III. Background Information

Nick D. Panagiotacopulos and Francisco Padilla

1. Electromyography

Electromyography (EMG) is a technique used for measuring, recording and evaluating the physiological, biochemical, and electrical activity of skeletal muscles during contraction. The EMG signal reflects the effect of an external excitation on a skeletal muscle. If it is done properly and interpreted correctly (based on muscle physiology), the outcome from the study of S-EMG signal can be very useful in the fields of biomechanics, ergonomics and kinesiology. More specifically, S-EMG results can be useful in the evaluation of muscle activity and its performance [87].

The Central Nervous System (CNS)

Figure 1 shows a simplified functional diagram of the central nervous system and how an EMG signal is acquired (Curtsey of Dr. Nick P.).

Anatomical structure of Skeletal Muscle

The muscle is surrounded by a connective tissue called epimysium and is divided by fascicles by perimysium which contain several muscle fibers. The structural unit of skeletal muscle is the muscle fiber, or cell (Fig. 2). A muscle cell is a thin structure ranging from 10 to 100 microns in diameter and from a few millimeters to 30 cm in length [5]. The muscle fibers do not extend the entire length of the muscle. Instead, the cells are attached to either the origin or insertion tendon at one end and connective tissue septa at the other end [87]. The muscle fiber is further subdivided into myofibrils where thick and thin filaments are arranged longitudinally. A muscle fiber produces force by contracting.

Figure 2. The organization of skeletal muscle

A muscle fiber is surrounded by the sarcolemma. The sarcolemma is a thin semi permeable membrane composed of a lipid bilayer that has channels by which certain ions can move between the intracellular and the extra cellular fluid [37, 71]

There are two types of skeletal muscle fibers: Type I and Type II fibers. Type I are slow twitch fibers with high endurance (long-endurance such as distance running,), while Type II are fast twitch fibers with lower endurance (these muscles fatigue faster but are used in powerful bursts of movements like sprinting).

Muscle fibers are always grouped together sharing the same nerve fiber. This nerve fiber (axon), refer to Fig 1 is the transmission line of a nerve cell and runs from the spinal cord to the peripheral muscle. A nerve cell which innervates a group of muscle fibers is known as motor-neuron. Every cell is located close to the spinal cord.

Muscle fatigue is the inability of a muscle to generate force. It can be a result of vigorous exercise, but abnormal fatigue may be caused by barriers to or interference with the different stages of muscle contraction.

Action Potential

There is a difference in the composition of the extra cellular fluid and intra cellular fluid in resting state [87]. The resting membrane potential is the voltage difference across the plasma membrane induced by the electrochemical potential difference. The major ions that maintain the resting potential are Na⁺ K⁺, Cl^–, etc. When substantially large stimulus is applied, an action potential is triggered. This takes place only when the depolarization is sufficient to reach a threshold value (Fig 3).

Electrical muscle activity, electromyography (EMG), is the result of an external excitation of the muscle fiber (cell) which usually runs along the whole muscle from tendon to tendon. In its resting state, there is a potential of approximately -90mV across the cell membrane (the fiber wall) with zero reference on the outside. The fiber is an ‘on-off device’ in the sense that mechanical work is produced in twitches only, which have durations ranging from 35 to 75 ms (1).

Fig 3. Action potential

An action potential is a rapid change in the membrane potential followed by a return to the resting membrane potential. An action potential is propagated with the same shape and size along the whole length of a nerve or muscle cell [9].

Discussion on Motor Unit (MU) Action Potential (AP)

The smallest functional unit of muscle contraction is the motor unit (Figs 1 and 4). A motor unit consists of a group of muscle fibers innervate by a single motor neuron (in other words, a motor unit consists of one motor neuron and all the muscle fibers it stimulates). Within a single motor unit, individual muscle fibers discharge nearly at the same time.

Fig 4. Motor Neuron

Motor unit recruitment (motor unit activatin) refers to the activation of additional motor units to accomplish an increase in contractile strength in a muscle. The higher the recruitment the stronger the muscle contraction will be. In physiology, an action potential occurs when the membrane potential of a specific cell location rapidly rises and falls: this depolarization then causes adjacent locations to similarly depolarize.

