect. The frequency of B1 and the Larmor frequency must be exactly equal, typical of a resonance phenomenon. If the frequency of rotation of B1 is indeed equal to the Larmor frequency, but has opposite sense of rotation, again B1 will not be at rest in the rotating frame and will rotate with frequency 2ω1, again producing only minor perturbations.

We see therefore that what is needed for nuclear magnetic resonance is the application of a radiofrequency magnetic field B1, circularly polarized, rotating at the same frequency and in the same sense as the Larmor frequency of precession.

Although the generation of a high-frequency rotating magnetic field is quite practicable, it is usually much simpler to provide a linearly oscillating field by supplying radiofrequency current to a solenoidal coil. Fortunately for most purposes linear polarization is quite adequate, since, as in the theory of rotary polarization in optically active materials, a linearly oscillating field may be regarded as the superimposition of two counterrotating fields. As shown in Fig. 11, if the linear oscillating field has amplitude 2B1, it may be decomposed into two circularly polarized fields each of amplitude B1, but rotating in opposite senses in a plane perpendicular to B. Resonance will be obtained with the component which has the correct sense, the other component having a negligible effect. It is not generally possible to tell which component is utilized and it is not usually necessary to know this. The information is only necessary in special cases for example to find the sign of the nuclear moment μ; from Eqs. (1) and (2) one sees that it is the sign of the gyromagnetic ration y, and therefore of μ, which determines the sense of the Larmor precession.

FIGURE 11 If the two equal vectors B1 shown rotating in opposite senses, are superimposed, the resultant on the right is a linear vibration of amplitude 2B1. The static field B of the laboratory magnet is perpendicular to the diagram.

IX RELAXATION

In section V we considered the effect of a 90° pulse on the proton magnetization M from a 1-ml vial of water. Suppose now we apply the radiofrequency pulse for twice as long. This will turn the magnetization through 180° and we call it a 180° pulse. The magnetization M is now pointing in the opposite direction to the applied field B and is clearly not in equilibrium (see Fig. 12a). Indeed, as Eq. (8) indicates, the proton spin system is formally at a negative temperature. The return of the magnetization to equilibrium is called relaxation. It is found experimentally that this return is exponential as indicated in Fig. 12b and the time constant T1, which characterizes this relaxation process, is called the longitudinal relaxation time. It is sometimes also called the spin–lattice relaxation time, a name which originates in early studies on crystalline solids. If the initial magnetization after the 180° pulse is –M0, its return to +M0 is thus given by

FIGURE 12 Longitudinal relaxation.

(9)

A tangent at the origin (the broken line in Fig. 12b) intersects the asymptote M0 at time T1. The recovery passes through M = 0 when

(10)

The value of T1 in pure water at room temperature is about 3 s.

How does the magnetization regain its equilibrium direction? In every water molecule each proton has a near neighbor proton about 1.5 × 10−10 m away. Since each proton has a magnetic moment it generates a small local magnetic field at its neighbor of about 5 × 10−4 T (5 G). This local field is not constant because the water molecules are continually reorienting and tumbling in a random fashion, being buffeted by neighboring water molecules. Thus this local field is a fluctuating field, randomly varying by ±5 G on a time scale of order 10−11 s, called the correlation time of the molecular motion.

If we carry out a Fourier analysis of this random motion we find that it covers all frequencies evenly out to 10¹¹ Hz (the inverse of the correlation time) as illustrated in Fig. 13. Suppose our NMR frequency is 100 MHz. Then there will be a significant frequency component of the local magnetic field at 10⁸ Hz, and this can cause protons to turn around just as the applied radiofrequency field at this frequency does, and thus promotes relaxation. The relaxation time T1 can be calculated from this theory and gives excellent agreement with the observed value (Bloembergen et al. 1948; Kubo and Tomita, 1954). We should note that since the proton spectral density depicted in Fig. 13 is constant up to about 10¹⁰ Hz, we might expect that T1 for pure water should be the same for all NMR frequencies; experimentally this is found to be so (see Chap. 4, Koenig and Brown, this volume).

FIGURE 13 Spectral density of interproton magnetic field of water molecules.

