Chapter 2

Biosignal
The signal generated by different portions of the biological organs is commonly termed as
biosignal. Every organ, tissue and cell of living body produces its own monitoring signal to
indicate its activity. The signal is classified broadly into two categories:

Electrical signal: This type of signals is due to the electrochemical changes in the cells. The
biochemical action produces different types of positive and negative ions inside and outside
of the cells and ionic gradient produces ionic voltages which are converted to electric
potentials by means of electrodes. Notable electrical signals are electrocardiogram (ECG),
electroencephalogram (EEG), electromyogram (EMG), etc.

Nonelectrical signal: Nonelectrical variables such as pressure, temperature, position, fluid

flow, etc. are converted to corresponding electrical signal by the appropriate transducers.

In measuring biological signals, the following problems must be address due to involvement
of living subjects:
• Inaccessibility of variables to measurement
• Variability of data
• Lack of knowledge about interrelationship
• Interaction among physiological systems
• Effect of electrode and/or transducer on measurement
• Artifacts
• Energy limitations
• Safety considerations
• Ethical issues

The design of signal measurement instruments involves the following factors:

• Range: amplitude, frequency
• Sensitivity
• Linearity
• Hysteresis
• Frequency response
• Accuracy
• Signal to Noise Ratio
• Stability
• Isolation
• Simplicity
• Cost

Due to some definite advantages of electrical signals in transformation, transmission and

storage, it is widely used. This chapter briefly describes the formation of bioelectric potential
in cells and different types of biosignals.

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2.1 Bioelectric Potential
Surrounding the cells of the body are the body fluids. These fluids are conductive solutions
containing charged atoms known as ions. The principal ions are sodium (Na+), potassium
(K+), and chloride (Cl-). The cell membrane is semi-permeable, it can block any ion
depending on its condition, either in rest or excited.

Cells at Rest: When a cell is at rest, the membrane of excitable cells readily permits entry of
K+ and Cl- ions but effectively blocks the entry of Na+ ions. With respect to concentration and
electric charge imbalance this results in two conditions. First, lower concentration of Na+
inside the cell makes the outside more positive than inside. Second, additional K+ ions enter
the cell for making balance. However this cannot be achieved because of the concentration
imbalance of K+ ions. Equilibrium is reached with a potential difference across the
membrane, negative on the inside and positive on the outside. This potential is termed as
resting potential. The cell is said to be polarized.

Figure 2.1: Polarized cell with its resting potential

Since measurement of membrane potential is generally made from inside the cell with respect
to the body fluids, the resting potential of a cell is given as negative value ranging from -60
to -100 mV with the nominal value of -70 mV.

When a section of the cell membrane is excited by the flow of ionic current from other cells
or by some form of externally applied energy, the membrane changes its characteristics and
begins to allow some of the Na+ ions to enter. The net result is an avalanche effect in which
ions literally rush into the cell to try to reach a balance with the ions outside. At the same
time K+ ions, try to leave the cell but are unable to move as rapidly as the Na+ ions because
the rate of Na+ pumping is around 2-5 times that of K+ pumping.

As a result, the cell has a slightly positive potential on the inside due to the imbalance of K +
ions. This potential is known as the action potential and is approximately +20 mV. A cell that
displays an action potential is said to be depolarized; the process of changing from the resting
state to the action potential is called depolarization.

Once the rush of Na+ ions through the cell membrane has stopped, the ionic currents that
lowered the barrier to Na+ ions are no longer present and the membrane reverts back to its
original, selectively permeable condition, wherein the passage of Na+ ions from the outside
to the inside of the cell is again blocked.

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 Figure 2.2: Depolarization of a cell

Figure 2.3: Depolarized cell during an action potential

Were these the only effects, however, it would take a long time for a resting potential to
develop again. But such is not the case. By an active process, called a Na+ pump, the Na+
ions are quickly transported to the outside of the cell, and the cell again may become
polarized and assumes its resting potential. This process is called repolarization.

Figure 2.4: Waveform of the bioelectric potential

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Following the generation of an action potential, there is a brief period of time during which
the cell can not respond to any new stimulus. This period is called the absolute refractory
period.

It may be noted that for active cells, the repolarization stops in a negative potential of around
-10 mV, called threshold potential and refractory period starts when the cell becomes non-
responsive to external stimuli. So the potential swings between threshold potential to action
potential.

To measure bioelectric potentials, a transducer capable of converting ionic potentials and

currents into electric potentials and current is required. Such a transducer consists of two
electrodes which measure the ionic potential difference between their respective points of
application. Although measurement of individual action potentials can be made in some types
of cells, such measurements are difficult because they require precise placement of an
electrode inside a cell. The more common form of measured biopotentials is the combined
effect of a large number of action potentials as they appear at the surface of the body, or at
one or more electrodes inserted into a muscle, nerve, or some part of the brain. Many
attempts have been made, for example, to explain the biopotentials from the heart as they
appear at the surface of the body. According to one theory, the surface pattern is a summation
of the potentials developed by the electric fields set up by the ionic currents that generate the
individual action potentials. This theory, although plausible, fails to explain a number of the
characteristics indicated by the observed surface patterns. A closer approximation can be
obtained if it is assumed that the surface pattern is a function of the summation of the first
derivatives of all the individual action potentials, instead of the potentials themselves.

2.2 Electrocardiogram
The electrocardiogram (ECG or EKG) is the graphical recording or display of the time
variant biopotentials produced by the myocardium during the cardiac cycle. The term
electrocardiograph means the instrument used for this type of recording and
electrocardiography means the technique used for such measurement. Each action potential
in the heart originates near the top of the right atrium at a point called the pacemaker or
sinoatrial (SA) node. The pacemaker is a group of specialized cells that spontaneously
generate action potentials at a regular rate, although the rate is controlled by innervation. To
initiate the heartbeat, the action potentials generated by the pacemaker propagate in all
direction along the surface of both atria. The wavefront of activation travels parallel to the
surface of the atria toward the junction of the atria and the ventricles. The wave terminates at
a point near the center of the heart, called the atriventricular (AV) node. At this point, some
special fibers act as a “delay line” to provide proper timing between the action of the atria
and the ventricles. Once the electrical excitation has passed through the delay line, it is
rapidly spread to all parts of both ventricles by the bundle of His .The fibers in this bundle,
called Purkinje fibers, divide into two branches to initiate action potentials simultaneously in
the powerful musculature of the two ventricles. The wave front in the ventricles does not
follow along the surface but is perpendicular to it and moves from the inside to outside of the
ventricular wall, terminating at the tip or apex of the heart. As indicated earlier, a wave of
repolarization follows the depolarization wave by about 0.2 to 0.4 second. This
repolarization, however, is not initiated from neighboring muscle cells but occurs as each cell
returns to its resting potential independently.