When an action potential is evoked in a motor nerve by a critical depolarization, an endplate, or the neuromuscular junction, potential is produced by acetylcholine release. Acetylcholine stimulates the membrane of the muscle fiber, or sarcolemma. This triggers rapid depolarization (about +20mV) and repolarization of the muscle fiber (Fig.3). The action potential is propagated along the sarcolemma and into the muscle fiber through the transverse tubules. The generation of an action potential takes place in all the muscle cells in the motor unit and is followed by synchronous contractions of all the muscle cells in the motor unit [71]. A motor-neuron together with its muscle fibers is called motor unit (MU). The number of muscle fibers coupled in one MU range from 3 (in small muscles) up to 1000 (in large muscles). All fibers belonging to the same MU are triggered simultaneously. The AP seen in by an electrode recording is the superposition of all the APs of the muscle fibers belonging to the triggered MU (MU-AP). The MU is usually activated repeatedly with a firing frequency of 10-50 Hz (3). A whole muscle usually consists of several hundreds of MUs.

The force development of the muscle is modulated by the CNS by recruitment (activation) of an appropriate number of MUs and by varying the firing frequencies of the recruited MUs (3). The MUs seem to be recruited in a fixed order according to the reversed size order of their motor-neuron (4).

The depolarization generates an electric field near the muscle fibers which can be detected by a skin surface electrode located near this field. The resulting signal is called the muscle fiber action potential. The combination of the muscle fiber action potential from all the muscle fibers of a single motor unit is the motor unit action potential.

When an electrical impulse originated from the central nervous system (CNS) reaches a point on the muscle (a point at which a nerve fiber comes in contact with a muscle fiber called innervation point) of the muscle fiber, a twitch is triggered via a biochemical transmission process. The biochemical process of transferring chemical energy into mechanical energy is elicited on both sides of the innervation point and spreads in both directions towards the ends of the muscle fiber (2). The biochemical process is accompanied by a transient drop of the membrane potential called depolarization. The electrical activity of the depolarization is detected with a electrode in the vicinity of the fiber. This signal is referred to as an action potential (AP). The AP resembles the transient membrane potential in Fig. 3.

EMG Electrodes

In general, EMG can be sensed by two types of apparatus. One is a needle electrode that has high selectivity in deep muscles and the other is a surface electrode that is useful in detecting the integrated behavior of large muscle groups.

Needle Electrodes

The action potential recorded during voluntary muscle contraction depends to a great extent on the type of recording electrode [16, 33]. Isolated motor unit action potentials can be recorded with the use of a needle electrode. The standard and bipolar concentric needle electrodes (Fig 5) as well as monopolar needle electrodes are commonly used in routine electromyography. Less commonly used special purpose electrodes include the multielectrode and flexible wire electrode. A flexible wire electrode [20] is used for kinesiology examination. The wires permit freedom of movement while they stay within the muscle.

Surface Electrodes

Surface EMG ( S-EMG) is a non-invasive technique that measures signals containing certain temporal characteristics useful for understanding the muscle’s response. Surface electrodes (Fig 6), place over the muscle, and register summated activities from many motor units [50]. S- EMG can be obtained by using electrodes affixed to the surface of the skin. With the development of very sophisticated electrodes, S-EMG is now being used in many areas of ergonomics, sports medicine and even in clinical applications. S-EMG is a very useful tool in detecting integrated muscular behavior.

One advantage of the S-EMG technique is the convenience in terms of relative ease of application and lessened discomfort of the subject. On the other hand, there are few disadvantages in using the S-EMG technique. The surface electrodes do not have selectivity to a specific muscle fiber because of the wide pickup are from the muscle as compared to fine wire electrodes. The signals detected from the pickup area may originate in a deep muscle, or even worse, from different muscle groups.

Therefore, there are concerns about the validity of a recording. Although efforts have been made to quantify and determine the effect of cross talk, there is no established or easy way for S- EMG to eliminate this problem. In general, however, S-EMG is satisfactory for the analysis of temporal, force, or fatigue relationships [87]. The main advantage of surface electrode can be most effectively utilized when the simultaneous activity of many muscles is being studied in a large group of muscles.

In summary, the EMG signal can be detected either by intramuscular electrodes (needle or wire) or by surface electrodes attached to the skin over the studied muscle. In both cases the EMG is the sum of the contributions from a large number of MU-APs in the muscle. The MU-AP is attenuated substantially on its way through the tissue. Hence, with intramuscular electrodes, MU-APs adjacent to the electrode, strongly dominate over contributions from more distant MU-APs. With surface electrodes, a larger number of MUs contribute. Since the amplitude of the MU-AP rapidly decreases with distance, the main contribution to a S-EMG comes from the superficial part of the muscle (2). Hence, S-EMG is restricted to superficial muscles.