Now let us return to the effect of a 90° pulse as described in section V and in Figs. 5 and 10. After the 90° pulse the magnetization M is left precessing in a plane perpendicular to B. Here again it is no longer in equilibrium with B. The magnetization decays transversely and regrows longitudinally. The decay of the transverse magnetization and the corresponding decay of the NMR signal is often approximately exponential. The time constant T2 which characterizes this transverse relaxation process is called the transverse relaxation time. In pure water T1 and T2 are equal, both having the value 3 s at room temperature. In other simple pure liquids T1 and T2 are equal, but with other materials this is not generally the case.

Here again we can understand the mechanism for this transverse relaxation process from a molecular viewpoint. We remember that the nuclear magnetization is the result of many millions of proton moments. Each experiences a local magnetic field from its nuclear neighbors in the liquid and these local fields are all slightly different. As a result all the protons precess at slightly different frequencies. Consequently they slowly dephase; in the rotating frame they spread out around the Z′ axis, decaying to zero in the time T2. It is the near-zero frequencies in the molecular motion which are effective in dephasing. However, we see from Fig. 13 that the proton spectral density near zero is the same as that at 10⁸ Hz. Consequently T2 = T1, as shown more rigorously by Bloembergen, Purcell, and Pound (1948).

Quite often, when dealing with liquids, another mechanism of transverse relaxation is operative. The magnetic field provided by the NMR magnet is never perfectly uniform and over the 1-ml sample there will be a spread of magnetic fields δB. Consequently there will be a spread of Larmor frequencies of precession δω = γδB over various domains within the sample. The components of magnetization from these domains will de-phase; in the rotating reference frame they are seen to spread out and the overall magnetization M decays in a time of order 1/δω or (γδB)−1. We note that this decay time is an instrumental artifact, reflecting the nonuni-formity of the magnetic field provided by the magnet and is not a property of the sample. To distinguish this effect from the true transverse relaxation time T2, the decay time (γδB)−1 is called T*2; very often with liquids T*2 < T2 and dominates the transverse decay. Shimming the magnet, discussed section VII, reduces δB and therefore increases T*2. A further trick is to spin the specimen about a vertical axis which has the effect of averaging the residual field nonuniformities over planes normal to the axis of spin, so increasing T*2 still further (Bloch, 1954).

Later in this chapter we see that in NMR imaging a magnetic field gradient is applied which deliberately spoils the uniformity of the magnetic field over the object of interest. In such cases the transverse decay of the NMR signal is strongly dominated by T*2.

A fuller discussion of relaxation is given in Chap. 4 by Koenig and Brown, this volume.

X SPIN ECHOES AND PULSE SEQUENCES

We now describe the remarkable phenomenon of spin echoes discovered and explained by Hahn (1950), which has many applications in NMR. Consider our 1-ml vial of water in the field of a magnet which, as usual, is not perfectly homogeneous. We apply a 90° pulse and observe a free induction decay lasting for about 2T*2. The components of magnetization from different domains in the sample precess at different frequencies dependent on their precise value of magnetic field. In the rotating frame these components of magnetization fan out all around the Z′ axis. After time τ we apply a 180° pulse which turns all magnetization components through 180°. They are still all precessing in the xy plane, but those that were behind are now ahead and vice versa as illustrated in the rotating frame in Fig. 14a. Consequently after another time τ they come back into phase and reconstitute an NMR signal, as shown in Fig. 14b. This reconstituted signal is called a spin echo.

FIGURE 14 The formation of spin echoes, (a) Components of magnetization viewed in the rotating frame spread out, but after a 180° refocusing pulse they return to form an echo, (b) The echo signal forms at time 2τ.

Hahn (1953) provided an instructive analogy. Consider runners on an athletic race track. After several laps they are spread all around the track. After a time τ the starter fires his gun again (180° pulse), whereupon each runner turns around and runs back at exactly the same speed as before. The fastest runners are now behind and the slower runners are in front and after an equal time τ they all come together at the starting line simultaneously (echo). We note that they do not stay together for long. The reconstitution of the echo takes a time T*2 and decays again in 2T*2. The more inhomogeneous the magnetic field the sharper the echo.