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 Figure 2.5: A typical ECG pattern

Figure 2.6: Defining waves and intervals of ECG

Figure 2.7: Defining segments and intervals of ECG

Alphabetic designations have been given to each of the prominent features. These can be
identified with events related to the action potential propagation pattern. To facilitate
analysis, the horizontal segment of this waveform preceding the P wave is designated as the
baseline or the isopotential line. The P wave represents depolarization of the atrial
musculature. The QRS complex is the combined result of the repolarization of the atria and
the depolarization of the ventricles, which occur almost simultaneously. The T wave is the

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wave of ventricular repolarization, whereas the U wave, if present, is generally believed to be
the result of after-potentials in the ventricular muscle. The P-Q interval represents the time
during which the excitation wave is delayed in the fibers near the AV node. The shape and
polarity of each of these features vary with the location of the measuring electrodes with
respect to the heart, and a cardiologist normally bases his diagnosis on readings take from
several electrode locations.

The P-R Interval is taken from the start of the P wave to the start of the QRS complex. It is
the time taken for depolarization to pass from the SA node via the atria, AV node and His-
Purkinje system to the ventricles. The QRS represents the time taken for depolarization to
pass through the His-Purkinje system and the ventricular muscles. It is prolonged with
disease of the His-Purkinje system. The Q-T interval is taken from the start of the QRS
complex to the end of the T wave. This represents the time taken to depolarize and repolarize
the ventricles. The S-T segment is the period between the end of the QRS complex and the
start of the T wave. All cells are normally depolarized during this phase. The ST segment is
changed by pathology such as myocardial ischemia or pericarditis.

Normal values of ECG: The values of amplitudes and durations of different waves,
complex, intervals and segments vary widely from person to person, time to time for the
same person due to natural variability. The following shows typical values of a normal adult
human.
Amplitudes: P wave: 0.25 mV R wave: 1.60 mV
Q wave: 0.40 mV T wave: 0.1 to 0.5 mV
Durations: P wave duration: 0.11 sec QRS duration: 0.09 sec
T wave duration: 0.12 sec
Intervals: P-R interval: 0.12 to 0.20 sec Q-T interval: 0.35 to 0.44 sec
S-T interval: 0.05 to 0.15 sec

2.3 Electroencephalogram
Electroencephalogram (EEG) refers to the recording of the brain's spontaneous electrical
activity over a period of time, as recorded from multiple electrodes placed on the scalp.
Diagnostic applications generally focus on the spectral content of EEG, that is, the type of
neural oscillations that can be observed in EEG signals. Electroencephalography is typically a
non-invasive (however invasive electrodes are often used in specific applications) method to
record electrical activity of the brain along the scalp. EEG measures voltage fluctuations
resulting from ionic current within the neurons of the brain. In clinical contexts, EEG is most
often used to diagnose epilepsy, which causes abnormalities in EEG readings. It is also used
to diagnose sleep disorders, coma, encephalopathies, and brain death. EEG used to be a first-
line method of diagnosis for tumors, stroke and other focal brain disorders, but this use has
decreased with the advent of high-resolution anatomical imaging techniques such as magnetic
resonance imaging (MRI) and computed tomography (CT). Despite limited spatial resolution,
EEG continues to be a valuable tool for research and diagnosis, especially when millisecond-
range temporal resolution (not possible with CT or MRI) is required.

Derivatives of the EEG technique include evoked potentials (EP), which involves averaging
the EEG activity time-locked to the presentation of a stimulus of some sort (visual,
somatosensory, or auditory). Event-related potentials (ERPs) refer to averaged EEG
responses that are time-locked to more complex processing of stimuli; this technique is used
in cognitive science, cognitive psychology, and psychophysiological research.

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The brain's electrical charge is maintained by billions of neurons. Neurons are electrically
charged (or "polarized") by membrane transport proteins that pump ions across their
membranes. Neurons are constantly exchanging ions with the extracellular milieu, for
example to maintain resting potential and to propagate action potentials. Ions of similar
charge repel each other, and when many ions are pushed out of many neurons at the same
time, they can push their neighbors, who push their neighbors, and so on, in a wave. This
process is known as volume conduction. When the wave of ions reaches the electrodes on the
scalp, they can push or pull electrons on the metal on the electrodes. Since metal conducts the
push and pull of electrons easily, the difference in push or pull voltages between any two
electrodes can be measured by a voltmeter. Recording these voltages over time gives us the
EEG.

The electric potential generated by an individual neuron is far too small to be picked up by
EEG or MEG (magnetoencephalogram). EEG activity therefore always reflects the
summation of the synchronous activity of thousands or millions of neurons that have similar
spatial orientation. If the cells do not have similar spatial orientation, their ions do not line up
and create waves to be detected. Pyramidal neurons of the cortex are thought to produce the
most EEG signal because they are well-aligned and fire together. Because voltage fields fall
off with the square of distance, activity from deep sources is more difficult to detect than
currents near the skull.

Scalp EEG activity shows oscillations at a variety of frequencies. Several of these oscillations
have characteristic frequency ranges, spatial distributions and are associated with different
states of brain functioning (e.g., waking and the various sleep stages). These oscillations
represent synchronized activity over a network of neurons. The neuronal networks underlying
some of these oscillations are understood (e.g., the thalamocortical resonance underlying
sleep spindles), while many others are not (e.g., the system that generates the posterior basic
rhythm). Research that measures both EEG and neuron spiking finds the relationship between
the two is complex, with a combination of EEG power in the gamma band and phase in the
delta band relating most strongly to neuron spike activity.

