Biofeedback instruments supplement our awareness of biological processes
ranging from the contraction of the tibialis anterior muscle in the leg
to the synchronous firing of cortical neurons. Hardware provides immediate and accurate information about our performance. Signals
are detected, processed and quantified, and displayed (Peek, 2016). Beginners often view
biofeedback equipment as intimidating "black boxes" that stand between them
and treating their patients.
Why should a psychologist or nurse understand filter settings or
skin-electrode impedances? Practical knowledge about these black boxes
allows us to make informed purchases, use instruments effectively, and
recognize malfunctions.
Technicians and their supervisors need to understand the science, instrumentation concepts, and protocols that are the foundation of biofeedback. A technician who doesn't know what a box does cannot provide safe and effective training.
Although this unit uses the electromyograph to illustrate instrumentation
concepts, most of this discussion also applies to
electroencephalographs. We
will discuss instrumentation for each of the major biofeedback modalities
in later units. Masseter placement graphic created by Devid Abd on fiverr.com.
BCIA Blueprint Coverage
This unit addresses Descriptions of the most commonly employed biofeedback modalities: SEMG
(III-A,Sources of artifact (III-B), and Essential electronic terms and concepts for biofeedback applications
(III-D).
This unit covers
Biological Signals, Electrodes, Site Preparation, Impedance Testing, Signal Processing, Biofeedback Display Settings, Display Options, Shape Performance Using a Threshold, Artifacts, Tracking Tests, Normal Values, and Computers in Biofeedback.
Please click on the podcast icon below to hear a full-length lecture.
Biological Signals
Biofeedback instruments measure performance directly or indirectly. While a
muscle sensor directly monitors voltage sources
like skeletal muscles, it indirectly monitors muscle contraction since an electromyograph does not measure force, movement, or range of motion. The electrical signal is linearly related to isometric muscle contraction, during which muscle length is constant (Peek, 2016; Stern et al., 2001).
Signals we measure directly are:
In contrast, a skin sensor registers temperature changes indirectly as
shifts in its electrical resistance. Signals we measure indirectly
are:
Electrodes transform a current of
ions into a current of electrons that flows through the cable into an
electromyograph's input jack. Check out the YouTube video Muscle Contraction, It's so Easy!!!
Signal strength is lost during volume conduction. Whereas a muscle
action potential is measured in millivolts
or thousandths of a volt, a volume-conducted signal that
reaches SEMG electrodes has been reduced to
microvolts or millionths of a volt, particularly by the
absorption of its higher frequencies by intervening tissue (Montgomery,
2004).
A recessed metal disk is filled with an electrolyte, a
conductive gel or paste. The electrolyte maintains contact between
electrode metal and skin even during moderate patient movement.
When a SEMG electrode is filled with
an electrolyte, the electrode metal donates ions to the electrolyte. In
turn, the electrolyte contributes ions to the metal surface. Signal
conduction succeeds as long as electrode and electrolyte ions are freely
exchanged.
However, conduction breaks down when chemical reactions produce separate positive and negative charge regions where the electrode and gel make contact. When an electrode is polarized, ion exchange is reduced, and impedance increases, weakening the signal reaching the electromyograph. This problem can develop during routine clinical use. Silver/silver-chloride or gold electrodes resist polarization.
Why Do We Prefer Three Electrodes?
At least two electrodes, designated active and reference, are needed to
measure the SEMG signal. These resemble a dipole antenna used to receive
FM broadcasts.
Electrode placement near a muscle's insertion into a tendon or offset to the side will reduce the strength of the EMG signal. Graphic adapted from AdvancerTechnologies.com.
Active placement over the wrong muscle or at an angle from the muscle
body will result in misleading signals (Sherman, 2002). Reference electrode (black) positioning is less critical since it may be
located within 6 inches of either active. Note that the color code for active electrodes varies with the manufacturer. For Thought Technology Ltd., the active electrodes are blue and yellow. The photograph shows a bipolar frontal placement.
SEMG recording can be monopolar or bipolar.
Monopolar recording
uses one active and one reference electrode. Since the active sees more
SEMG activity than the reference, a voltage develops between them.