The electrically conducting part of a surface electrode usually consists of a silver-covered surface (10-50 mm2) which is taped or glued to the skin. The impedance between electrode and skin is reduced by applying some kind of electrode paste. The standard procedure is to use two electrodes, 10-20 mm apart, placed over the belly of the muscle and aligned in the muscle fiber direction. The two electrodes are connected to a differential amplifier with a third electrode placed somewhere else on the body as a reference. The input impedance and common mode rejection should be as high as possible.

Features of EMG

Considerable progress has been made when EMG analysis has been used in ergonomics to investigate topics like musculoskeletal injury, low-back pain, and muscle fatigue from overexertion. Although modern instrumentation has greatly facilitated the acquisition of EMG data, many issues remain unresolved in the interpretation of EMG signals [87]. The basic features of EMG signals are first discussed in this section followed by the problems regarding each feature.

The information obtainable from EMG analysis can be generally divided into the following three categories. (1) The relationship between temporal aspects of EMG and anatomically associated movement. (2) The relationship between EMG and production of force. (3) The relationship between EMG and muscle fatigue.

Temporal Information

The most basic information obtainable from an EMG recording is whether the muscle was on or off during an activity or at a particular time. In order for a muscle to be considered on, the EMG recording must exceed a certain threshold, whether defined by an arbitrarily of statistically predetermined level or by the noise level of the equipment responsible for the measurement. It often is more difficult to determine that a muscle is off because a muscle may infrequently be in a state of total relaxation.

Some researchers describe the threshold in an absolute value. This measure simply relates how active the muscle was during the experimental conditions. The measure is not an indication of muscle force, but simply a function of muscle usage. The signal can be quantified in several ways. Quantification may include peak activity, mean activity, activity as a function of a given position or posture, and rate of muscle activity onset [87].

To determine the on-off state, force, or fatigue present within a muscle, some form of EMG signal treatment usually is recommended and often required. If the raw or processed signal exhibited activity, the muscle was in use during the exertion [87]. Differences do exist, however, between the temporal characteristics of the EMG and the produced tension. The most apparent is the pure time delay. Initial tension levels in the muscle also influence the delay times seen at onset.

Most studies that now investigate the on-off state of the muscle are interested in the phasing of the EMG activities under various experimental conditions. A quantitative evaluation of muscle on-off state was performed by Marras and Reilly [65], using statistical analysis of muscle event time’s derived form processed EMG. They were interested in how the patterns of trunk muscle activation changed as the angular velocity of the trunk increased during controlled simulated lifting motion.

EMG-Force Information

An EMG-force measurement seeks to quantify the average number and firing rate of motor units contributing to a particular muscle contraction, and to relate the quantity to the actual force produced. With increasing intensity of a contraction, more and more units will be recruited, and the unit firing frequency will also be increased. The observed motor unit activity reflects these changes as the resulting interference pattern becomes denser and of greater amplitude. The signal can be processed to estimate a numerical value (usually a percentage of a maximum voluntary contraction) to the level of EMG activity associated with the generation of a corresponding force.

However, a great deal of confusion exists regarding the relationship between processed EMG and produced force. Researchers have long suggested that EMG could be used to represent the active control input of the muscle, and that some relationship must exist. Some researchers have presented EMG as a direct indication of muscle force while others have presented very complex models using these signals to predict force. Many researchers have attempted to investigate the force of a muscle by simply observing the rectified and averaged (in some cases integrated) EMG signal in terms of the absolute number of micro volts generated and associated with a particular activity [87]. A direct comparison of the relationship between the EMG signal and force output has been reported by Lawrence and De Luca [56]. One aspect of this study investigated was whether or not the normalized surface EMG signal amplitude versus normalized force relationship was dependent on exercise level. The RMS value of the signal amplitude was used as the variant parameter because it is the parameter that more completely reflects the physiological correlates of the motor unit behavior during a muscle contraction. (This result showed almost linear relationship between normalized RMS and force) [7].

EMG-Fatigue Information

Very little is known about general physical fatigue that is experienced after heavy work. Metabolic changes, such as an increased blood lactate concentration and a fall in pH, may contribute to the perception of fatigue but cannot fully explain the phenomenon [71].

Fatigue can be experienced slowly or rapidly depending on the type of work performed by a muscle. When the workload is relatively small, the slow motor units are initially excited. If the task requires fast and forceful contractions, fast motor units are also recruited for maximum output. Rapid muscle fatigue can be observed in the latter case.

Localized Muscle Fatigue

We are interested in fatigue on a local level within the body. This is true particularly in musculoskeletal exertions. Typical externally visible symptoms of fatigue are loss of force production capabilities and localized discomfort and pain. This type of fatigue has become known as localized muscle fatigue (LMF) [17]. Muscle exertion levels do not necessarily need to be high to cause LMF. Isometric contractions of as low as 10% of maximum voluntary contraction have shown signs of LMF [87].