2.3.1 Normal EEG

The EEG is typically described in terms of (1) rhythmic activity and (2) transients. The
rhythmic activity is divided into bands by frequency. To some degree, these frequency bands
are a matter of nomenclature (i.e., any rhythmic activity between 8–12 Hz can be described as
"alpha"), but these designations arose because rhythmic activity within a certain frequency
range was noted to have a certain distribution over the scalp or a certain biological
significance. Frequency bands are usually extracted using spectral methods (for instance
Welch) as implemented for instance in freely available EEG software such as EEGLAB or
the Neurophysiological Biomarker Toolbox. Computational processing of the EEG is often
named Quantitative electroencephalography (qEEG).

Figure 2.8: A typical single-electrode EEG for 1 second

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Most of the cerebral signal observed in the scalp EEG falls in the range of 1–20 Hz (activity
below or above this range is likely to be artifactual, under standard clinical recording
techniques). Waveforms are subdivided into bandwidths known as alpha, beta, theta, and
delta to signify the majority of the EEG used in clinical practice.

2.3.2 EEG Waves

EEG consists of mainly 5 waves. The waves are briefly described below.

Delta waves: Delta (δ) is the frequency range up to 4 Hz. It tends to be the highest in
amplitude and the slowest waves. It is seen normally in adults in slow wave sleep. It is also
seen normally in babies. It may occur focally with subcortical lesions and in general
distribution with diffuse lesions, metabolic encephalopathy hydrocephalus or deep midline
lesions. It is usually most prominent frontally in adults (e.g. FIRDA - Frontal Intermittent
Rhythmic Delta) and posteriorly in children (e.g. OIRDA - Occipital Intermittent Rhythmic
Delta).

Theta waves: Theta (θ) is the frequency range from 4 Hz to 7 Hz. Theta is seen normally in
young children. It may be seen in drowsiness or arousal in older children and adults; it can
also be seen in meditation.[47] Excess theta for age represents abnormal activity. It can be seen
as a focal disturbance in focal subcortical lesions; it can be seen in generalized distribution in
diffuse disorder or metabolic encephalopathy or deep midline disorders or some instances of
hydrocephalus. On the contrary this range has been associated with reports of relaxed,
meditative, and creative states.

Alpha waves: Alpha (α) is the frequency range from 7 Hz to 14 Hz. Hans Berger named the
first rhythmic EEG activity he saw as the "alpha wave". This was the "posterior basic
rhythm" (also called the "posterior dominant rhythm" or the "posterior alpha rhythm"), seen
in the posterior regions of the head on both sides, higher in amplitude on the dominant side. It
emerges with closing of the eyes and with relaxation, and attenuates with eye opening or
mental exertion. The posterior basic rhythm is actually slower than 8 Hz in young children
(therefore technically in the theta range).

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Beta waves: Beta (β) is the frequency range from 15 Hz to about 30 Hz. It is seen usually on
both sides in symmetrical distribution and is most evident frontally. Beta activity is closely
linked to motor behavior and is generally attenuated during active movements. Low
amplitude beta with multiple and varying frequencies is often associated with active, busy or
anxious thinking and active concentration. Rhythmic beta with a dominant set of frequencies
is associated with various pathologies and drug effects, especially benzodiazepines. It may be
absent or reduced in areas of cortical damage. It is the dominant rhythm in patients who are
alert or anxious or who have their eyes open.

Gamma waves: Gamma (γ) is the frequency range approximately 30–100 Hz. Gamma
rhythms are thought to represent binding of different populations of neurons together into a
network for the purpose of carrying out a certain cognitive or motor function.

Sensorimotor rhythm aka mu rhythm: In addition to the posterior basic rhythm, there are
other normal alpha rhythms such as the mu rhythm (alpha activity in the contralateral sensory
and motor cortical areas) that emerges when the hands and arms are idle; and the "third
rhythm" (alpha activity in the temporal or frontal lobes). Alpha can be abnormal; for
example, an EEG that has diffuse alpha occurring in coma and is not responsive to external
stimuli is referred to as "alpha coma". Mu ranges 8–13 Hz., and partly overlaps with other
frequencies. It reflects the synchronous firing of motor neurons in rest state. Mu suppression
is thought to reflect motor mirror neuron systems, because when an action is observed, the
pattern extinguishes, possibly because of the normal neuronal system and the mirror neuron
system "go out of sync", and interfere with each other.

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 Comparison of EEG Waves
Band Frequen Location Normal Pathologic
cy (Hz)
frontally in Subcortical lesions
adults, Slow-wave in adults Diffuse lesions
Delta posteriorly in Sleep in babies Metabolic
<4
children; high- Has been found during some encephalopathy
amplitude continuous-attention tasks Hydrocephalus
waves Deep midline lesions
Higher in young children
Drowsiness in adults and
Focal subcortical
teens
lesions
Found in Idling
Metabolic
locations not Associated with inhibition of
Theta 4–7 encephalopathy
related to task elicited responses (has been
Deep midline disorders
at hand found to spike in situations
Some instances of
where a person is actively
hydrocephalus
trying to repress a response or
action).
posterior
Relaxed/reflecting
regions of
Closing the eyes
head, both
Also associated with
sides, higher in
Alpha 8 – 15 inhibition control, seemingly Coma
amplitude on
with the purpose of timing
dominant side.
inhibitory activity in different
Central sites
locations across the brain.
(c3-c4) at rest
both sides,
symmetrical Range span: active calm ->
distribution, intense -> stressed -> mild
Beta 16 – 31 most evident obsessive Bbenzodiazepines
frontally; low- Active thinking, focus, hi
amplitude alert, anxious
waves
A decrease in gamma-
Displays during cross-modal
band activity may be
sensory processing
associated with
(perception that combines
cognitive decline,
two different senses, such as
Gam Somatosensory especially when related
32 + sound and sight)
ma cortex to the theta band;
Also is shown during short-
however, this has not
term memory matching of
been proven for use as
recognized objects, sounds, or
a clinical diagnostic
tactile sensations
measurement
Mu suppression could
indicate that motor
mirror neurons are
Sensorimotor Shows rest-state motor working. Deficits in
Mu 8 – 12
cortex neurons. Mu suppression, and
thus in mirror neurons,
might play a role in
autism.