Bipolar recording uses two actives and a shared reference electrode.
Since each active is paired with the reference, bipolar recording
produces two voltages.
Bipolar is superior to monopolar recording because it allows us to obtain a cleaner signal.
Clinicians and researchers prefer bipolar recording because it
monitors a wider surface area, which is ideal for relaxation training and allows a differential amplifier
stage to remove contamination appearing at both active electrodes. Note that the colors white and red designate active electrodes in the graphic below. Trapezius placement graphic created by Devid Abd on fiverr.com.
SEMG scanning allows clinicians to test muscles with a movable sensor sequentially. Instead of attaching multiple sets of EMG electrodes, a clinician can quickly move a SEMG scanning electrode from muscle to muscle.
Telemetry
Telemetry systems, like the NeXus-10
and Thought
Technology Ltd.'s Tele-Infiniti telemetry systems shown below, solve two problems. First, telemetry protects clients from electrical shock since there is no wired connection to a computer powered by a wall outlet. Second, telemetry allows us to monitor clients during unrestricted movement.
Thought Technology Ltd. generously provided the diagrams below from their Basics of Surface
Electromyography Applied to Physical Rehabilitation and Biomechanics (2009).
Measure the quality of skin-electrode contact, which is
called impedance. An impedance meter,
which sends a nonpolarizing AC
signal through the skin, provides the most valid impedance measurement.
In bipolar
recording, we measure impedance between each active electrode and the
shared reference. Impedance testing results in two measurements in the Kohm
(thousand-ohm) range.
Low and Balanced Skin-Electrode Impedances Are Critical
A conservative rule is that each measurement should be less than 10 Kohms
and within 5 Kohms of each other.
Why? Two reasons. First, high skin-electrode impedance reduces SEMG signal strength and makes it harder to differentiate from contamination. Second, a differential amplifier subtracts voltages that look identical because they are probably artifacts like 50/60Hz. Imbalanced impedance makes voltages look different (the signal with higher impedance will appear smaller), allowing artifacts to "sneak" through.
Inadequate skin preparation and the application of insufficient electroconductive gel can produce high and imbalanced impedances.
Impedance imbalance can lower SEMG values and the signal-to-noise ratio and increase signal contamination.
This problem is illustrated by the recording shown below adapted from Peper, Gibney, Tylova, Harvey, and Combatalade (2008).
Revised caption: Recording of electrode contact artifact. When the trainee tried to relax, SEMG activity never dropped near zero due to poor skin/electrode contact. After applying electroconductive gel and the Triode electrode was reattached to the trainee's non-dominant arm, the skin/electrode contact improved. The signal dropped to near zero during relaxation and appropriately increased during tension.
Sherman (2002) cautions that high impedance due to poor skin preparation can make a highly-contracted muscle look virtually silent by reducing its amplitude to one-tenth of its actual value.
An impedance test can be performed manually with a separate
impedance meter or voltohmmeter or automatically by data acquisition system software like the MyoScan-Z
using BioGraph Infiniti 5.0 and later software.
Gell-Bridge Artifact Short-Circuits the EEG and SEMG Signal
Gel-bridge artifact occurs when electrodes are closely spaced and the electrode gel
smears, creating a bridge between the active and reference electrodes. A gel-bridge creates a short circuit and results in
abnormally low readings. Disposable pre-gelled electrodes avoid this problem. For permanent electrodes, remove the electrodes, clean the skin, remove the smeared gel, and reapply the electrodes to fix this problem.
Signal Processing
Biological signals enter the black box via an electrode cable or Bluetooth.
SEMG signals entering an electromyograph are dropped
across an input impedance, amplified, filtered, rectified, integrated,
measured by a level detector, and finally displayed as shown in the diagram below from Thought Technology
Ltd.'s Basics of Surface Electromyography Applied to Physical Rehabilitation and Biomechanics (2009).
The biological signals monitored in biofeedback are very weak. The SEMG
signal, for example, is measured in microvolts (millionths of a volt).
These signals must first be amplified over several stages to isolate the
activity we are interested in and drive displays. Stereo amplifiers perform
the same tasks when boosting audio signals above their noise floor to levels that
can drive loudspeakers.