Returning to our 1-ml sample let us apply a 90°–180° pulse sequence and generate an echo at time 2τ as before. Now let us apply a second 180° after time 3τ. This inverts the magnetization components again and a second echo is formed at time 4τ. This procedure can be repeated as illustrated in Fig. 15, with 180° pulses applied at τ, 3τ, 5τ, … (2n – l)τ, and echoes formed at 2τ, 4τ, 6τ … 2nτ (Carr and Purcell, 1954). The amplitudes of the echoes decay, reflecting the true transverse decay of the magnetization, namely, with the time constant T2. This Carr–Purcell pulse sequence therefore provides an excellent method for measuring the time T2 of a liquid.

FIGURE 15 Carr–Purcell sequence. After an initial 90° pulse a series of 180° pulses at τ, 3τ, Sτ, … successively refocuses the transverse magnetization yielding echoes at 2τ, 4τ, 6τ, …. The amplitude of the echoes (dashed line) decays with the true transverse relaxation time T2.

There is another cause of decrease of the echo amplitude in a spin–echo pulse sequence. In the interval 2τ between the 90° pulse and the echo some water molecules will diffuse to new locations where the magnetic field is different. These molecules will not fully contribute to the echo. This additional decrease of echo amplitude can be used to measure diffusion coefficients. On the other hand in the Carr–Purcell pulse sequence the overall decay time is broken up into many short time intervals, each 2τ, which reduces the effects of diffusion in the measurement of T2.

So far we have encountered two pulse sequences. First the 90°–τ–180° spin-echo sequence (Fig. 14) and second the 90°–[τ–180°–]n Carr-Purcell sequence (Fig. 15). Another useful sequence is the 180°–τ–90° inversion-recovery sequence, illustrated in Fig. 16a, which is useful for measurements of T1. Then there is the saturation sequence, a series of 90° pulses, which is also useful for measuring T1, particularly in solids and tissues where TT1, (Fig. 16b). Another useful sequence is the stimulated echo sequence which requires three pulses (Fig. 16c), which is useful when the system to be investigated is to be subjected to three successive manipulations, as in volume localization. There is a wide variety of other pulse sequences used for specific purposes. All these pulse sequences can be programmed on the dedicated computer which, as we saw in section VI, is an integral part of any NMR system.

FIGURE 16 Some other pulse sequences: (a) Inversion-recovery pulse sequence. The time between the 180° inversion pulse and the 90° inspecting pulse is TI. (b) Saturation-recovery pulse sequence. TR is the repetition time. (c) Stimulated echo sequence. Three 90° pulses are applied at times 0, τ, T. The stimulated echo appears at τ + T ( Hahn, 1950). Other echoes (not shown) also appear.

XI LIQUIDS, SOLIDS, AND TISSUES

One of the most characteristic features of NMR is the great difference in behavior of liquids and solids. Water and ice provide good examples. The proton NMR signal from pure water is a very sharp single response with an intrinsic width of about 0.1 Hz; T1 and T2 are both about 3 s. By contrast the proton NMR response from ice at 100K is a broad response about 50 kHz wide, with T2 about 10 μs and T1 about 600 s (Pake and Gutowsky, 1948). Thus the NMR spectral response is almost six orders of magnitude broader in ice than in water; this is reflected in T2 which is almost six orders of magnitude shorter in ice than in water. The details for other liquids and solids differ, but it is rather generally true that

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The explanation for this highly characteristic behavior lies in the dynamical behavior of the molecules of which the liquids and solids are composed. In the liquid state the molecules rapidly and randomly reorient and diffuse with a correlation time of order 10−11 s. In solids the molecular correlation time may be as short as 10−10 s near the melting point but may be as long as 1 s at a low temperature. Such a long correlation time means the molecules effectively form a rigid array in the solid and each proton has a static local field from its neighbor of order ±5 G. A spectral spread of 10 G corresponds to 42 kHz for protons which explains the broad NMR response.

It is possible to narrow the broad NMR response from solids by rapidly spinning them (Andrew et al., ), the so-called magic angle (Andrew, 1959; Andrew and Wynn, 1966). Magic angle spinning (MAS) has become a standard technique for enabling high-resolution NMR spectra to be obtained from solids.