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Some features of the EEG are transient rather than rhythmic. Spikes and sharp waves may
represent seizure activity or interictal activity in individuals with epilepsy or a predisposition
toward epilepsy. Other transient features are normal: vertex waves and sleep spindles are seen
in normal sleep.

Note that there are types of activity that are statistically uncommon, but not associated with
dysfunction or disease. These are often referred to as "normal variants." The mu rhythm is an
example of a normal variant.

The normal Electroencephalography (EEG) varies by age. The neonatal EEG is quite
different from the adult EEG. The EEG in childhood generally has slower frequency
oscillations than the adult EEG.

The normal EEG also varies depending on state. The EEG is used along with other
measurements (EOG, EMG) to define sleep stages in polysomnography. Stage I sleep
(equivalent to drowsiness in some systems) appears on the EEG as drop-out of the posterior
basic rhythm. There can be an increase in theta frequencies. Santamaria and Chiappa
cataloged a number of the variety of patterns associated with drowsiness. Stage II sleep is
characterized by sleep spindles—transient runs of rhythmic activity in the 12–14 Hz range
(sometimes referred to as the "sigma" band) that have a frontal-central maximum. Most of the
activity in Stage II is in the 3–6 Hz range. Stage III and IV sleep are defined by the presence
of delta frequencies and are often referred to collectively as "slow-wave sleep." Stages I-IV
comprise non-REM (or "NREM") sleep. The EEG in REM (rapid eye movement) sleep
appears somewhat similar to the awake EEG.

EEG under general anesthesia depends on the type of anesthetic employed. With halogenated
anesthetics, such as halothane or intravenous agents, such as propofol, a rapid (alpha or low
beta), nonreactive EEG pattern is seen over most of the scalp, especially anteriorly; in some
older terminology this was known as a WAR (widespread anterior rapid) pattern, contrasted
with a WAIS (widespread slow) pattern associated with high doses of opiates. Anesthetic
effects on EEG signals are beginning to be understood at the level of drug actions on different
kinds of synapses and the circuits that allow synchronized neuronal activity.

2.3.4 EEG Measurement

The 10–20 system or International 10–20 system is an internationally recognized method to
describe and apply the location of scalp electrodes in the context of an EEG test or
experiment. This method was developed to ensure standardized reproducibility so that a
subject's studies could be compared over time and subjects could be compared to each other.
This system is based on the relationship between the location of an electrode and the
underlying area of cerebral cortex. The "10" and "20" refer to the fact that the actual distances
between adjacent electrodes are either 10% or 20% of the total front–back or right–left
distance of the skull.

Each site has a letter to identify the lobe and a number to identify the hemisphere location.
The letters F, T, C, P and O stand for frontal, temporal, central, parietal, and occipital lobes,
respectively. Note that there exists no central lobe; the "C" letter is used only for
identification purposes. A "z" (zero) refers to an electrode placed on the midline. Even
numbers (2,4,6,8) refer to electrode positions on the right hemisphere, whereas odd numbers
(1,3,5,7) refer to those on the left hemisphere. In addition, the letter codes A, Pg and Fp
identify the earlobes, nasopharyngeal and frontal polar sites respectively.

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Two anatomical landmarks are used for the essential positioning of the EEG electrodes: first,
the nasion which is the distinctly depressed area between the eyes, just above the bridge of
the nose; second, the inion, which is the lowest point of the skull from the back of the head
and is normally indicated by a prominent bump.

Figure 2.9: Electrode placement in EEG measurement

Figure 2.10: Normal EEG by 20-40 system

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 Figure 2.11: EEG with the onset of epilepsy

2.4 Electromyogram
An electromyogram (EMG) is the graphical representation of electrical potential generated by
muscle cells when these cells are electrically or neurologically activated. The signals can be
analyzed to detect medical abnormalities, activation level, or recruitment order or to analyze
the biomechanics of human or animal movement. Electromyography is an electrodiagnostic
technique for evaluating and recording the electrical activity produced by skeletal muscles.
EMG is performed using an instrument called an electromyography.

The electrical source is the muscle membrane potential of about –90 mV. Measured EMG
potentials range between less than 50 μV and up to 20 to 30 mV, depending on the muscle
under observation. Typical repetition rate of muscle motor unit firing is about 7–20 Hz,
depending on the size of the muscle (eye muscles versus seat (gluteal) muscles), previous
axonal damage and other factors. Damage to motor units can be expected at ranges between
450 and 780 mV.

There are two kinds of EMG: surface EMG and intramuscular EMG. Surface EMG assesses
muscle function by recording muscle activity from the surface above the muscle on the skin.
Surface electrodes are able to provide only a limited assessment of the muscle activity.
Surface EMG can be recorded by a pair of electrodes or by a more complex array of multiple
electrodes. More than one electrode is needed because EMG recordings display the potential
difference (voltage difference) between two separate electrodes. Limitations of this approach
are the fact that surface electrode recordings are restricted to superficial muscles, are
influenced by the depth of the subcutaneous tissue at the site of the recording which can be
highly variable depending of the weight of a patient, and cannot reliably discriminate
between the discharges of adjacent muscles.
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 Figure 2.12: A typical EMG

Figure 2.13: Muscle contraction resulting EMG

Intramuscular EMG can be performed using a variety of different types of recording

electrodes. The simplest approach is a monopolar needle electrode. This can be a fine wire
inserted into a muscle with a surface electrode as a reference; or two fine wires inserted into
muscle referenced to each other. Most commonly fine wire recordings are for research or
kinesiology studies. Diagnostic monopolar EMG electrodes are typically stiff enough to
penetrate skin and insulated, with only the tip exposed using a surface electrode for reference.
Needles for injecting therapeutic botulinum toxin or phenol are typically monopolar
electrodes that use a surface reference, in this case, however, the metal shaft of a hypodermic
needle, insulated so that only the tip is exposed, is used both to record signals and to inject.