Electromyographs boost incoming signals in stages. The first amplification stage is called a preamplifier. A manufacturer may
place the preamplifier in the sensor shell to reduce signal loss, as in Thought Technology Myoscan-Z shown below. The "Z" designation means that software can perform an impedance (Z) check.
While AC signals can be amplified using single-ended or differential amplifiers, differential amplifiers yield cleaner signals.
A single-ended amplifier is used during monopolar recording, producing
only one signal. This circuit indiscriminately boosts the signal, whether
SEMG
activity or artifact, resulting in excessive contamination.
A differential amplifier is used during bipolar recording, producing
two separate signals. This design combines two single-ended amplifiers, 180o out of phase, so only the two signals' difference is boosted. How does this reduce artifact? When there is no SEMG activity,
identical noise signals reach each amplifier. The differential amplifier
subtracts these signals, canceling out the artifact. A
perfect differential amplifier's output would be 0.
The graphic below was redrawn from John Demos' BCIA-recommended Getting Started with EEG Neurofeedback (2nd ed.). A differential amplifier rejects the common voltage (e.g., 3 feet) and outputs the voltage difference (e.g., 4 feet). A single-ended amplifier outputs the entire voltage (e.g., 7 feet, EEG artifact and signal value).
Common Mode Rejection Reduces Artifact
A common-mode rejection ratio (CMRR) specification shows us how well a differential amplifier rejects noise. Since differential amplifiers are imperfect, they will boost the signal and some noise. The CMRR
specification compares the degree to which a differential amplifier
boosts signal (differential gain) and artifact (common -ode gain). CMRR =
differential gain/common-mode gain.
Unlike impedance, you are not expected to measure CMRR. Manufacturers should measure it at 50/60Hz where the most energetic artifacts, like
power line (50/60Hz) noise, are found. The smallest acceptable ratio is
100
dB (100,000:1). This ratio means that signal is boosted 100,000 times more
than competing noise. State-of-the-art equipment achieves 180-dB ratios or higher.
Lower ratios could result in unacceptable contamination of biological
signals.
How to Record Cleaner Signals
You can take four steps to maximize common-mode rejection. First,
identify artifact sources. If you have a portable electromyograph, use it like a
Geiger counter. Move the unit around the room with SEMG sensors connected
but held in your hand. Artifact sources should produce the largest
display values.
Second, remove the artifact sources you find. For example, fluorescent
lights can be replaced with fixtures that produce less 50/60Hz noise.
Third, position the electromyograph and electrode cable to reduce
artifact reception. Use the location and angle that yield the lowest
readings when not attached to a patient.
Fourth, keep skin-electrode impedances balanced within 5 Kohms. If both
actives receive identical noise signals, the imbalance will make the signals
look different and prevent the complete subtraction of artifacts.
Choose an Electromyograph with High Differential Input Impedance
An electromyograph drops the SEMG signal across a network of resistors to ensure that it receives 99% or more of the electrodes' signal. This resistor network produces differential input impedance in the Gohm (billion-ohm range) to minimize the effect of unequal skin-electrode impedances. State-of-the-art electromyographs achieve 10 Gohms.
Differential input impedance is another specification that your manufacturer measures for you.
Differential input impedance must be at least 100 times skin-electrode impedance.
Why? This ratio allows 99% or more of the SEMG signal to enter an electromyograph. Stronger signal voltages enable an electromyograph to differentiate SEMG activity from artifacts, resulting in more accurate measurement and feedback.
How do we achieve this? Ensure that skin-electrode impedance is low and balanced.
Operational Amplifiers
DC signals, like temperature, are amplified using an
operational
amplifier (op-amp). This circuit is a very high gain DC amplifier that
uses external feedback to perform computations like addition,
subtraction, and averaging biological signals. Like the integrated circuit below, operational amplifiers are used in feedback thermometers (skin temperature) and electrodermographs (skin electrical activity).
Filters
Filters can help differential amplifiers reduce contamination of the SEMG signal by artifacts. Filters select the frequencies we want to measure and essentially ignore the ones we don't. Let's unpack the term frequency.
Bandpass filters and notch
filters perform this function in an electromyograph.
