Magnetic Resonance Imaging (5 May Version)

Purpose: The goal of this experiment is to introduce the fundamental principles and practice of magnetic resonance imaging (MRI), a form of NMR spectroscopy. A two-dimensional (2D) image of the stem or bud from a plant will be obtained and the utility of NMR relaxation as a means of enhancing contrast will be examined. This experiment was developed by Cyrus Maher, Class of 2006, as his senior thesis project in chemistry.

References* denotes those references which should be read as preparation for the experiment.

1)* S. L. Smith, "Nuclear Magnetic Resonance Imaging", Anal. Chem., 57, 595A-608A (1985).

2)* H. Friebolin, Basic One- and Two-Dimensional NMR Spectroscopy, 4th. ed., Wiley-VCH, Weinheim, 2005. (Examine the material on MRI, relaxation, and the spin-echo exoperiment.)

3) P. T. Callaghan, Principles of Nuclear Magnetic Resonance Microscopy, Oxford Univ. Press, Oxford, 1991. (This monograph is one of the 3 great treatises in NMR.)

4) Z.-P. Liang and P. C. Lauterbur, Principles of Magnetic Resonance Imaging, IEEE Press, New York, 2000. (Liang and Nobel Laureate Lauterbur provide a careful mathematical treatment of MRI. The text was written to accompany a course at the University of Illinois.)

5) K. R. Brownstein and C. E. Tarr, "Importance of Classical Diffusion in NMR Studies of Water in Biological Cells", Phys. Rev. A, 19, 2446-2453 (1979).

Introduction and Basic Theory

In the following sections, vectors are printed in bold face and operators in Italics.

NMR spectroscopy is based on the magnetic properties of the atomic nucleus. Any nucleus with non-zero spin (s>0) is a magnetic dipole with a magnetic dipole moment = gNS where the nuclear magneton, N = eh/4πmH = 0.50504 x 10-26 J/T = 0.50504 x 10-23 erg/Gauss. In the case of the proton which will be the spin examined in this experiment, s, the spin quantum number, is ½ and g, a quantum mechanical correction factor to classical physics, is 5.585486. When a single nucleus with spin (spin angular momentum) is placed in a magnetic field with strength B, the Hamiltonian energy operator is given by

H = -·B = -gNS·B (1)

If the magnetic field is entirely along the z axis, i.e. B = B0 k, the Hamiltonian reduces to

H = -gNB0Sz (2)

Recall that the Sz operator yields the z component of the spin angular moment and its eigenvalue in units of h/2π is given by the quantum number m. m is +½ and -½ for a proton. Therefore, the magnetic energy of the nucleus is given by

E = -gNB0m (3)

In an NMR experiment, the spins are excited by a pulse of radiofrequency radiation. The selection rules for the excitation are Δs = 0 and Δm = 1. Therefore, the frequency of the transition is given by

ν = ΔE/h = gNB0/h (4)

NMR spectroscopists often measure frequency as angular frequency ω in radian/s sec where ω = 2πν.

ω = [gN(2π/h)]B0 = γB0 (5)

γ is called the gyromagnetic ratio; its value in cgs-esu units for the proton is 26753 radian Gauss-1 s-1. In a conventional NMR experiment, the instrument is adjusted for a homogeneous magnetic field. That is, B0 is constant across the sample and all protons with the same chemical shift exhibit the same NMR frequency. In the case of our instrument, B0 = 9.3974 Tesla or 93974 Gauss so ω = 2.5473 x 109 radian/s and ν = 400.13 MHz.

Imaging via NMR spectroscopy, i.e. MRI, is achieved by the use of gradients. Their application with be first explained via imaging along the z axis. In MRI the magnetic field generated by the superconducting magnet, B0, is supplemented by a gradient, a linear variation in the magnetic field.

B = B0 + Gzz where Gz = (B/z) (6)

A gradient is generated by passing a current through a special coil whereby Gz = ЖI. In the case of our spectrometer, the current supply for the gradients consists of 3 channels. Each channel can produce upon computer control a current in the range -10 A to 10 A. The conversion factor Ж, the so-called coil constant, is 5.56 Gauss/cm-A. It follows from equations (5) and (6) that the NMR frequency ω is now a function of position.

ω = ω(z) = γB0 + γGzz (7)

Recall that a spectrum is a graph of proton density versus frequency. In the case of no gradient, the case in conventional NMR spectroscopy, the spectrum will consist of a sharp line centered at ω = γB0. However, with MRI, the spectrum becomes a continuous function. The Fourier transform of the signal yields proton density, ρ, as a function of

2

frequency. Since the frequency depends linearly on position, the spectrum, a graph of ρ versus ω, is easily converted with the aid of equation (7) to an image, a plot of ρ versus z.

Multi-dimensional MRI is achieved by the use of a triple-axis probe, one with coils that can independently create gradients along 3 orthogonal directions. Our new probe which can only detect and excite protons has this functionality. Our other probes, while quite versatile for chemical purposes, can only apply a gradient along the z or axial direction and only permit one-dimensional imaging.