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Slightly more complex in design is the concentric needle electrode. These needles have a fine
wire, embedded in a layer of insulation that fills the barrel of a hypodermic needle, that has
an exposed shaft, and the shaft serves as the reference electrode. The exposed tip of the fine
wire serves as the active electrode. As a result of this configuration, signals tend to be smaller
when recorded from a concentric electrode than when recorded from a monopolar electrode
and they are more resistant to electrical artifacts from tissue and measurements tend to be
somewhat more reliable. However, because the shaft is exposed throughout its length,
superficial muscle activity can contaminate the recording of deeper muscles. Single fiber
EMG needle electrodes are designed to have very tiny recording areas, and allow for the
discharges of individual muscle fibers to be discriminated. To perform intramuscular EMG,
typically either a monopolar or concentric needle electrode is inserted through the skin into
the muscle tissue. The needle is then moved to multiple spots within a relaxed muscle to
evaluate both insertional activity and resting activity in the muscle. Normal muscles exhibit a
brief burst of muscle fiber activation when stimulated by needle movement, but this rarely
lasts more than 100ms. The two most common pathologic types of resting activity in muscle
are fasciculation and fibrillation potentials. A fasciculation potential is an involuntary
activation of a motor unit within the muscle, sometimes visible with the naked eye as a
muscle twitch or by surface electrodes. Fibrillations, however, are only detected by needle
EMG, and represent the isolated activation of individual muscle fibers, usually as the result of
nerve or muscle disease. Often, fibrillations are triggered by needle movement (insertional
activity) and persist for several seconds or more after the movement ceases.

After assessing resting and insertional activity, the EMG assesses the activity of muscle
during voluntary contraction. The shape, size, and frequency of the resulting electrical signals
are judged. Then the electrode is retracted a few millimetres, and again the activity is
analyzed. This is repeated, sometimes until data on10–20 motor units have been collected in
order to draw conclusions about motor unit function. Each electrode track gives only a very
local picture of the activity of the whole muscle. Because skeletal muscles differ in the inner
structure, the electrode has to be placed at various locations to obtain an accurate study.

Single fiber EMG assesses the delay between the contractions of individual muscle fibers
within a motor unit and is a sensitive test for dysfunction of the neuromuscular junction
caused by drugs, poisons, or diseases such as myasthenia gravis. The technique is
complicated and typically only performed by individuals with special advanced training.
Surface EMG is used in a number of settings; for example, in the physiotherapy clinic,
muscle activation is monitored using surface EMG and patients have an auditory or visual
stimulus to help them know when they are activating the muscle (biofeedback). A review of
the literature on surface EMG published in 2008 concluded that surface EMG may be useful
to detect the presence of neuromuscular disease (level C rating, class III data), but there are
insufficient data to support its utility for distinguishing between neuropathic and myopathic
conditions or for the diagnosis of specific neuromuscular diseases. sEMG may be useful for
additional study of fatigue associated with post-poliomyelitis syndrome and
electromechanical function in myotonic dystrophy (level C rating, class III data).

2.5 Electroretinogram
The electroretinogram (ERG) is a diagnostic test that measures the electrical activity
generated by neural and non-neuronal cells in the retina in response to a light stimulus. The
electrical response is a result of a retinal potential generated by light-induced changes in the
flux of transretinal ions, primarily sodium and potassium. Most often, ERGs are obtained

15
using electrodes embedded in a corneal contact lens, which measure a summation of retinal
electrical activity at the corneal surface. The International Society for Clinical
Electrophysiology of Vision (ISCEV) introduced minimum standards for the ERG in 1989.
The ERG can provide important diagnostic information on a variety of retinal disorders
including, but not limited to congenital stationary night blindness, Leber congenital
amaurosis, and cancer-associated retinopathy. Moreover, an ERG can also be used to monitor
disease progression or evaluating for retinal toxicity with various drugs or from a retained
intraocular foreign body.

2.5.1 Components of ERG

A typical ERG signal is presented in the following figure.

Figure 2.14: Maximal response ERG waveform from a dark adapted eye

a-wave: initial corneal-negative deflection, derived from the cones and rods of the outer
photoreceptor layers. This wave reflects the hyperpolarization of the photoreceptors due to
closure of sodium ion channels in the outer-segment membrane. Absorption of light triggers
the rhodopsin to activate transducin, a G-protein. This leads to the activation of cyclic
guanosine monophosphate phosphodiesterase (cGMP PDE) eventually leading to a reduction
in the level of cGMP within the photoreceptor. This leads to closure of the sodium ion
channels resulting in a decrease of inwardly directed sodium ions, or a hyperpolarization of
the cell. The a-wave amplitude is measured from baseline to the trough of the a-wave.

b-wave: corneal-positive deflection; derived from the inner retina, predominantly Muller and
ON-bipolar cells. The hyperpolarization of the photoreceptor cells results in a decrease in the
amount of neurotransmitter released, which subsequently leads to a depolarization of the
post-synaptic bipolar cells. The bipolar-cell depolarization increases the level of extracellular
potassium, subsequently generating a transretinal current. It is this transretinal current that
depolarizes the radially oriented Muller cells and generates the corneal-positive deflection.
The b-wave amplitude is generally measured from the trough of the a-wave to the peak of the
b-wave. This wave is the most common component of the ERG used in clinical and
experimental analysis of human retinal function.

c-wave: derived from the retinal pigment epithelium and photoreceptors. The c-wave is a
reflection of the resulting change in the transepithelial potential due to the hyperpolarization
at the apical membrane of the RPE cells and the hyperpolarization of the distal portion of the

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Muller cells. The c-wave generally peaks within 2 to 10 seconds following a light stimulus,
depending on flash intensity and duration. Due to the c-wave response developing over
several seconds, it is susceptible to influences from electrode drift, eye movements, and
blinks.

Latency of response refers to the onset of the stimulus to the beginning of the a-wave.
Implicit time is a measure of the time interval from onset of the stimulus to the peak of the
b-wave.

2.5.2 Types of Recording Electrodes

Burian-Allen Electrode: commonly used electrode for flash ERG. It uses variable lens sizes
consisting of an annular ring of stainless steel surrounding the central
polymethylmethacrylate (PMMA) contact-lens core with a lid speculum.

Dawson-Trick-Litzkow Electrode: low-mass conductive Mylar thread consisting of

individual fibers impregnated with metallic silver.

ERG-Jet Electrode: a disposable plastic lens with a gold-plated peripheral circumference.

Mylar Electrode: aluminized or gold-coated Mylar.

Skin Electrode: may be used as a replacement for corneal electrodes by placing an electrode
on the skin over the infraorbital ridge near lower eyelid; due to decreased amplitudes and
variable responses, the skin electrode is primarily used for screening purposes only.