Spectral Charges in EMG during Fatigue

During LMF, changes occur in the surface recorded EMG signal. Two of the most commonly cited changes are a shift in the frequency content of the signal toward the low end [17] and an increase in the amplitude. Many researchers have applied spectrum analysis methods to show the presence of fatigue [38]. Lindstrom [60] and DeLuca [27] contend that the spectrum shift and amplitude increase is related. They state that tissue acts as low pass filter. As the frequency content of the original signal shifts to the lower frequencies, more energy is transferred through the tissues to the electrodes. This energy transfer, in turn, increases the amplitude of the recorded signal.

A group of investigators have demonstrated a decrease of power density in the high frequency region of the EMG signal and increase in the low frequency region during fatigue contractions [46, 47, 51, and 54]. Many researchers have proposed physiologic explanations for the changes in amplitude and spectral characteristic [87]. Lindstrom et al. has demonstrated that the frequency shifts were almost entirely dependent on the propagation velocity of the action potentials [60]. The reduced propagation velocities have been linked to the production and accumulation of acid metabolites [70].

Some investigators, however, showed that the use of spectral analysis does not provide significant differences with fatigue [66]. They may be related to the fact that the frequency of the EMG signal can be altered by many factors, for example, by the change in load [32] or by the change in muscle length [8].

Muscle Fatigue through microvolt

As described above, the EMG signal will increase its amplitude while the muscle is exerting a given amount of force in an isometric contraction. This may be due to the need to recruit more motor units to perform the same amount of work as the muscle fiber fatigue. Thus, by observing the processed EMG signal of a given portion of the muscle under constant force conditions, a quantitative indicator of the degree of muscle fatigue can be established. It is also important to note that this trend is evident only with surface electrodes.

Limitations in Fatigue Analysis

Although the use of EMG in measuring localized muscle fatigue is well established and frequently used, the technique has limitations. It is important to understand some of these limitations before undertaking an EMG analysis in the field of ergonomics or biomechanics. The first problem is in the basis definition of fatigue. Because there is no universal definition of fatigue, agreement on the validity and meaning of EMG measures will be questioned. Other factors in LMF such as pain tolerance, motivation, and synergistic accommodation are not included in EMG analysis and yet have been argued to be important. Additionally, spectral shifts have been used for short-term contraction fatigue, but the use of EMG in long-term fatigue is questioned. For muscle fatigue that occurs over a longer period of time, possibly hours, the use of EMG has not been well established. In addition, shifts in the various EMG indices have been shown to decrease rapidly during the initial stages of a contraction, but do not decay as rapidly toward the end of a long session of work [87].

For more detailed information on the EMG background see (2).

References:

Astrand P.O., Rodahl K, Textbook of work physiology, Second Edition. McGraw-Hill, New York, 1977.
Basmajian J., DeLuca C.J., Muscles Alive, Fifth Edition, Williams& wilkins, Baltimore, 1985.
Milne-Brown H. S. Stein R. B. , YemmR., Changes in firingrate of human motor units during linearly changing voluntary contraction. J. Physiol 230 (1973) 371-390.
Haenneman E., Somjen G.,Carpenter D. O., Excitability and ihitabitability of motorneurons of different sizes. J. Neurophysiology 28 (1965) 599-620.

2. Basics of Magnetic Resonance Spectroscopy

A bit simpler description of the proposed process : When MRS is used the patient is placed inside an homogeneous magnetic field. This field causes the spins of protons to align in a specific direction, designated the longitudinal direction. A short RF pulse transverse to this field is then used to synchronize the precession of these proton spins. When this pulse ends, the spins revert back to their original state, emitting radio signals in the process. The exact signal a proton emits depends on the specific chemical environment of the proton; nuclei in different environments emit radio signals at different frequencies. These signals are combined to form a free induction decay which is then analyzed through FFT. By examining the spectra in frequency space, it is possible to determine the concentrations of certain chemicals and metabolites in a given sample. This makes spectroscopy an extremely powerful diagnostic tool. Furthermore, because the fields used function at radio frequency, magnetic spectroscopy examinations are also extremely safe.