Experiments which utilize a triple-axis probe for 3D imaging are now based on multi-dimensional spectroscopy. Richard Ernst, our Robbins Lecturer in 1998, received the Nobel Prize in Chemistry for the development of Fourier-transform NMR and multi-dimensional NMR. In order to understand Ernst's contribution, consider the simplest 2D experiment. Ernst divides the experiment into preparation (P), evolution (E), and detection (D) periods. During the preparation period, a coherent oscillation of the magnetic dipoles is created by the application of a radiofrequency pulse or sequence of pulses. Following excitation, the magnetization is allowed to oscillate for a time period t1, the evolution period. Finally, the receiver and the A/D converter are gated on and the magnetization is measured TD(2) times during the detection period, t2. The experiment is completed TD(1) times. With each pass, the length of the evolution period is incremented. Upon completion of the experiment, the signal is a function of two independent time scales, t1 and t2. A two-dimensional Fourier transform yields a spectrum as a function of two angular frequencies, ω1 and ω2.

A 2D experiment can be converted into 3D imaging with the following modifications.

a) A selective radiofrequency pulse is used during the preparation period along with a gradient along the z axis. Selectivity is understood by referring to the Heisenberg Uncertainty Principle. If a wave form is applied for a very short period, Δt, there is a large spread in the wave form's frequency, Δν. That is, (Δt)(Δν) ~1. This would the case of a hard pulse, e.g. 5-10 μs, where the radiation covers the entire spectrum. Conversely, if Δt, the pulse length, is sufficiently long, the radiation seen in the probe covers a very narrow range of frequencies. We use in our MRI methods pulse lengths of 1.5 to 3.0 ms. The selectivity of the preparation pulse is further enhanced by adjusting the shape of the pulse. Hence, the radiation is centered at a frequency ω' and covers the range ω' - (Δω/2) < ω < ω' + (Δω/2). Because of equation (7), the selective pulse only excites protons in a narrow slice along the z axis, z' - (Δz/2) < z < z' + (Δz/2), and only these protons contribute to the signal during the detection period. For obvious reasons, the gradient applied during the preparation period is called the Slice gradient.

b) During the evolution period, a second gradient is applied along the y axis. Hence, the oscillation frequency of the magnetization varies along

3

the y axis and information about the structure of the specimen along the y axis is encoded in the signal. This information is not detected directly as the signal is only detected during the detection period. However, the state of the magnetization at the start of the detection period depends on its state or phase at the end of the evolution period. In the vector model of spin angular momentum, the angular momentum vector precesses about the z axis at an angular frequency defined by equation (7). At the end of the evolution period t1, the phase of the signal is given by φ = ω(y)t1 where the notation for the angular frequency, ω(y), emphasizes the application of a y gradient, often called the Phase gradient.

c) Finally during the preparation period, a third gradient, the Read gradient, is applied along the x axis. Therefore information on the position along the x axis is directly encoded in the signal collected during t2. A double Fourier transformation is followed by a transformation of the axes from a frequency scale to a position scale. The result is a plot of proton density in the x-y plane in a slice perpendicular to the z axis.

For technical reasons. two important modifications are made in the experiment. A full discussion including several details not discussed here is provided in the comprehensive treatises by Callaghan and Liang & Lauterbur. A 180 selective pulse is inserted in the middle of the evolution period. The pulse sequence becomes (90)-(TE/2)-(180)-(TE/2). TE is called the echo period. This is the famous Carr-Purcell spin echo sequence. It is discussed in Friebolin and is employed to correct for the loss of signal resulting from dephasing of the spins caused by magnet inhomogeneity and the application of the Slice gradient. After application of the first pulse, the magnetization rapidly decays. However, after the second pulse, it recovers as an echo and peaks in amplitude at a time TE after the first pulse. One can apply a sequence of 180 pulses and delays, This will in fact be one of our approaches. Secondly, rather than varying t1 as a constant Gy as originally proposed by Ernst, most MRI units hold t1 constant and increment the gradient Gy. Note in either case, position and frequency information during the evolution period is encoded into the detected signal via the phase at the end of the evolution period.

In the execution of the modified Carr-Purcell pulse sequence, one generates a trail of echoes. You will observe that the intensity of the echoes decreases exponentially as a function of the total echo time, nTE where n = 1, 2, etc. That is,

I = I0exp(-nTE/T2) (8).

In equation (8), T2 is the time constant for the decay of the signal. This decay results from the dephasing of the spins contributing to the signal. In other words, T2, often called the transverse or spin-spin relaxation time, is the time constant for the loss of coherence and is due to natural, irreversible processes. Relaxation is the term applied to the molecular basis for the loss of coherence. If the mechanism responsible for relaxation is known, values of T2 can provide additional information about the sample. Relaxation

4

is also used as a tool to optimize contrast as different populations of water can relax at different rates. At the macroscopic level, relaxation is also manifested in two other ways: the rate of return to equilibrium with a time constant T1 and the rate of energy change between dipoles. The energy exchange can be measured via the nuclear Overhauser effect (nOe).