Cotton-Wick Electrode: Burian-Allen electrode shell fitted with a cotton wick which is
useful for minimizing light-induced artifact.

Hawlina-Konec Electrode: Teflon-insulated thin metal wire (silver, gold, platinum) with
three central windows, 3 mm in length, molded to fit into the lower conjunctival sac.

2.5.3 Normal ERG responses under scotopic and photopic conditions

The rod and cone photoreceptor function responses can be separated using a variety of ERG
techniques. Scotopic (rod) responses are isolated by dark-adaptation for a minimum of 20
minutes per ISCEV standards followed by a short wavelength stimulus as a single flash or 10
Hz flicker. Although the resulting response has rod and cone components, the rod component
is dominant and the primary contributor to the increased amplitude and increased implicit
time. Photopic (cone) responses can be obtained either before or after dark-adaptation. Since
rods cannot follow a flicker stimulus greater than 20 Hz, cone photoreceptor function is
primarily measured under light-adapted conditions for at least 10 minutes with either single
flash (stimulus wavelength greater than 680 nm) or 30 Hz flicker stimulus. Photopic
responses result in small b-wave amplitudes with a short latency (30-32 ms), whereas
scotopic (rod) conditions produce much larger b-wave amplitudes with a longer latency (60
ms).

Oscillatory potentials (OP) are high-frequency, low-amplitude wavelets on the ascending

limb of the b-wave with a frequency of about 100 to 160 Hz. Although it is not known for
certain, it is suspected that OPs are generated from the amacrine cells located in the inner
retina.

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2.5.4 Factors affecting the ERG
• Duration of stimulus
• Size of retinal area illuminated
• Interval between stimuli
• Size of pupil
• Systemic circulation and drugs
• Development of Retina
• Clarity of Ocular Media
• Age, Sex, and Refractive Error
• Anesthesia
Diurnal Fluctuations

2.5.5 Types of ERG

The focal ERG (fERG; also known as the foveal ERG) is used primarily to measure the
functional integrity of the fovea and is therefore useful in providing information in diseases
limited to the macula. A variety of techniques have been described in the literature for
recording fERGs. Differing field sizes varying from 3 degrees to 18 degrees and light
stimulus frequencies have been used in the various methods, however each technique deals
with the challenge of limiting amount of light scattered outside the focal test area. Focal ERG
is useful for assessing macular function in conditions such as age-related macular
degeneration, however requires good fixation from the subject. The full-field ERG (ffERG)
measures the stimulation of the entire retina with a flashlight source under dark-adapted
(scotopic) and light-adapted (photopic) types of retinal adaptation. This is useful in detecting
disease with widespread generalized retinal dysfunction i.e. cancer associated retinopathy,
toxic retinopathies, and cone-rod dysfunction. Due to the massed retinal electrical response,
small retinal lesions may not be revealed in ffERG recordings.

The multifocal ERG (mfERG) simultaneously measures local retinal responses from up to
250 retinal locations within the central 30 degrees mapped topographically. This new
technology was developed by Erich Sutter in the early 1990s and involves powerful
computers and high –intensity display monitors. The light stimuli are presented on a video
monitor in one of a large number of arrays consisting of hexagonal elements. The hexagonal
elements in the array are distributed so that the focal retinal responses have an approximately
equal signal-to-noise ratio. The central hexagons are smaller than those in the periphery. The
elements are stimulated in a pseudo-random sequence of light and dark, called a maximum
length sequence (or m-sequence). The resulting waveforms are similar to those of the ffERG:
initial negative deflection (N1 or a-wave), followed by a positive deflection (P1 or b-wave),
and a second negative deflection (N2 or c-wave). MfERGs are useful in detecting localized
abnormalities within the retina in conditions such as retinitis pigmentosa, branch retinal
artery occlusion, fundus flavimaculatus, and Stargardt’s disease. Degree of retinal toxicity
related to certain drugs such as hydroxychloroquine or ethambutol is better detected using
mfERG compared to ffERG. Early visual field defects due to glaucoma may also be detected
sooner using mfERG compared to automated perimetry.

The pattern ERG (PERG) uses pattern-reversal stimuli similar to VEP testing and captures
retinal ganglion cell activity predominantly in the N95 waveform component. The PERG is
used to detect subtle optic neuropathies. In demyelinating optic neuropathy, the PERG is
relatively normal, while it may be abnormal in ischemic optic neuropathies.

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 Abnormalities in ERG with various disease states
Disease entity full-field ERG findings
Multiple evanescent white dot initially depressed a- and b-wave responses with return to
syndrome (MEWDS) normal values
marked rod dysfunction and elevated threshold of rods and
Vitamin A deficiency
cones on dark adaptation
markedly depressed photopic response and less affected
Cone dystrophy
scotopic response
Cancer associated retinopathy
significantly reduced a-wave and b-wave amplitudes
(CAR)
Melanoma associated extinguished rod responses, normal a-wave, reduced b-wave
retinopathy (MAR) (electronegative ERG)
minimal or sub-normal a- and b-wave amplitudes (response
Retinitis pigmentosa
primarily from cone system)
Congenital Achromatopsia non-detectable cone response, normal or subnormal rod
(typical (rod) monochromat) response
Congenital Achromatopsia
normal ERG responses
(atypical (cone) monochromat)
Congenital Red-Green Color
normal ERG responses
Deficiency
Congenital Hereditary normal scotopic a-wave with selectively reduced b-wave;
Stationary Night Blindness implicit time of b-wave is approximately the same under
(Schubert-Bornschein type) scotopic and photopic conditions
normal photopic responses with predominantly reduced
Oguchi disease
scotopic b-wave amplitudes
reduced scotopic amplitudes which improve to normal values
Fundus Albipunctatus
after longer (variable) perior of dark adaptation
reduced b-wave amplitudes relative to a-wave in scotopic and
Fleck Retina of Kandori
photopic conditions
extent of reduced a- and b-wave amplitudes depends on extent
Stargardt Macular Dystrophy of fundus pigmentary changes; longer duration of dark-
(Fundus Flavimaculatus) adaptation may be necessary for scotopic amplitudes to reach
normal values
Best Vitelliform Macular
normal ERG responses with an abnormal EOG
Dystrophy
Pattern dystrophies normal ERG responses
North Carolina Macular
normal ERG responses
Dystrophy
Progressive Bifocal
subnormal 30-Hz flicker and single-flash scotopic responses
Chorioretinal Atrophy
Fenestrated Sheen Macular initially normal ERG responses with reduced scotopic and
Dystrophy photopic responses in later stages
Familial Internal Limiting
reduced b-wave amplitudes
Membrane Dystrophy