Bandpass Filters
A bandpass filter combines two filters called high-pass and low-pass
filters. A high-pass filter selects frequencies above a cutoff. In
contrast, a low-pass filter selects frequencies below a cutoff.
When you combine high-pass and low-pass filters, this selects signals between the upper and
lower cutoffs. We call this region the bandpass.
For example, the
100-200 Hz bandpass filter commonly used in electromyographs combines a
100-Hz high-pass filter and a 200-Hz low-pass filter.
The bandpass filter's bandwidth is the 100-Hz difference between the high-pass and low-pass frequencies (200 Hz-100 Hz).
A bandpass filter is defined by its center frequency, cutoff frequencies,
and slope.
The center frequency lies in the middle of the bandpass. For a
100-500 Hz bandpass filter, the center frequency is 300 Hz. Send a 300-Hz
signal into this filter, and 100% will reach the next stage.
Cutoff or corner frequencies are the
points where voltage is reduced to
0.707 of its initial strength. For a 100-500 Hz bandpass filter, the
cutoffs are 100 Hz and 500 Hz. Send a 100-Hz signal into this filter, and
only 71% will get through. This graphic was adapted from Schwartz and Andrasik (2016).
Caption: This diagram shows a 100-500 Hz bandpass superimposed on a 10-1000 Hz surface EMG spectrum. A 58-62 Hz notch filter is shown in red. The left and right borders of the 100-500 Hz bandpass are their cutoff frequencies.
Filter slope is the rate by which voltage is reduced as frequency
changes. The slope is expressed as a ratio of
decibels (logarithmic ratio of
signal strength) per octave (doubling of frequencies).
Filter slopes determine how rapidly an electromyograph excludes
frequencies outside the bandpass. Steeper slopes, expressed as higher
ratios, are needed when artifacts occupy frequencies near the cutoff
frequencies (50/60Hz noise).
How to Select the Right Bandpass
Electromyographs can process frequencies from 20-1000 Hz. Most signal power lies below 200 Hz; 80% of power ranges from 30-80 Hz.
There is minimal power above 500 Hz (Bolek, Rosenthal, & Sherman, 2016; Cram, 1991).
You can select a bandpass by a switch on stand-alone electromyographs and in the sensor housing or software on data acquisition systems.
Two choices are
narrow (100-200 Hz) or wide (20-500 Hz) bandpasses. The actual ranges will differ across manufacturers.
Consider a narrow
bandpass when there are high environmental noise levels or when nearby muscles contaminate the measurement of the muscle you're monitoring. A narrow bandpass will result in lower SEMG values than a wide bandpass (Peek, 2016). Select a wide bandpass when noise is acceptable.
Use a wide bandpass when artifact and muscle cross-talk are not problems. Under ideal conditions, a wide bandpass will represent SEMG activity more accurately.
Wide bandpasses can better measure proportional increases in muscle contraction than
narrow bandpasses (Sherman, 2002). Wide bandpasses measure SEMG activity more accurately because greater muscle contraction recruits more motor units and more
fast-twitch fibers, increasing the amount of signal
power in the higher frequencies.
While a wide bandpass can easily detect an increase in power in the 200-500 Hz
range, a narrow bandpass can be insensitive to this change because it attenuates frequencies over 200 Hz.
As a client moves from muscle relaxation to strong contraction in the diagram below, a wide bandpass shows an increase from 2 to 9 microvolts, while a narrow bandpass only shows an increase from 1 to 2 microvolts. Diagram adapted from Richard Sherman.
Below is a BioGraph ® Infiniti
display
of the raw and integrated SEMG signal and the power spectrum. Power shifts to the higher frequencies, increasing
the median frequency as the client contracts the frontales muscles.
Notch Filters Help Control Artifact
Bandpass filters reduce signal voltages above and below the cutoff frequencies.
The degree of signal attenuation depends on the filter slope. Engineers developed notch filters because artifacts can contaminate SEMG signals within the bandpass.
A notch filter rejects a narrow range of frequencies within the bandpass that contain the most energetic artifacts like 50/60Hz.