Basics of Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Spectroscopy (MRS) [2-9]

MRS is a tool that has been used by chemists for many years in the analysis of chemical compounds. A chemical compound consists of molecules, and molecules are composite systems of atoms. An atom consists of a small nucleus and a cloud of electrons. Basically, the nucleus is made up of two types of subatomic particles, the protons (p), and the neutrons (n). According to quantum mechanics these subatomic particles are intrinsically spinning. If a (large) number of these particles (p, n) are grouped together, their respective spins will add and the nucleus will have a net nuclear spin. The net nuclear spin is zero for all the nuclei except those with an odd number of protons and an even number of neutrons. These are the nuclei of importance to a magnetic field. As the nuclei spin, their charges circulate and generate a magnetic field. Such magnetic nuclei, which have north and south magnetic poles, have no preferred orientation in space. But if we put them in a uniform static magnetic field, H, they tend to line up with the field (favorable state). The next thing we do is to change the orientation of the nuclei (perturb the nuclei) in the field (turn them over to make them point in the opposite direction). To achieve this (less favorable state) state we have to apply energy into the system. This energy can be obtained from the application of a precisely tuned pulsed radiofrequency (RF) field orthogonal to the static field H. RF is generated from a radio transmitter by changing its frequency. When the RF of the transmitter becomes equal to the frequency of the spinning nucleus, then we achieve resonance and the RF at which resonance occurs is known as resonance or Larmor frequency ω. The equation:

ω = -γH,

where γ is the gyromagnetic ratio (which is associated with each nucleus), is the key equation in nuclear magnetic resonance (NMR) and MRS. It is the connection to particular nuclei that gives MRS and NMR its ability to look at a specific atomic species in a complex system.

is the key equation in NM / MRS, where Η is the magnetic field strength ,and γ is the gyromagnetic ratio (which is associated with each nuclei).

In MRS experiments, it has been observed that high resolution spectral analysis requires relatively low molecular weight compounds(otherwise the spectra become too complex), very pure homogeneous samples, and extremely homogeneous magnetic fields of high strengths (4 Tesla or more).

For example, the tissue -water signals is typically four orders of magnitude more intense than that of the metabolites, making it difficult to observe the weak metabolite signals in the present of the intense water signal.

Clearly, the above imposed limitations by the biological systems require strong magnetic fields (i.e. 3 Tesla or more).

Clearly, one can select which nuclei to observe by using the appropriate frequency ω. The resonant response signal of the nuclei is known as free induction decay (FID). It occurs exactly when resonance is achieved, then the RF field is removed and the nuclei are coming back to their initial favorable state responding with the FID signal. The FID¹ signal is then detected by a receiver coil placed around the chemical substance under study. If we apply Fourier transform on the FID signal, we obtain its frequency spectrum, which gives a convenient representation of the resonant frequency content of the FID signal. For example, phossphorus-31 (31P) spectroscopy provides information such as the cellular energy-state and intracellular pH, as well as phospholipid metabolism, whereas water-suppressed proton spectroscopy can quantify the concentrations of various intermediary metabolites including amino acids and lactate. Most of the spectroscopy techniques critically depend upon the quality of the spectral resolution that can be obtained. For instance, good frequency resolution can be achieved by optimizing the magnetic field homogeneity over the sample volume under investigation. One way to achieve good homogeneity is by limiting the volume of interest by some form of volume selection mechanism (localization). Along this line a number of volume selection methods are used such as: SPARS (spatially resolved spectroscopy) and STEAM (stimulated echo acquisition mode).

Magnetic Resonance Imaging (MRI) images consist of a series of T1 and T2 weighted images (T1 and T2 are known as relaxation times). They are used to guide the localization of area of interest for MRS studies. That is, from these images, a cube like region (a “voxel” or volume element) is chosen for specific MRS examination. For example, in the case of: an Alzheimer’s disease examination, we might choose to examine an homogeneous area in the occipital grey matter; or for an examination of a tumor, one would obviously choose the site of the tumor. Actually, MRS performs this localization through frequency and phase encoding in the presence of a magnetic gradient inside the homogeneous magnetic field of the MRI machine. This allows us to excite the specific region of the body that we are interested in by a radio frequency (RF) pulse. When one strikes that specific region of the body with a RF pulse, this region begins to resonate based on the chemicals within that region. Then, the resultant resonances are read out using radio frequency detectors. Note that in the brain multiple chemicals resonate at multiple frequencies.