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Gyrate Atrophy
Choroideremia
X-linked Juvenile
Retinoschisis
Goldmann-Favre Syndrome
Wagner Disease
Autosomal Dominant
Neovascular Inflammatory
Vitreoretinopathy
Autosomal Dominant
Vitreoretinochoroidopathy
Familial Exudative
Vitreoretinopathy
Birdshot Retinochoroidopathy scotopic than photopic responses; delayed photopic and
Acute Zonal Occult Outer
Retinopathy
Pseudo-Presumed Ocular
Histoplasmosis Syndrome
(Multifocal Choroiditis)
Behcet Disease
Sickle Cell Retinopathy
Takayasu Disease
Carotid Artery Occlusion
Central and Branch Artery and
Vein Occlusions
Hypertension and
Arteriosclerosis
Chloroquine and
Hydroxychloroquine toxicity
Thioridazine
Quinine
significantly reduced or extinguished rod and cone responses
reduced a- and b-wave amplitudes in both scotopic and
photopic conditions; increased rod and cone b-wave implicit
times
reduced photopic and scotopic b-wave amplitudes
non-detectable ERG response
normal or mild to moderately reduced photopic and scotopic
responses (variable depending on extent of fundus
involvement)
selective reduction of b-wave amplitude
initially normal ERG with only mild to moderately reduced
amplitudes in older patients
normal ERG responses (diminished oscillatory potentials may
be observed)
selective reduction in b-wave amplitude more prominent in
scotopic b-wave implicit times
ERG amplitudes vary from normal to subnormal with
prolonged b-wave implicit times
moderate to severely reduced rod and cone responses
initially loss of oscillatory potentials in flash ERG with
subsequent reduction of b-wave amplitude
normal ERG in the absence of peripheral retinal
neovascularization, reduced amplitudes of ERG components
when peripheral retinal neovascularization is present
initially decreased oscillatory potentials, later stages involve
reduced a- and b-wave amplitudes
reduced b-wave greater than a-wave amplitudes depending on
extent and severity of occlusion
reduced scotopic b-wave amplitudes in CRAO and CRVOs;
slight b-wave reduction or normal ERG in branch retinal artery
or vein occlusions
initially reduced oscillatory potentials followed by reduced a-
and b-wave amplitudes
normal ERG responses unless presence of advanced
retinopathy; cone function initially more affected than rod
function
decreased photopic and scotopic a- and b-wave responses
depending on degree of fundus changes
transiently decreased a- and b-wave amplitudes under both
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 photopic and scotopic conditions with recovery after 24 hours
Methanol reduced a- and b-wave amplitudes
Gentamicin significantly reduced ERG amplitudes
Vitamin A deficiency reduced ERG amplitudes particularly under scotopic conditions
Kearns-Sayre Syndrome reduced ERG amplitudes
initially a transient supernormal response then negative pattern
followed by non-detectable response in severe cases (rod
Siderosis
function more affected than cones; reduction of b-wave
amplitude more than a-wave)

2.6 Electrooculogram
Electrooculography (EOG) is a technique for measuring the corneo-retinal standing potential
that exists between the front and the back of the human eye. The resulting signal is called the
electrooculogram. Primary applications are in ophthalmological diagnosis and in recording
eye movements. Unlike the electroretinogram, the EOG does not measure response to
individual visual stimuli. To measure eye movement, pairs of electrodes are typically placed
either above and below the eye or to the left and right of the eye. If the eye moves from
center position toward one of the two electrodes, this electrode "sees" the positive side of the
retina and the opposite electrode "sees" the negative side of the retina. Consequently, a
potential difference occurs between the electrodes. Assuming that the resting potential is
constant, the recorded potential is a measure of the eye's position.

Figure 2.15: Electrooculograms for the period of REM sleep

2.6.1 Principle
The eye acts as a dipole in which the anterior pole is positive and the posterior pole is
negative.
1. Left gaze: the cornea approaches the electrode near the outer canthus of the left eye,
resulting in a negative-trending change in the recorded potential difference.
2. Right gaze: the cornea approaches the electrode near the inner canthus of the left eye,
resulting in a positive-trending change in the recorded potential difference.

2.6.2 Ophthalmological diagnosis

The EOG is used to assess the function of the pigment epithelium. During dark adaptation,
resting potential decreases slightly and reaches a minimum ("dark trough") after several

21
minutes. When light is switched on, a substantial increase of the resting potential occurs
("light peak"), which drops off after a few minutes when the retina adapts to the light. The
ratio of the voltages (i.e. light peak divided by dark trough) is known as the Arden ratio. In
practice, the measurement is similar to eye movement recordings (see above). The patient is
asked to switch eye position repeatedly between two points (alternating looking from center
to the left and from center to the right). Since these positions are constant, a change in the
recorded potential originates from a change in the resting potential.

2.7 Electroneurogram
An electroneurogram (ENG) is a method used to visualize directly recorded electrical activity
of neurons in the central nervous system (brain, spinal cord) or the peripheral nervous system
(nerves, ganglions). The acronym ENG is often used. An electroneurogram is similar to an
electromyogram (EMG), but the latter is used to visualize muscular activity. An
electroencephalogram (EEG) is a particular type of electroneurogram in which several
electrodes are placed around the head and the general activity of the brain is recorded,
without having very high resolution to distinguish between the activity of different groups of
neurons.

Figure 2.16: Measurement of ENG

An electroneurogram is usually obtained by placing an electrode in the neural tissue. The

electrical activity generated by neurons is recorded by the electrode and transmitted to an
acquisition system, which usually allows to visualize the activity of the neuron. Each vertical
line in an electroneurogram represents one neuronal action potential. Depending on the
precision of the electrode used to record neural activity, an electroneurogram can contain the
activity of a single neuron to thousands of neurons. Researchers adapt the precision of their
electrode to either focus on the activity of a single neuron or the general activity of a group of
neurons, both strategies having their advantages.