Practically, these signals may still contaminate measurements to some
degree. A notch filter is designed to reject a narrow range of
frequencies (containing artifact) admitted by the bandpass
filter. Fifty/sixty-Hz artifact removal is illustrated below by a recreated screen from Thought Technology Ltd.'s
Basics of Surface Electromyography Applied to Physical Rehabilitation and Biomechanics (2009).
Engineers design electromyographs with selectable notch filters for your region’s power line frequency, which
will generally be 50 Hz or 60 Hz. Power grid reversal 50 or 60 times a
second generates these frequencies. Your powerline frequency is the most potent source of environmental SEMG contamination.
Notch filters
significantly reduce signals from about 48-52 for 50-Hz power line frequencies and 58-62 Hz for 60-Hz frequencies.
Although a notch filter preserves
common-mode rejection in high-noise environments, there is no free lunch in biomedical engineering. Notch filters suffer two main limitations.
First, a 50/60Hz notch filter does not affect harmonics or multiples of
50/60Hz artifact (analogous to spreading ripples on a still pond) at 100/120 Hz, 150/180 Hz,
and 200/240 Hz.
Second, biological signals we want to measure may be eliminated along with artifact
since the bulk of the SEMG signal lies between 10 and 150 Hz (Andreassi, 2007). The decision to enable a notch filter should depend on the degree to which artifact would contaminate a recording. While
imperfect, notch filters strike a reasonable compromise between artifact
reduction and signal preservation.
Signal-to-Noise Ratio and Sensitivity
Skin preparation, differential amplification, bandpass selection, and
notch filters collectively determine the amount of artifact that contaminates the SEMG signal. The signal-to-noise ratio compares
SEMG and artifact
voltages. This specification should exceed 60 dB (1,000:1)at 50/60Hz for
adequate sensitivity or detection of weak signals.
Sensitivity allows an electromyograph to discriminate a low
SEMG value
during relaxation from background noise. State-of-the-art instruments
achieve ratios exceeding 100 dB (100,000:1).
Dummy Subjects Allow Us to Measure Total Noise
You can measure
total noise in your clinic or laboratory environment using a dummy subject.
Attach an EMG electrode cable to a circuit board
called a dummy subject. Its two 10-Kohm resistors (shown below) simulate the impedance of a
human subject. Simply insert the cable with the dummy subject
connected into an electromyograph channel in your encoder box and record the resulting activity in microvolts for 1 minute.
The average value calculated by the electromyograph will represent total noise: environmental and hardware-generated noise. The value should be under 0.25 μV so that you can distinguish muscle relaxation from background noise. Most muscles should be under 3 μV (Khazan, 2019).
Calibrate Your SEMG Sensor
Some data acquisition systems like Thought Technology Ltd.'s Infiniti system allow you to calibrate a specific SEMG sensor for a particular SEMG channel by
inserting a zeroing clip.
A zeroing clip shorts the sensor's inputs to allow
the system to adjust for offset errors (readings that are too high or low). For example, if the SEMG amplitude for a specific sensor in one particular
channel is 0.1 μV too high, the software will subtract 0.1 μV
from all its future readings. You will need to repeat calibration whenever you replace the original SEMG sensor.
Caption: Inserting a zeroing clip into the SEMG sensor to calibrate the equipment.
Rectification
The filtered SEMG signal is an AC waveform with mirror-image positive and
negative halves. If we simply added them together, the sum would be
0 since the positive and negative voltages would cancel each other.
A rectifier solves this problem by converting the filtered AC signal into
a positive DC signal.
Positive voltages can be added together.
Manufacturers use one of two circuits. A half-wave rectifier changes an AC signal’s upper or lower half into a positive DC signal. In
contrast, a full-wave rectifier converts both halves into a positive DC
signal. Graphic adapted from Schwartz and Andrasik (2016).
Raw, rectified, and integrated SEMG signals are shown below. Graphic adapted from AdvancerTechnologies.com.
Based on the Nyquist theorem, the sampling rate should be at least twice a
signal's highest frequency to accurately represent the original analog signal. For SEMG, the lowest sampling rate should be 1,000 samples per second since there is minimal SEMG power above 500 Hz. The lowest sampling rate should be 32 Hz (32 samples per second) for skin conductance, and temperature since their maximum frequencies are around 16 Hz.