MRS in Diagnostic Medicine and its Significance

The standards for diagnosing brain tumor are medical imaging and stereotactic biopsy. The disadvantage to imaging tests such as x-ray, PET, and MRI are that although they were proficient at recognizing large lesions in the brain smaller lesions were often much more difficult to ascertain whether they were tumors or not . Even in cases of well-defined lesions, the non-quantifiable observations could lead to an inaccurate diagnosis. In the case of suspected malignancy, and when imaging alone could not determine malignancy with certainty, patients could only turn to biopsies in the form of surgical removal or fine needle aspirates. Such surgeries obviously carry the risk of neurological deficit due to post-surgical hemorrhaging and are restricted to areas of the brain that are relatively easy to access. Needle aspirations or stereotactic biopsies are used to remove only a small portion of tissue, while induce less trauma in the brain. However, in several cases they have been shown to be inaccurate because they were not adequate or were obtained from the wrong location. Both problems may be caused by the restricted degrees of freedom in manipulating the needle. It is thus not unusual that an accurate and reliable diagnosis is difficult to be obtained, and therefore the need for a better diagnostic method is clear.

MRS may resolve these issues by providing a safe (non-invasive), quantitative, and accurate measurement, allowing clinicians to determine concentrations of chemicals or metabolites (chemical markers) in regions of interest within the human body and assist them in the diagnosis of various pathologies. It has been proved to be of great diagnostic value in the evaluation of lesions and tumors especially in the brain.

Using MRS, the biochemistry of a region of interest can be determined. The differences in the biochemistry of diseased brain and normal brain can then be used as a diagnostic tool to screen for pathologies such as brain tumors. In brain tumors, certain biochemical markers are used. For example, the biochemical marker NAA is decreased due to a loss of neurons in the area of the lesion, the biochemical marker Cho is increased dramatically due to the proliferation of tissue in cell death, and the biochemical marker LAC/Lip is often increased due to this cell death. This biochemical “signature” is very sensitive information and requires specific tests to determine if the lesion within the region of interest is a tumor or not.
In MRS, it has been observed that high resolution spectral analysis requires relatively low molecular weight compounds (otherwise the spectra become too complex), very pure homogeneous samples, and extremely homogeneous magnetic fields of high strengths (i.e. 3 Tesla or more).
______
1
The resultant signal, detected by the detectors, is termed free-induction decay (FID). The FID is a signal generated by the alterations of the local magnetic field, with its amplitude decaying gradually as the magnetization of the region returns to its baseline and is losing its strength. An example of an FID can be seen on the top left figure of research activity 2.
The FID is a sum of all resonances. Then, by applying Fourier transformation (FFT) on FID, it is possible to determine which chemicals are present (from the signal’s frequency or ppm content) in the region being examined. However, Wavelet Transforms can provide a higher resolution frequency spectrum which with proper calibration can provide a whole new world of neurochemical markers that remain to be characterized, and assist in a better diagnosis of neurological and other diseases.
The following is a list of the currently known neurochemical markers available at short-echo times (TE: 15 to 35 ms) using standard single-voxel PRESS sequence and includes:

1. NAA (N-Acetyl-Aspartate), a neuronal marker. NAA peak resonates at about 2.0 ppm.
2. Cho (Choline), a marker for myelination, indicates axons break up and degradation. Cho peak resonates at about 3.2 ppm.
3. Cr (Creatine), an energy marker. Cr peak resonates at about 3.00 ppm.
4. Myo (Myoinositol), a marker for gliosis and glial activity. Myo peak resonates at about 3.5 ppm.
5. Lac (Lactate), reflects cell death or anaerobic respiration. Lac (doublet) peak resonates at 1.33 ppm. Lactic acid levels get higher when strenuous exercise or other conditions such as heart failure, a severe infection (sepsis), or shock are taken place. Then, blood flow and oxygen throughout the body becomes lower. Very high levels of lactic acid cause a serious, a sometimes life-threatening condition.
6. Glu (Glutamate and glutamine), neurochemical markers. Glu/Gln peak resonates at about 2.2-2.4 ppm. Glutamate is a neurotransmitter that is released by nerve cells in the brain. It is responsible for sending signals between nerve cells, and probably very important in the learning and memory processes. If glutamine is too much this may lead to seizures and the death of brain cells, if it is too little, it can cause psychosis, coma and death. Excess Glu, not only over stimulates the nervous system, it is also toxic to the brain and can age/degenerate it too quickly and can cause brain damage after stroke. Glutamine is an important amino acid with many functions in the body. It is a building block of protein and critical part of the immune system. Furthermore, it has a special role in intestinal health. Our body produces this amino acid, and it is also found in many foods.
.7 Lip (Lipids), fat in head in pediatrics indicate poor outcome. They are usually associated with necrosis, growth arrest, inflammation, malignancy, apoptosis, and craniopharyngioma which are connected to high amounts of cholesterol in the cyst fluid. Lip (CH2n) peak resonates at about 1.3 ppm and Lip (CH3) at 0.9 ppm.
8. (Ala) Alanine (doublet) peak resonates at 1.47 ppm.
9. GABA (Gamma-aminobutyric acid). GABA peaks resonate at 2.2-2.4 ppm.
10. Citrate peak resonates at 2.6 ppm.