Relaxation is a spectroscopic phenomenon that affects line shapes, intensities, and in MRI contrast. At the molecular level, relaxation is caused by any process that produces a randomly varying magnetic field. In bulk water the most important mechanism for relaxation is the interaction between magnetic dipoles. However, in this experiment the water is confined in biological structures such as cells and a different relaxation mechanism which was developed by Brownstein and Tarr dominates. When more than one mechanism, e.g. dipole-dipole (DD) and Brownstein-Tarr (BT), contributes to relaxation, the net, experimental value of T2 is given by equation (9).

1/T2 = 1/T2,DD + 1/T2,BT (9).

You will show that T2,DD, i.e. T2 from the dipole-dipole mechanism, is orders of magnitude greater than the experimental value of T2. It follows from equation (9) that the experimental value of T2 is dominated by T2,BB and therefore T2 T2,BT. This is an important result as it enables you to use the Brownstein-Tarr mechanism to interpret the data.

In order to draw the conclusion made above, one must be able to estimate T2,DD

from the dipole-dipole relaxation mechanism. To this end, consider the rotation of a water molecule, a process described by a random-walk model. The time scale for this random-walk is set by the rotational correlation time, c. As the molecule rotates, one hydrogen spin, a magnetic dipole, perceives a vector change in the magnetic field due to the movement of the other spin, also a dipole. In the case of water, the correlation time for this rotational modulation of the magnetic field is in the range of picoseconds. One can estimate the correlation time for water by applying hydrodynamic theory at the molecular level and assuming that the water molecule is spherical. This assumption of spherical symmetry works very well for globular proteins. The result derived originally by Einstein and Debye is

c = V/kBT (10)

where V is the molecular volume, a quantity generated by Spartan, and is the viscosity of the solvent. With an estimate of c in hand, T2 is calculated via equation (10). Note that cgs units are used in equation (11). The details for this celebrated result can be found in Callaghan.

1/T1 = 1/T2 = (3/2)[h2γ4/4π2rHH6]c (11)

5

In equation (11), rHH is the proton-proton distance in water. A good estimate of rHH and V can be obtained via molecular modeling, e.g. a Hartree-Fock calcuation in Spartan with a 3-21G* basis set. The sixth-power dependence on the relation between the molecular rotation and observables is the basis for the application of the nuclear Overhauser effect to the determination of protein structure.

From the perspective of the physical chemist, relaxation and the value of T2,DD are important as they provide a window on molecular mobility on the picosecond time scale. If protons on a molecule, e.g. a protein in bulk water, have different mobilities, their T2's will also be different. Note that a decrease in mobility, e.g. a longer correlation time, leads to a decrease in T2. If one lengthens the echo time, the signal will be dominated by the more mobile proton. If the dipole-dipole model dominates, the structure containing this water would stand out in the MRI image. However, there is a cost for this enhancement. As one lengthens the echo time, the signal for all waters will decrease. Obtaining good contrast will require an increase in the number of scans in order to restore the lost signal-to-noise.

As part of the calculations for the experiment, you will calculate T2,DD and show that it is ca. 3 s. In contrast, you will find that the experimental value of T2 is ca. 20-30 ms. Consequently, a mechanism other than dipole-dipole, e.g. the Brownstein-Tarr mechanism, makes the dominant contribution to T2 and the value of T2 provides information about the size of the space in which the water molecule is confined. Translational diffusion is a random-walk process described quantitatively by the relationship

rrms = <r2>0.5 = [6Dt]0.5 (12)

where rrms is root-mean-square distance and D is the translational diffusion constant. During a period t, 68% of the molecules will travel up to rrms from the position at t = 0. For the MRI experiment, t equals the total echo time and one finds that rrms is of the order of magnitude of the cell dimensions. That is, during the execution of the pulse sequence, a typical water molecule has a high probability of diffusing to the cell wall. The encounter between the water molecule and the cell wall abruptly changes the phase of its Larmor precession and is the origin of relaxation. After working out the mathematical details, Brownstein and Tarr showed that T2 is directly proportional to the size of the enclosure. The constant of proportionality, which depends on the detailed geometry of the enclosure and the details of the interaction between water and the cell wall, cannot be determined in the MRI experiment. Therefore, we can only draw qualitative conclusions from the value of T2. Water populations in different spaces will exhibit different values of T2. Water in a smaller space will on the average require less time to diffuse to the cell wall and will have a smaller T2.