This book is frequently concerned with NMR of living tissues. These tissues are not homogeneous and the NMR signal is an integrated response from all the nuclei present in various cells and other environments. The relaxation properties differ from one compartment to another. Nevertheless the overall return of nuclear magnetization to equilibrium often approximates to an exponential behavior so that effective relaxation times T1 and T2 can be ascribed. In live human tissues the proton T1 varies from one tissue to another in the range 200–1000 ms, while T2 is typically five times shorter, in the range 40–200 ms. For a given tissue T1 increases with NMR frequency. Summarizing,

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The presence of a wide variety of solutes and organelles in cellular water provides a basis for understanding the reduction of T1 compared with pure water, the further reduction of T2, and the dependence of T1 on operating frequency (Andrew et al., 1990, Chap. 3).

XII FINE STRUCTURE

Although the proton NMR response from water is a single sharp resonance line, other liquids are often more interesting. For example the response from ethyl alcohol (Fig. 17) consists of three resonances close together. The separation is proportional to the magnetic field strength B and encompasses 3 ppm, namely, about 300 Hz if measured at 100 MHz. The integrated intensity of the three responses is in the ratio 1:2:3, which suggests that they arise from protons in the hydroxyl, methylene, and methyl groups, respectively. This can be proved by repeating the experiment with CD3CH2OH which leads to the disappearance of the strongest group, and then with CH3CD2OH which causes the middle response to disappear. In fact the protons in every chemical group have their own position, slightly different from water, an effect called the chemical shift.

FIGURE 17 Proton NMR spectra of ethyl alcohol (a) under moderate resolution displaying three chemically shifted resonances and (b) under higher resolution displaying the multiplet structure of the methyl and methylene resonances.

The chemical shift has its origins in the extranuclear electrons which act as a weak magnetic screen, so that the magnetic field experienced by the nucleus is not the value B which a bare nucleus would experience but is (1 – σ)B, 1. Our basic resonance Eq. (4) must therefore be modified to

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The separation of the resonance frequencies for different molecular groups from a well-chosen reference group is termed the chemical shift. Both σ and the chemical shift are expressed in dimensionless units of parts per million. All NMR nuclei exhibit the chemical shift. As one proceeds through the Periodic Table the shielding constants increase with the number of extranuclear electrons, and values of σ range from 10−6 to 10−2.

Besides the chemical shift there is another source of fine structure. Suppose we look at the NMR spectrum of ethyl alcohol again, but using a much more uniform magnetic field. In this situation the two main lines of Fig. 17a each break up into a multiplet as shown in Fig. 17b. The separation of the components in the multiplets is independent of the magnetic field strength B. The multiplets have their origin in a coupling between the protons in one molecular group and those in the adjacent molecular group, mediated by the molecular electrons. It is called spin-spin coupling, scalar coupling, or simply J coupling. The separation of lines is the same in both adjacent groups and is given by the coupling constant J, expressed in Hz.

(one up and two down) which shift the resonance one-third to the left and to the right. We thus have an equally spaced quartet. There are three permutations for two spins up and one down and three for one spin up and two down, so the two central lines of the quartet have three times the intensity of the outer two. In fact their intensities are given by the binomial coefficients 1331. Similarly if we consider the effect of the two methylene protons on the methyl group, we find a triplet with intensities 121. It will be asked why the hydroxyl proton resonance is not also broken into a triplet. In fact in completely dehydrated ethanol this is found. But a trace of water leads to rapid exchange of hydroxyl and water protons, faster than the coupling constant J and the breakdown of the multiplet.

XIII NMR SPECTROSCOPY

The spectra of ethanol in Fig. 17 illustrate the main features of an NMR spectrum from a liquid: the main chemically shifted responses which tell us which chemical groups are present and their multiplet structures which tell us their connectivity to specific neighbors. Extensive tabulations have been made of chemical shifts for protons in a wide range of compounds providing excellent diagnostic information for determining the groups present in an unknown liquid; similar tabulations have been made for ¹³C, ³¹P, and other nuclei (See Appendix). Likewise extensive tabulations and empirical rules have been made of J coupling constants in compounds of known molecular geometry enabling internuclear distances and bond angles to be determined with some precision. NMR spectra can therefore be used to identify unknown compounds from their characteristic NMR signatures, to analyze mixtures of compounds, and to determine molecular structures in quantitative 3D detail. NMR spectroscopy can determine the 3D structures of peptides and proteins of medium length, nucleic acids, carbohydrates, and other macromolecules. It is one of the most useful experimental techniques in structure determination for the chemist, biochemist, and molecular biologist. It rivals X-ray crystallography in ab initio structure determination and precision and has the merit that it does not require a single crystal; moreover, the structure determined is that found in solution which in many cases corresponds more closely to the natural habitat of the molecule than that found in a crystal. In living systems NMR spectroscopy can monitor the relative and absolute concentrations of metabolites in various anatomical regions, providing a noninvasive measure of physiology and pathology. In medicine it provides the clinician with a noninvasive indicator of disease and continuous assessment of response to therapy.