Note that, these markers are identified by the frequencies (or ppm) at which they occur.
Looking at the list of neurochemical markers just presented, it is surprising that different chemical markers seem to resonate at the same resonance frequencies² (ppm). We believe that this is due to (i) the MRI/MRS machine’s inability to differentiate them and obtain finer data and (ii) the weakness of FFT to differentiate frequencies that are too close together. More specifically, several frequencies (which correspond to the different chemicals) are close together and fall under an envelope and appear to be as one frequency (but actually there are more). We believe that the new MRI scanners (3 Tesla and above) will resolve the first problem and that Wavelets, can easily resolve the second problem.

Note: Head injuries are perhaps the most damaging of all traumas, because of the variability in cause, extent, and effect. They are also, one of the most difficult types of trauma to diagnose. Not too long ago, MRS has emerged as a new and more accurate method in both the diagnosis of the severity of head trauma, and in the prediction of the outcome of patients, especially in cases where patients are comatose.

At the Huntington Magnetic Resonance Spectroscopy Unit, in Pasadena CA, Dr. B. Ross and his collaborators have reported that in traumatic head injury, many chemical markers have been analyzed and are used for diagnosis. More specifically: They reported that:

NAA, in most cases of head trauma is reduced to some degree. They observed that if this reduction is slight, this indicates optimistic prediction. However, if it is high, this indicates permanent brain damage and severe mental retardation,
Cho: If elevated levels of Cho are observed, seen during the breakdown of myelin in axons and in membrane degeneration, associated with diffuse axonal injury, the prognosis is good if Cho is the only abnormal metabolite in the spectrum,
Lac: the presence of Lac, is a sign of hypoxia, and in general predicts a very poor outcome for a patient.

In MRS studies of humans we wish to determine accurately, the chemical composition of a specific region in the human body containing the tumor under study In general, these regions are tissues comprised of very complex molecules, are highly non – homogeneous, and contain high levels of water and fat as well as small amounts of metabolites (which have been reported to be useful in tumor characterization). Furthermore, the high intensity signals from water and fats severely interfere with the observation of the weak signals from low molecular weight metabolites.
Through examination of these metabolites and others, it is possible to obtain a much more quantitative diagnosis of head injury and a much more accurate prediction of future outcome than other clinical standards. Note that in the above studies, the data were phase corrected and the signals from pure water were suppressed by post process.
For example, the tissue – water signals is typically four orders of magnitude more intense than that of the metabolites, making it difficult to observe the weak metabolite signals in the present of the intense water signal.
_____________
² Some comments on dopamine detection using H1-MRS.
Dopamine in the brain is difficult to be detected because:
The concentration of dopamine is low
The concentration of other protons is very high
The magnetic field is not homogeneous.
The brain cannot be rapidly rotated the way a laboratory solution state NMR sample is to average magnetic field inhomogeneity
Compare to laboratory solution state NMR, the viscosity in the brain would be high causing further line broadening.

If you look at the spectra in H1 – NMR Probe for in Situ Monitoring of Dopamine Metabolism and Its Application to Inhibitor Screening J. Am. Chem. Soc., 2012, 134 (30), pp 12398–12401, you’ll see that even in tissue samples, there are too many other stronger signals for conventional H1- NMR to be successful, so C-13 labeling is used in combination with H1- NMR.
Again, we repeat here comments that we made earlier. That is: We believe that this is due to (i) the MRI/MRS machine’s inability to differentiate them from other neurochemical markers and obtain finer data and (ii) the weakness of FFT to differentiate frequencies that are too close together. More specifically, several frequencies (which correspond to the different chemicals) are close together and fall under an envelope and appear to be as one frequency (but actually there are more). We believe that the new MRI scanners (3, 7, and 11 Tesla) will resolve of the first problem and that Wavelets, can easily resolve the second one.

A bit simpler description of the MRS process : When MRS is used the patient is placed inside an homogeneous magnetic field. This field causes the spins of protons to align in a specific direction, designated the longitudinal direction. A short RF pulse transverse to this field is then used to synchronize the precession of these proton spins. When this pulse ends, the spins revert back to their original state, emitting radio signals in the process. The exact signal a proton emits depends on the specific chemical environment of the proton; nuclei in different environments emit radio signals at different frequencies. These signals are combined to form a free induction decay which is then analyzed through FFT. By examining the spectra in frequency space, it is possible to determine the concentrations of certain chemicals and metabolites in a given sample. This makes spectroscopy an extremely powerful diagnostic tool. Furthermore, because the fields used function at radio frequency, magnetic spectroscopy examinations are also extremely safe.