Experimental Procedure

You will use Version 2.1.1 of ParaVision to acquire and process your images. ParaVision is Bruker's MRI software. You will be using the same software as that found

6

on the large medical instruments. We have modified the Chemistry Department's NMR spectrometer so that it can perform authentic MRI. To this end, we replaced our single-channel gradient amplifier with a triple-channel gradient amplifier and purchased a proton only, triple-axis gradient probe. We retained the original 9 Tesla magnet which has a bore size of only 52 mm. This narrow bore must accommodate the probe with its compact electronics and the NMR tube. You will insert your biological specimen in the same 5.00 mm OD, 4.22 mm ID NMR tube that you used in organic chemistry and the barrier experiment. Hence the diameter of your specimen must not exceed 4.2 mm. Furthermore, the receiver/transmitter coil can only excite protons over a maximum range of 16 mm and an effective range of 11 mm. Furthermore, in contrast to the large medical units which use water-cooled gradient coils and large currents (up to 200 A!!), our gradient coils are air cooled. Hence, we must be conservative in the currents used and the length of the periods when gradients are applied. As a result of these constraints, we are limited to the imaging of small specimens with axial symmetry and must devote 30 minutes or more to the acquisition of a good image. However, we can achieve far superior resolution, 0.05 mm or better, with our arrangement. Resolution on a medical unit is 1 mm or less.

In this section, boldface marks xwinnmr, ParaVision, and Unix commands. Italics will be used to highlight xwinnmr and ParaVision buttons.

A) Preliminary steps done in xwinnmr.1) Secure a biological specimen and load it into a 5 mm NMR tube. The specimen should contain mobile water. Its outer diameter, OD, should not exceed the inner diameter, ID, of the NMR tube. Plant materials have been found to work well. A desiccated fly would not work as most of the hydrogen is tied up in relatively immobile macromolecules that give very broad signals. In a hydrated animal specimen, both water and fat molecules which differ in chemical shift by ca. 3 ppm, are present and contribute significantly to the signal. Unless the contribution of the fat to the fid is suppressed, its presence limits resolution in the MRI image. Special techniques are required to image solids. Something that wiggles will give a very poor image since long acquisition times are required on our spectrometer.

In order to facilitate retrieval of the specimen from the NMR tube, attach a short piece of dental floss which will serve as a leash. A piece of narrow glass rod can be used to gently push the specimen into the tube. Cover the top of the tube with Parafilm.

2) You will be using the proton-only triple-axis gradient probe, probe 31. The instructor will load the probe, connect the cables, and check the DC balance on the 3 channels of the gradient amplifier. The identity of the probe that has been installed is shown on the white board.

3) Turn off the auxiliary cooling air and eject the sample in the probe. Insert the NMR tube with your specimen into the rotor. Use the depth gauge. The section of the specimen that you wish to image should be centered in the black strip marked 5 mm. Insert the sample into the probe and then turn on the auxiliary cooling air.

7

3) Log on to the SGI O2 workstation ernst which services the spectrometer. Use the "student" account. We have two versions of the xwinnmr software; we shall use the version tied to ParaVision, the MRI software. First load xwinnmr by entering xwinnmr and perform these initial configuration steps.

a) Indicate the change of the probe at the software, i.e. xwinnmr, level. To this end, enter at the xwinnmr command line the command edhead. Scroll down the list of probes and highlight the line for probe 31, the triple-axis gradient probe. Click on the button Define as Current Probe and leave the procedure by clicking on the Exit button..

b) Load the generic shim set for the probe by entering rsh xyz_6Apr2006. This file might be updated. Check the white board adjacent to the O2 work station for any updates. This will get you in the ball park but you will refine the shimming later in the procedure.

c) You will be running at ambient temperature and will not be using the temperature controlled. Even so, it is useful to know the temperature of the probe. To discover this, enter edte. The window for the temperature controller will open. Record the ambient temperature and close the window.

d) Check the keypad. The sample should not be spinning. The lock and sweep lights should also be off.

e) Tune the probe so that the frequency at its maximum sensitivity corresponds to the transmitter frequency. In the step you will also set the impedance of the probe so that it matches the impedance of the transmitter. This step called matching minimizes reflection of power at the connection of the two devices. To this end, turn on the display window in xwinnmr by entering acqu and initiate the tune/match process by entering wobb. The tuning curve for the probe will appear on the screen. The minimum on the curve should line up with the vertical line at the spectrometer frequency. If it does not, one carefully adjusts TUNE rod at the bottom of the probe. The instructor will demonstrate this procedure. Never turn the tuning rod against the end of its range. This will damage the probe, a $25,000 item. The minimum in the tuning curve should be close to zero, the probe's impedance is correctly matched to the transmitter. If not, the MATCH stub has to be adjusted as well. The instructor will perform this step. When the tune/match procedure has been completed, terminate the process by clicking on the STOP button at the left of the xwinnmr window. Alternatively, one can enter the kill command and click on the wobb line in the list of active processes.

8

f) As a first step, you will record a one-dimensional proton spectrum of the specimen without gradients. Following the procedure employed in the Barrier experiment, use the File and Load command to load the template file for the procedure: MRI_1D. Next, make a copy of the template with the File and New commands. Provide a unique file name for the copy and click on Save. Next as in the Barrier experiment, set the gain on the proton receiver by entering rga and then execute the pulse sequence by entering zg. After the fid (free induction decay) has been acquired, Fourier transform the fid by entering ft and then phase the resulting spectrum.