Sampling at rates that are too slow results in aliasing, where a slower
beat frequency is produced by a
signal near the sampling rate (Lubar & Gunkelman, 2003; Teplan, 2002;
Thompson & Thompson, 2016).
A/D Converter Resolution
Bit Depth
Bit depth is the number of
voltage levels that an A/D converter can represent. Using the equation, N = 2X, where N is the number
of discrete values and X is the number of binary digits or bits.
An A/D converter with a bit depth of 16
calculates 65,536 values, and one with a bit depth of 24 calculates 16,722,216 values. The greater the bit depth,
the smaller the physiological changes detected.
For SEMG biofeedback training, 16-bit processing is
recommended, while for research, 24-bit resolution may be preferred (Bolek, 2013). For neurofeedback training,
16-bit resolution is preferred (Lubar & Gunkelman, 2003). Mind Media's NeXus-10 claims 24-bit resolution and a sampling rate of 2048 samples per second, while
Thought Technology Ltd.'s ProComp Infiniti claims 14-bit resolution and a
sampling rate of 2048 samples per second.
Integration Allows Us to Measure SEMG Voltage
Now that we have a rectified signal that has been converted into 0's and 1's, we can add SEMG voltage over time to determine whether a muscle is relaxed or tense.
The rectified SEMG signal is sent to an integrator to measure signal
amplitude in microvolts (μV).
The peak-to-peak method provides the largest estimate, equivalent to
the
energy between the positive and negative peaks of the original
AC waveform, which is 2 times the peak value.
Peak voltage is 0.5 of the peak-to-peak
value.
Root mean square (RMS) voltage is 0.707 of the peak value and 20% higher than the average voltage.
Average voltage is 0.637 of the peak value.
Conversion among these methods is straightforward. If peak-to-peak voltage is 20
μV,
peak voltage is 10 μV, root mean square voltage is
7.07
μV, and average
voltage is 6.37 μV.
EMG signal integration using the RMS method is shown below by a graphic adapted from Thought Technology Ltd.'s Basics
of Surface Electromyography Applied to Physical Rehabilitation and Biomechanics (2009).
Biomedical engineers mainly use the root mean square and average methods
to measure EEG and SEMG signals. Basmajian and DeLuca recommended the
root mean square method for quantifying the SEMG.
Below is a NeXus-10 ® BioTrace+ display of the raw and integrated 100-500 Hz SEMG signal generously provided by John S. Anderson.
SEMG Voltages Are Relative--Not Absolute
Whereas two different feedback thermometers should register the same room temperature, we can't assume the same for two electromyographs' measurement of SEMG voltage. Another elephant in the room is that SEMG amplitude is a relative measurement of skeletal muscle electrical activity because measurement depends on a surprising number of parameters, some controllable and others intrinsic to your client or electromyograph. Graphic Aleksandr_Kuzmin/Shutterstock.com.
The relativity of SEMG voltages characterizes all biofeedback modalities. Temperatures are no more absolute than SEMG values. When you attach a temperature probe to a client, factors like cold exposure before entering the clinic, stabilization period, sensor placement, task, and your relationship with the client can greatly affect temperature readings. All psychophysiological measurements are affected by hardware, environmental, procedural, and client factors. Experienced clinicians and researchers manipulate controllable factors and standardize those not controllable to increase measurement validity.
To compare SEMG values across session pre-baselines, standardize the electrodes, placement, skin-electrode impedance, baseline conditions, location in the room, bandpass and notch filter settings, and rectification and integration methods.
Consider Your Client's Adiposity When Interpreting SEMG Values
Adipose tissue filters the SEMG signal and reduces its amplitude.
Consider your client's subcutaneous fat when interpreting SEMG measurements from different sites on the same
individual or the same location between individuals (Shaffer & Neblett, 2010).
A reading of 5 μV obtained from one electromyograph could easily be
8 μV on another due to different bandpasses and integration methods. In contrast,
temperature measurements are absolute because two feedback thermometers
should register the same room temperature.
Now that we can integrate SEMG voltages, we must display this information to a client. We will need to choose an appropriate time constant, vertical scale, and display speed.