Acknowledgments: At this point, it is appropriate to acknowledge the services of the following volunteers:
During the last 4.5 years, my nephew Dimitrios Panayotakopoulos of Greece, an Architect, helped me to develop the existing Web page, with dedication, and great care. Without his help, FKW’s web page will not be what it is today.
During the last 2.5 years Alexia Escobar, an attorney, helped me create the FKW organization, and encourage me to continue working when I was ready to give up.
Since 1.5 years ago, Dr. Francisco A. Padilla, a physician, provided useful medical advice when this was needed, and also helped to improve the appearance of the Web page.
For activity number 5, Dr. Qi Junyu, an expert in Soil-Temperature interaction, and a scientist from the University of Maryland, he will collaborate with FKW for that portion of the project.
The Activity on G5 , was motivated from a discussion with Hamid Naghieh, a chemical engineer/ physicist

References

1. Spoiler Alert, by M. Farrell, Consumer Report, September 2016.

On NMR, MRI, MRS:

2. ABCs of FT-NMR, John D. Roberts, University Science Books, 2000
3. All About NMR Physics Notes by Moriel Ness Aiver, University of Maryland, School of Medicine
4. Spin Dynamics, Basics of Nuclear Magnetic Resonance, Malcolm H. Levitt, John Wiley & Sons, Second Edition,
2008
5 Principles of Magnetic Resonance Imaging, Zhi-Pei-Liang and Paul C. Lauterbur, IEEE Press, 2000
6. Principles of Magnetic Resonance Imaging Dwight G. Nishimura, Department of Electrical Engineering, Stanford
University, 2010
7. Magnetic Resonance Spectroscopy, R. K. Harris, Longman Scientific & Technical, John Wiley Sons, 1986.
8. Magnetic Resonance Spectroscopy of Human Brain, Brian Ross, Stefan Bluml, Wiley Online Library, 2001 Anatomical
Record Vol. 265. Issue 22001
9. Pros/cons of ultra high field MRI/MRS for human applications, M. Ladd., Progress. NMRS, Vol.109 Dec 2018, p. 1-50
On Wavelets:
12. A Wavelet Tour of Signal Processing, Stephane Mallat, Academic Press, 1998.
On Physics, and high frequency EM fields:
13. The Feynman Lectures on physics, Vol 2. Addison Wesley
14. Waves, Berkley Physics Course, Volume 3 (On Standing Waves)
15. Exposure to high frequency electromagnetic fields, biological effects, and health consequences (100 kHz –100GHz)
Editors: Paolo Vecchia and al., International Commission on Non-ionizing Radiation Protection, ICNIRO, 16/2009
16. Radiofrequency EM Wave and Paramagnetic Particle Effects on the Red Blood Cells, C, Nadejde, and al., 3rd National
Conference of Applied Physics, Romania, 9-10 June 2007, PUBLISHED in the Romanian Journal of Physics, Vol54, Nos1-2,
p.105 -114. Bucharest- Romania
17. Elimination of standing wave Effects in Ultrasound Radiation Force excitation in Air using Random CARRIER Frequency
Packets, Thomas Huber and al., The Journal of Acoustical Society of America, 130, issue 4, 1838 2011, 10.1121/1.3628336
18. The largest Unethical Medical Experiment in human history, Ronald N. Kostoff, Ph.D, school of Public Health Policy,
Georgia Institute of Technology, 2020 (1,086 pages).

On Digital Image Processing:
19. Digital Image Processing, R. C. Gonzalez and R. E. Woods, Addison Wesley, 1992
20. Digital Image Processing, Kennneth R. Castlemen, Prentice Hall, 1996 (JPL’S point of view)

On Soil Properties:
21. Wang, Q., Qi, Junyu, Li, J., Cole, J., Waldhoff, S.T. and Zhang, X., 2020. Nitrate loading projection is sensitive to freeze-
thaw cycle representation. Water Research, 186, p.116355.
22. Qi, Junju, Li, S., Li, Q., Xing, Z., Bourque, C.P.A. and Meng, F.R., 2016. A new soil-temperature module for SWAT
application in regions with seasonal snow cover. Journal of Hydrology, 538, pp.863-877.
23. Tisserat Brent, U.S.A Department of Agriculture, Functional Foods Research Unit, National Center, Peoria, IL
Fabrication of Natural Fiber Seed Floor Reinforced with Non-woven Help Mats, Journal of Polymers and the Environment,
Springer, 09, 07, 2018,