How are these NMR spectra obtained? We have seen that water gives a single sharp NMR response and has a FID which is a simple radiofrequency signal decaying within an exponential envelope of time constant T2. If the spectrum consists of two sharp responses close together in frequency, the FID will be the superposition of two radiofrequency signals both within the exponential envelope, the two rf signals exhibiting a characteristic beat pattern at their difference frequency. If the spectrum consists of a cluster of several lines of different amplitudes, the FID will be a more complicated rf decay pattern in which all the separate NMR contributions from the different nuclei in the molecules interfere with each other, decaying within an overall envelope characterized by T2, which will persist for several seconds. The classic procedure in physics for sorting out all the frequency components in a complicated waveform is Fourier analysis. So we apply a Fourier transform to the acquired FID to yield the NMR spectrum. Indeed it has been shown rather generally by Lowe and Norberg (1957) that the Fourier transform of the NMR free induction decay following an excitation pulse is the NMR spectrum (see also Ernst et al., 1987).

We therefore take a glass tube containing about 1 ml of pure liquid or solution, place it in the rf probe, excite it with a 90° pulse, collect the FID, store it in the computer, calculate the NMR spectrum with a fast Fourier transform program, and display it on a computer screen or a paper trace.

An example of a proton NMR spectrum of moderate complexity is shown in Fig. 18 (see also Chap. 14, Bell et al., this volume). For molecules of larger molecular weight the problem of assigning all the chemically shifted protons and their spin multiplets is very considerable, particularly as the spectral components overlap. There are a number of procedures which are helpful in clarifying spectra (for example double-resonance excitation and the nuclear Overhauser effect). However, there are two major ways of improving matters. The first is to work in a higher magnetic field B. The separation of chemically shifted spectral lines increases in proportion to B and since the intrinsic width of the lines is not very dependent on B the spectrum opens up, the resolution of the NMR spectrum increasing in proportion with B. This is the driving force behind the steady upward march to NMR magnets at 17.6 T (750 MHz for protons) and onward toward 23.5 T (1 GHz). Signal/noise ratio also improves at least in proportion to increase of B which is also very beneficial.

FIGURE 18 Part of the proton NMR spectrum of a steroid at 400 MHz (Sanders and Mersh, 1982).

The second major development which has produced a dramatic development in the usefulness of high-resolution NMR is the introduction of 2D NMR spectroscopy. The essential idea was advanced by Jeneer (1971) and strongly developed by Ernst and his colleagues (Aue et al., 1976; see also Ernst et al., 1987). In its simplest form two NMR pulses are applied separated by a time t1. After the first pulse the nuclear spin system evolves under its influence. After the time t1 the second pulse is applied and the spin system develops further and decays during the subsequent time t2 during which it is acquired and stored in the computer. The experiment is repeated for a different time t1 and again the NMR signal is recorded during t2. Indeed the pulse pairs are applied many times with progressively incremented values of t1 and the NMR signal is in each case recorded during t2 so that a matrix of data S(t1,t2) is acquired for many values of both t1 and t2. To this array of data, a double-Fourier transform is applied yielding a spectrum S(ωl,ω2) in two frequency dimensions. An example of such a spectrum is shown in Fig. 19; the signals are displayed in a third dimension either using a stacked plot or a contour plot. One immediate advantage is that the data are spread out over a large area instead of along a congested ID line. This opens up the spectra enormously and improves effective resolution. In addition by suitable choice of pulses it is possible to observe off-diagonal correlated nuclei which greatly assists assignment.