For the record, the following parameters are defined in the template: pulse program, zg30; SOLVENT, select H2O+D2O; power level, pl1 = -4 dB; pulse time, p1 = 4.0 s; td, 4k.

g) Next refine the shimming of the probe for the specimen that you have loaded. In routine spectroscopy, this is done by maximizing the deuterium lock signal. However, in the case of MRI, the specimen contains very little deuterium and you cannott lock the spectrometer. Magnet drift is very slight with superconducting magnets so locking is not required. In the absence of a lock signal, one employs a different approach to improve the shimming. The approach employed in this experiment is to exercise repetitively a pulse sequence such as zg and adjust the shimming while looking at the spectrum. This is called shimming on the spectrum.

To initiate shimming on the spectrum, enter gs. The pulse sequence will be executed repetitively and the result of each acquisition rather than the accumulated signal will be displayed on the screen. Next enter frq in the command line. As a result, a phased spectrum will appear on the screen after each pass through the pulse sequence. Observe the effect on the spectrum of adjustments in the most important shimming parameters: z, z2, z3, x, and y. Large changes may be needed. The z shim is the most important. When you have optimized the spectrum, terminate the procedure by clicking on the STOP button.

4) Now that you have optimized the homogeneity, measure the proton spectrum of your specimen. Gradients will not be used in this step so all protons, e.g. water protons in the case of a plant specimen, in the receiver coil of the probe will contribute to the signal. One can determine the chemical identity and net concentration of the compounds containing hydrogen but not their location. (This approach is often done in the application of NMR to the diagnosis of disorders of the brain. In this medical application, phosphorus-31 is used.) The proton spectrum can also be measured in ParaVision but xwinnmr is easier to use.

9

a) Perform an acquisition by entering zg. After the completion of the acquistion, Fourier-transform the data by entering ft and phase the spectrum by entering apk. You might want to phase manually.b) Observe the spectrum and determine the chemical shifts in ppm and Hz of the major peaks. Does water make the dominant contribution to the spectrum? The presence of several important components in the spectrum can degrade the quality of an MRI image. The simpler MRI methods work best when one chemical species contributes to the signal and therefore the variation of the signal with frequency depends solely on location of the molecules. Also measure the full width at half height. Recall that you used the macro hwcal in the barrier experiment. Before employing hwcal, you have to set the parameters for the spectral display. Print via xwinplot the parameters, the fid, the spectrum and possibly expanded sections. Annotate the printout with the identity of your specimen. First capture the parameters by entering xwp_lp and then enter xwinplot. The window for wxinplot will open and you can plot and print from there.

c) When you are done with these steps, exit from xwinplot and xwinnmr.

B) Imaging via ParaVision:

1) Start ParaVision by entering pv from a Unix shell. Be patient. ParaVision will open a sequence of windows including one for the ParaVision version of xwinnmr. You may close the window that gives the address for Bruker. Do not close the xwinnmr window directly! Report any system crashes to the instructor. An increase in the O2 memory from 64MB to 256 MB seems to have solved this problem. ParaVision employs a number of windows. You will make extensive use of 4 of them.

a) The master window is the Systems Control window, small window that normally appears in the upper right of the screen. Use this (only this one!) menu via the File and exit entries to exit from ParaVision. You can open the xwinnmr window under Tools. Important messages appear in the message box. Note that ParaVision users an older version of xwinnmr than the chemical applications even though we just purchased ParaVision.

b) You will make extensive use of the ParaVision Scan Control to set up an imaging experiment and define most parameters for the run. One can open the Spectrometer Control Tool under the Scanner entry in the menu. ParaVision is written for physicians and denotes the spectrometer as a scanner. Information on files and the status of tasks appears in the various message boxes.

c) You will set some parameters and directly control the spectrometer using the Spectrometer Control Tool. Not all functions of this tool

10

function properly under Version 2.1.1 of ParaVision, the most recent release for Unix work stations.

d) The images are displayed and manipulated by the Image Display & Processing window. The underlying program is called xtip.

2) To set up a scan, ParaVision's term for the acquisition of an MRI image, define your specimen by clicking on the New Patient button at the top of the Scan Control window. A new window will appear. Provide the name of the specimen in the field for patient name and a unique experiment number. Press the Enter key on the keyboard after you have typed each entry. The experiment number becomes part of the file name at the Unix level. Use the current date for the experiment number. For example, if the date were April 1, 2006, enter 4012006. The other fields such as the weight and sex of the patient do not matter. Exit this menu by clicking on Accept.

3) The successful completion of the previous step leads automatically to the Edit Study menu which can also entered directly by clicking on the Edit Study button in the Scan Control. Provide a name for the type of measurements that you will make in the "study" line and select POMONA in the "location" line. The latter is very important as it sets the path to the protocols developed for our spectrometer. Exit this menu by clicking on Accept.

4) Completion of the last step should open the Edit Scan menu. If not, click on the on the New Scan button. At this step, you will define the type of experiment, i.e. the method, and the associated parameters. Click on the Load button. You will be presented with a list of protocols. Select MSME_Chem160 and click on Accept.

The method you have selected will generate images in 3 slices and measure T2 for selected populations of water molecules in your biological specimen. You will use the Multi Slice Multi Echo (MSME) method which a variation of the classic Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with gradients. A graphical summary is given in Appendix I. The CPMG pulse sequence employs a train of 180 pulses with echo periods TE and acquisition periods.