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2.8 Heart Rate
The cardiac cycle completed in one minute is termed as heart rate (HR). It is normally
represented as beats per minute (BPM). HR is a noninvasive measurement and a primary
indicator of cardiac function. There are two types of HR normally measured to assess
possible cardiac abnormality: mean heart rate (MHR) and instantaneous heart rate (IHR).

2.8.1 Mean Heart Rate

The average or mean HR is the count of cardiac cycles normally in one minute. It can be
calculated from ECG, blood pressure, heart sound, blood volume and any such activity of
heart. The MHR can be calculated from measured ECG either by direct count of the number
of R peaks or from the autocorrelation function of ECG. Normal value of MHR for an adult is
60 – 120 bpm. The MHR gives very little information, better information can be obtained
from mean and standard deviation (SD) or from coefficient of variation (CV = SD / Mean).
Two types of MHR are considered for assessment.

Resting Heart Rate: Resting heart rate (Resting HR) is the number of beats in one minute
when you are at complete rest. Your resting heart rate indicates your basic fitness level. The
more well-conditioned your body, the less effort and fewer beats per minute it takes your
heart to pump blood to your body at rest.

Recovery Heart Rate: This is the heart rate that our body will decrease to after an exercise
session. For example, you exercise for a 1/2 hour at 155. Two minutes after you stop
exercising, your heart rate decreases to 95. The 95 would be your recovery heart rate. It is
used to evaluate your fitness level after exercise. It is good to set a two minute time frame
and see how many beats you recover in that time frame. Compare this recover heart rate
between exercise sessions.

2.8.2 Instantaneous Heart Rate

IHR represents beat to beat interval of heart. It is calculated from each ECG. If the interval
between two consecutive R-peaks is Δt, then IHR for that beat is 60/Δt bpm. Alternatively,
the time interval can also be represented as a function of time and is termed as Tachogram.
In this way we can get a time-series of HR from which we can calculate MHR, SD, CV, etc.

Figure 2.17 Tachogram and IHR calculation

The autonomic nervous system (ANS) is the portion of the nervous system that controls the
body's visceral functions, including action of the heart, movement of the gastrointestinal tract
and secretion by different glands, among many other vital activities. It is well known that

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mental and emotional states directly affect the ANS. Many of research studies have examined
the influence of emotions on the ANS utilizing the analysis of heart rate variability, or heart
rhythms, which serves as a dynamic window into autonomic function and balance. Thoughts
and even subtle emotions influence the activity and balance of the ANS. The ANS interacts
with our digestive, cardiovascular, immune and hormonal systems. Negative reactions create
disorder and imbalance in the ANS. Positive feelings such as appreciation create increased
order and balance in the ANS, resulting in increased hormonal and immune system balance
and more efficient brain function.

While the rhythmic beating of the heart at rest was once believed to be monotonously regular,
we now know that the rhythm of a healthy heart under resting conditions is actually
surprisingly irregular. These moment-to-moment variations in heart rate are easily
overlooked when average heart rate is calculated. Heart rate variability (HRV), derived from
the electrocardiogram (ECG), is a measurement of these naturally occurring, beat-to-beat
changes in heart rate. Systems-oriented models propose that HRV is an important indicator of
both physiological resiliency and behavioral flexibility, reflecting the individual's capacity to
adapt effectively to stress and environmental demands. It has become apparent that while a
large degree of instability is detrimental to efficient physiological functioning, too little
variation can also be pathological. An optimal level of variability within an organism's key
regulatory systems is critical to the inherent flexibility and adaptability that epitomize healthy
function. This principle is aptly illustrated by a simple analogy: just as the shifting stance of a
tennis player about to receive a serve may facilitate swift adaptation, in healthy individuals,
the heart remains similarly responsive and resilient, primed and ready to react when needed.

The normal variability in heart rate is due to the synergistic action of the two branches of the
ANS, which act in balance through neural, mechanical, humoral and other physiological
mechanisms to maintain cardiovascular parameters in their optimal ranges and to permit
appropriate reactions to changing external or internal conditions. In a healthy individual, thus,
the heart rate estimated at any given time represents the net effect of the parasympathetic
(vagus) nerves, which slow heart rate, and the sympathetic nerves, which accelerate it. These
changes are influenced by emotions, thoughts and physical exercise. Our changing heart
rhythms affect not only the heart but also the brain's ability to process information, including
decision-making, problem-solving and creativity. They also directly affect how we feel.
Thus, the study of heart rate variability is a powerful, objective and noninvasive tool to
explore the dynamic interactions between physiological, mental, emotional and behavioral
processes. Figure 2.15 shows examples of IHR of 5 normal adult persons.
.
The mathematical transformation (Fast Fourier Transform) of HRV data into power spectral
density (PSD) is used to discriminate and quantify sympathetic and parasympathetic activity
and total autonomic nervous system activity. Power spectral analysis reduces the HRV signal
into its constituent frequency components and quantifies the relative power of these
components.

24
Figure 2.18: IHR of 5 normal adult subjects (in each panel, the alphabet indicates subject’s
identification while the numeral indicates the age of the person)

25
HRV refers to the regulation of the sinoatrial node, the natural pacemaker of the heart by the
sympathetic and parasympathetic branches of the autonomic nervous system. Our
assumption, when we assess HRV, is that the beat-to-beat fluctuations in the rhythm of the
heart provide us with an indirect measure of heart health, as defined by the degree of balance
in sympathetic and vagus nerve activity. Some examples of IHR are provided below:

IHR a 33-year-old male experiencing anxiety: the prominent spikes are due to pulses of
activity in the sympathetic nervous system

IHR of a healthy 30-year-old male driving car and then hiking uphill

26
IHR during a heart lock-in. Entrainment is reflective of autonomic nervous system balance
and is commonly experienced when using the freeze-frame and heart lock-in techniques

Heart rhythm of a heart transplant recipient: the lack of variability in heart rate is due to loss
of autonomic nervous system input to the heart

Heart rhythm of a 44-year-old female with low heart rate variability while suffering from
headaches and pounding sensation in her head.

27