FIGURE 19 Example of a 2D NMR spectrum. The protein seminal inhibitor IIA in H2O (Wider et al., 1984).

Even with the clarification of spectra which results from 2D NMR spectroscopy there remain congested areas of the spectrum. Using a third or fourth pulse these regions may be spread out in a third or fourth dimension yielding 3D and 4D spectra of still greater resolution.

XIV IN VIVO MAGNETIC RESONANCE SPECTROSCOPY

In this section we turn to applications of NMR spectroscopy to living systems, often simply called magnetic resonance spectroscopy and abbreviated MRS. More information on MRS may be found in the book by Andrew, Bydder, Griffiths, Iles, and Styles (1990). MRS spectra are most often from ¹H, ¹³C, or ³¹P nuclei from well-defined regions of live human beings or animals for fundamental investigations of metabolism in normal living systems or for diagnosis of disease or response to therapy. Examples are the brain, for example to investigate stroke or epilepsy, the heart, kidneys, liver, or other specific organs. For a human being it is usually necessary to employ a large horizontal bore magnet of bore diameter typically 1 m into which the whole body is inserted. For examination of human arms or legs a magnet of smaller bore, 0.3 to 0.4 m diameter, may suffice. Such small bore magnets may also be adequate for the examination of human babies and are also suitable for the investigation of smaller animals such as rats, rabbits, guinea pigs, and cats. Very small animals such as gerbils and mice can be examined in vertical bore magnets 50 or 89 mm diameter.

We have seen that high-resolution NMR spectroscopy requires the highest possible field strengths in order to obtain adequate spectral resolution and sensitivity. However, it is not yet possible to build wide-bore magnets of the very highest magnetic fields. Most human whole-body MRS studies have been carried out at 1.5 or 2 T, with some work at 3 and 4 T.

In vivo NMR spectra are rather weak. In the case of ¹H spectra strong lines are observed from water and fat, but these are not of great interest; the real interest centers on such components as choline, creatine, lactic acid, and N-acetyl aspartate and these are of low concentration. In the case of ¹³C and ³¹P the molecules of interest are again of low concentration and for ¹³C there is the additional problem of a low isotopic abundance (1.1%) unless enriched. It is therefore essential to gather the NMR signals as efficiently as possible, by coupling the receiver coil as closely as possible to the region of interest in the body. A receiver coil may be wound closely round the body part of interest (e.g., arm, leg, neck, head). A flat receiver coil may be laid on or over the region of interest, often called a surface coil. In the case of ¹H NMR a special pulse sequence is used which suppresses the water and fat resonances which would otherwise overshadow the weak resonances which are to be recorded. The NMR signals may be recorded many times and co-added, which substantially improves the signal/noise ratio; if N .

A typical in vivo ³¹P NMR spectrum is shown in Fig. 20. It shows the resonances from ADP, ATP, inorganic phosphate, phosphocreatine, and some weaker responses from other metabolites. There is some hint of multiplet structure but this is not clearly resolved. The heterogeneity of the living material may lead to distributions of chemical-shift values and local susceptibilities, which broaden the NMR lines and reduce resolution compared with pure material in solution.

FIGURE 20 ³¹P NMR spectrum of an in vivo human muscle.

It is clearly of great importance to record the in vivo NMR spectrum from the organ or region of interest precisely. We want no contamination from neighboring regions. This problem of precise localization of the spectra presents the greatest challenge to the in vivo NMR spectroscopist.

XV LOCALIZATION OF MRS SPECTRA

In this section we discuss several methods which have been used for the localization of MRS spectra. None of them are perfect and some may be more useful than others for a particular situation. In some cases more than one method can be applied simultaneously with advantage (see also Chap. 9, Alger, this volume).

Surface coil. The sensitive region of a surface coil of circular cross section is roughly speaking a hemisphere below the coil with a radius equal to the radius of the coil. This is of value for the examination of regions on or just below the surface such as superficial tumors. The extent of the region examined below the coil is not well-defined.

Field profiling. The static magnetic field of the magnet can be shaped in such a way that there is a region of homogeneous field close to the magnet center, surrounded by a very inhomogeneous field. The homogeneous field gives a good spectrum superimposed on a broad response from other regions which can be subtracted. The region of interest cannot