(90)-(TE/2)-(180)-(TE/2)-(acq) -(TE/2)-(180)-(TE/2)-(acq)-(TE/2)-(180)-(TE/2)-(acq)-...

If the sequence -(TE/2)-(180)-(TE/2)-(acq)- is repeated n times, one acquires an echo signal n times and with one pass through the entire pulse sequence acquires data for n images. The total echo time for the first, second,... image is 1(TE), 2(TE), ... Gradients are applied during execution of the pulse sequence to yield a 2D image of an axial slice.

It is worth mentioning at this point the hierarchy of the information associated with each experiment. At the hardware level, one starts with the pulse program which defines the sequence of RF pulses and their timing. The gradient program defines the gradients that are required for the execution of the pulse programs. Both of these require relationships between the physical experiment, e.g. gradient strength, and imaging parameters, such as the size of the image or field of view (FOV). The combination of pulse program,

11

gradient program, and programmed inter-relationship between parameters constitutes a method. The execution of a particular experiment also requires the specification of parameters. The combination of parameters and a method constitutes a protocol.

Normally methods, pulse programs, gradient programs, and most parameters are determined in a research lab or the applications lab of the manufacturer of the MRI. The protocols are carefully tested when the MRI unit is installed. A technician in a hospital uses the unit as a turn-key system and only makes small changes in the parameters. You will have more flexibility in this experiment.

5) At the next step, you will verify the parameters and make minor changes via the Scan Editor which is opened in the Scan Control window by clicking on the Edit Scan button. You can now view and modify most of the acquisition and processing parameters. These parameters are grouped into 6 sets: Standard, Geometry, Contrast, Mode, Special, and Research. A group is selected by clicking on the appropriate button in the panel on the left. The document will address the most important parameters whose values must be checked and changed as appropriate. Once you have done this, click on the Accept button. The Store button is used to save a Protocol under a new name.

a) Standard group You will produce a 2D image in a slice perpendicular to the z axis. The field of view (FOV) is the dimension of this image. In the first run, you will image in one slice. The following parameters are important:

Field of View: 5.0 mm (It is tempting to improve resolution by reducing the FOV. However a reduced FOV requires stronger gradients which our probe and gradient amplifier cannot handle. Therefore, don't reduce the value of FOV.

Slice Thickness: 1 mm (A smaller slice thickness requires higher gradient currents. We are sacrificing axial resolution for safety.)

Slices: 3 Interslice distance: 1 mm. Repetition Time: 2000 ms (This parameter gives the time between excitation

pulses. Its long value relative to that in a hospital environment is required to prevent damage to the gradient coils.)

Echoes: 7 (The signal will be acquired 7 times (once per echo) per pass through the pulse sequence. Another method will be used to acquire multiple echoes.)

TE1 Effective 1: 18.3 ms (This is the echo time, TE.) Averages: 4 (This corresponds to NS in xwinnmr and provides the number

of passes. This number must be even.)

b) Geometry groupSome of the information here duplicates that found in the Standard group. The new information will be discussed below. Note that the field of view (FOV) is given in both the Read (x) and Phase (y) directions but the field for the second is

12

grayed out and cannot be adjusted. This is because the FOV is declared to be isotropic in a later line.

Matrix Read: 128 (Values of the proton density are obtained at 128 points along the x axis. This corresponds to sampling the fid 2(128) = 256 times during the acquisition period. Note that the resolution along the z or Read axis is given by FOV/(Matrix Read). If you halve this value of Matrix Read, you will not degrade the resolution much but will reduce the length of the run. If you decide to halve Matrix Read, also halve Matrix P1.)

Matrix P1: 128 (P stands for phase. This sets the resolution along the y axis. The pulse sequence is completed 2(128) = 256 times; with each pass the y gradient is incremented. The Matrix Read and Matrix P values should be the same.

Offset: 0.00 mm (This value is given in mm are defined the location of the Read slice along the z axis. The origin is in the center of the receiver coil. There is nothing sacred about an offset of 0.00 mm. Other values are possible as long as they are less in magnitude than ca. 5 mm.)

Slice Orient: Axial (This very important parameter sets the Slice gradient along the z axis.)

Readout: L-R (This sets the direction of the Read gradient along the x axis. A-P for anterior to posterior would work as well since the specimen has axial symmetry.)

Anti Alias Read: 2 (Set to avoid foldover of the data.)

Anti Alias P1: 1

Isotropic: FOV (The value of Isotropic sets the x and y dimensions equal.)

c) Mode groupDimensionality: 2D (A two-dimensional image will be obtained. This

parameter defines in part the pulse program.)

d) Research groupSeveral important hardware parameters are set or reported here including the length of the run.

Measurement Method: MSME_TOMO (This very important line selects the

method and hence the pulse program, the gradient program, and the inter-relationship between the parameters. )

13

Sweep Width: 50125.3 Hz

Excitation Pulse Length: 1500 μs (This is the length of the selective 90 preparation pulse. It has a hermite pulse shape.)

Inversion pulse: 1500 μs (This is the length of the 180 pulse. It should have the same length as the excitation (90) pulse.)

6) You are now poised to control the spectrometer and acquire an image. Open the Spectrometer Control Tool by clicking on the icon with the hammer and screwdriver in the Scan Control Window.

a) Using the slide bar, set the Tx Attenuator 0 value to 38.1 dB. The Tx Attenuator 0 sets the RF power for this pulse. This value corresponds to pl1 in xwinnmr. [This value is calculated from the value for a hard pulse via the xwinnmr macro pulse. A correction of -11.4 dB is added since a shaped pulse rather than a rectangular pulse is used.] The power for the 180 pulse has to be doubled, doubled since the same pulse time is used for both pulses. Therefore the value of Tx Attenuator 1 has to be 6 dB less than Tx Attenuator 0.

If the probe is properly tuned and matched, the values of Tx Attenuator 0 and Tx Attenuator 0 (38.1 and 32.1) should work for all samples. The ParaVision manual suggests that these values be determined experimentally for each sample. Normally, there is no need for this additional step which is sometimes taken in 2D spectroscopy when well defined pulses are essential for good results.

b) Using the slide bar, set the Receiver Gain to 200. This is an estimate. In order to set the correct receiver gain, you want to start the pulse program in a repetitive mode. Click on the GSP button at the top of the Spectrometer Control Tool. In a few seconds, the acqDisplay window will open. The fid will appear in this window. The message "Digitizer Filling" and a number will appear at the bottom. The number is the fraction of the full scale of the A/D that is employed. The number should not exceed 100% or the signal will be clipped. Adjust the Receiver Gain so that the Digitizer Filling is around 50%. When this process is completed, stop it by clicking on the STOP sign.

c) Begin the acquisition of the data by clicking on the GOP button. You may have noticed an entry for your run (scan) in the Scan Overview window of the Scan Control. When the experiment is ready to be performed, the active entry will have the word READY at the end of the line. Once the run has begun, the message will change to IN PROGRESS and an X is drawn across the GSP and GOP buttons. Upon completion,

14

the message will be COMPLETED. While the run is in progress, note the yellow band in the Status line. The band is a measure of the time required for the completion of the run. Timing information is also given in the acqDisplay window. While the run is in progress, don't manipulate any images on the screen. Our Unix workstation might crash if it is asked to perform too many tasks.

7) Upon completion of the run, transfer the results to the Image Display and Processing window. Highlight the line for the run in the Scan Overview window of Scan Control. With the cursor over the line, hold down the middle mouse key. A special icon will appear that represents the image. Drag the icon to the Image Display and Processing window. The image for one echo time will be shown. The following instructions will show to examine an image for each echo time, define regions of interest (ROI), and extract intensities for each ROI. For each slice, one ROI should be an area where there is no signal. This is the background ROI.

a) Fill the window with one image by clicking onLayout

1 x 1 viewportb) Expand and shift the image as desired by clicking on the magnifying lens icon. While holding down the Left mouse button, moving the mouse shifts the image. While holding down the Middle mouse button, moving mouse expands or shrinks the image.c) Select a frame with a particular echo time by clicking on

Display Frame Select (Provide the number of the frame).

Buttons to the right also provide this function.d) Examine images at several echo times. Do the intensities in the images for all populations of water decay at the some rate or at different rates? With your specimen, can relaxation be used as a vehicle for enhancing contrast? To pursue these questions quantitatively, define several regions of interest (ROI's). To define a ROI, open the Region of Interest tool by clicking on

ROI Define

A new window will open. To create a new ROI, click on the New button. Then click on the button that defines the shape of the ROI, e.g. square or circle and click on the Graphic All button. Next move the cursor into the image; hold down the Left mouse button and move the mouse to define the ROI. Providing a name for the ROI is the last step for the definition process.

Each ROI created has a unique name. The current ROI will be highlighted. The change to a different ROI, simply click on its name in the table. To obtain data for the selected ROI in the image currently displayed, click on the Display button. Use the tool discussed in part (c) above to move from image to image. For each

15

ROI including the background ROI, record for each value of TE its integrated intensity and its area in pixel units (pu).

D) ShutdownOnce you have completed the experiment and recorded the values of all important imaging parameters, it is important to shutdown the spectrometer in an orderly fashion. First, exit ParaVision through the Systems Control window. You might close the Image Display & Processing window first but do not directly close the xwinnmr window. Ask the instructor if the probe will be changed. If not, simply remove your sample from the spectrometer and insert a replacement sample tube, e.g. the sealed chloroform sample. Extricate your biological specimen from the NMR that you used.

Use the following more involved process if the probe will be changed.1) Eject your sample. Keep the eject air flowing but do not add another sample yet.2) The instructor will physically exchange probes. Now add the replacement sample tube.3) Complete the switch at the software level. Enter XWINNMR (the chemist's version) and declare the new probe, probably the dual probe, number 3-via the edhead command. This is the reverse of the earlier application of edhead. You will not use edhead at the ParaVision level since probe 31 is the only probe used with MRI.4) While still in XWINNMR, load the default shim file via the rsh command. The name of the file for the probe is provided on the white board.5) Finally, re-initialize the interface by entering the ii command.6) Exit from XWINMR.7) Print any files that you need and log off the O2 work station.

Calculations and Preparation of the ReportYour report should be accompanied by copies of all images and entries from your laboratory notebook. Provide a summary of the key acquisition and processing parameters for the MSME run. You will select 2-3 regions in your MSME images, preferably ones that show contrast and exhibit differences in anatomical structure, and determine T2 for each from the a fit of integrated intensity I with echo time.

Your intensity data will be scaled and background corrected. For the background ROI, divide the integrated intensity by the pixel area. Average the 7 values of the scaled intensity and calculate the 95% confidence interval. Similarly, scale the intensities obtained for the other ROI's by dividing their intensities by their respective pixel areas. Subtract from each scaled value the average of the scaled background intensity. Compare each scaled, background-corrected intensity with the 95% confidence interval obtained from the averaging of the scaled background intensities. A datum is only useful if it is larger than the 95% confidence interval.

16

Fit the scaled, background-corrected data that are above the noise level to equation (8). The best approach is the use of the nonlinear regression routine of NCSS. A less satisfactory approach is linearization followed by linear regresssion. That is, one employs equation (12).

ln[I] = b -nTE/T2 (12)

Validate the use of the Brownstein-Tarr model for relaxation. Estimate T2 which would be obtained if the dipole-dipole mechanism were dominant. That is, using equations (10) and (11), calculate the rotational correlation time of water in bulk water and the associated value of T2. This result from modeling should be long compared with the experimental values of T2. The department purchased two licenses for ParaVision. The second copy has been installed on the Linux work station in Seaver North 205. The public copy of SYBYL is also installed on this workstation. The second copy of ParaVision is a more recent version and is available for processing and displaying data. The Linux work station cannot control the spectrometer. To start a ParaVision session, enter pv -l. (Please note, the l in the command is the letter "el" and not the number "one".)

In order to access your data on the Linux box, you will have to transfer the appropriate folder from the SGI work station. Make sure that the Ethernet cable for the SGI is connected to the port on the wall. Disconnect it when you are done with the transfer. A bit of Unix is required for this process.

1) While logged on to the SGI, go to the directory where the data are stored.cd /disk2/Pv2.1.1/data/student/nmr

2) Confirm that the directory is there. The first letters of the directory will be the registration number that you gave to the specimen, i.e. patient.

ls3) In what follows, dirname is the name of the directory. This directory contains a hierarchy of directories and files. In order to move it, it must first be converted in one file via the Unix tar command. This is equivalent to the Windows zip command.

tar cvf dirname.tar dirname4) Log on to the Linux box and check your directory structure.

cd /opt/PV3.0.2/data/your_user_name5) if the directory nmr does not exist, create it via the Unix command

mkdir nmrYou only have to do this once.6) Go the the nmr directory.

cd nmr7) Now use secure shell ftp to transfer the tar'd file from the SGI to the Linux box.

sftp student@ernst.pomona.edu(Provide the student pass word, sage_hen, as prompted.

cd /disk/Pv2.1.1/data/student/nmr [Go to the directory on the SGI

17

with the tar file.]ls *.tar [Confirm that the file is there.]get dirname.tar [Transfer your tar file to the Linux box.]bye [Exit from the ftp shell.]

8) Now untar the file. that is, extract the folders and files from the tar file.tar xvf dirname.tar

You are now ready to access the file from ParaVision. When you run ParaVision the first time[recall enter pv -l], you will have to use the file command and the patient option to access your file.

18

Appendix I. Graphical representation of the MSME method. The pulse sequence to generate one echo is shown.

Magnetic Resonance Imaging2.doc, WES, 5 May 2006

19

20

MAGNETIC RESONANCE IMAGING

NAME:_________________________________ Date experiment completed:________

1) Description of your biological specimen. (species, nature of the specimen, etc.)

2) Attach a copy of your 1D proton spectrum and the MSME images with a brief anatomical analysis on the back. The images should show the ROI's used in the determination of T2.

3) Analysis of the Relaxation Data from the MSME Images image total echo ROI # 1 ROI # 2 ROI # 3 # time (ms) intensity intensity intensity

____ ________ ___________ ___________ ___________

____ ________ ___________ ___________ ___________

____ ________ ___________ ___________ ___________

____ ________ ___________ ___________ ___________

____ ________ ___________ ___________ ___________

____ ________ ___________ ___________ ___________

____ ________ ___________ ___________ ___________ value of T2 ___________ ___________ ___________

T(probe): __________ viscosity of water at T(probe):__________________

molecular volume of water: ________________ rHH:________________________

predicted correlation time using equation (10):______________

predicted value of T2,DD using equation (11):________________

MSME parameters:

matrix size: __________________ digital resolution:__________________

receiver gain: ___________ Tx0:_____________ Tx1:____________

21

22