Time Constant
We have to decide how long to average them before displaying SEMG activity to a client. Software allows us to select a time constant that specifies the averaging period. Your choice of time constant will depend on the goal of SEMG training.
For neuromuscular rehabilitation, a short time constant (0.5 seconds)
that reveals minute, rapid changes could be valuable.
However, a longer time constant (2 seconds) that updates the display more slowly would be more appropriate for relaxation training.
Vertical Scale (Resolution)
We can automatically or manually adjust a biofeedback screen's
resolution (the degree of signal change) by changing its scale
(the range of values displayed).
The scale should provide sufficient information to enable
a client to recognize changes in signal strength and learn voluntary control. You can determine the vertical scale by examining the vertical axis range on the left of the display. In the BioGraph ® Infiniti
display below, a 0 - 10 μV scale is too narrow to show the two vigorous contractions, so it cuts off the 100-μV signal peaks.
In the BioGraph ® Infiniti
display below, a 0 - 150 μV scale accommodates the large voltage changes.
When a muscle is weak or when a client has reached a plateau, the SEMG signal may not show any change. An unresponsive display can be highly frustrating for a client. To remedy this problem, you can increase the display resolution to reveal smaller change increments. For example, instead of a vertical range of 0-5 μV, you might choose 0-2 μV. For SEMG training, a resolution of 0.1 to 0.25 μV may be desirable when signal voltage does not rapidly
change.
Select an Appropriate Display Speed
The display speed determines how quickly a screen refreshes.
We can determine the display speed by examining the horizontal timeline at the bottom of the screen, which runs from 12.46 to 13.16 (30 seconds).
A speed that is too slow (e.g., 2 minutes) is no longer real-time, making it harder for a client to associate sensations and strategies with physiological change. However, a speed that is too rapid (e.g., 10 seconds) can overwhelm a client with too much information, interfering with learning. Although clients and training goals differ, a Goldilocks zone may be around 30 seconds.
Display Options
Clinicians can now provide patients with an extensive selection of
informative and engaging displays for adults and children like those from BrainMaster Technologies.
Software provides analog, digital, binary, and
power spectral feedback.
Analog Feedback
Analog displays vary continuously in
proportion to change in signal strength.
Below is a NeXus-10 ® BioTrace+ screen generously provided by John S. Anderson. The flower opens as the client reduces SEMG activity.
The Thought Technology Ltd. Rehabilitation Suite screen below combines analog (0-100 μV) with digital feedback for absolute EMG in RMS for sensors A-D and EMG symmetry (A vs. B and C vs. D). The ball's position on the gorilla's back shifts in response to changes in left-right balance. Animations can increase the engagement of both adults and children.
Binary Feedback
A binary display shows whether the signal is above or below a threshold or goal.
Performance above or below a threshold can turn an animation, sound, or visual display on or off. A frequent goal in rehabilitation is to reduce asymmetry in the SEMG voltages of two muscles. In the Thought Technology Ltd. Rehabilitation Suite screen below, the red light turns off when asymmetry between left and right muscle sites falls below 35%.
The top BioTrace+ /NeXus-10 spectral display
shows a frequency range from 1-21 Hz (X-axis) with the amplitude in microvolts (Y-axis). Time progresses from the
back of the display to the front. The bottom raw EEG oscilloscope display of the same sample data shows
frequencies from 2-45 Hz. John S. Anderson generously provided this movie.
Shape Performance Using a Threshold
A biofeedback therapist routinely shapes patient performance by adjusting a
threshold or goal.
A therapist can manually select a threshold or allow a data acquisition system to calculate one based on client performance automatically. A therapist or software may choose a more challenging threshold after a client has succeeded more than 70% of the time.
Thresholds are depicted as orange lines on two vertical analog displays on the bottom right of the Thought Technology Ltd. Rehabilitation Suite screen below.
A
level detector decides
whether SEMG activity is above or below the threshold setting to adjust a
feedback display.
For example, tone feedback may disappear to signal reward when SEMG activity falls this value.
Artifacts
Artifacts are false values produced by the client's body (abnormal heartbeats) and actions (movement), the environment (50/60Hz), and hardware limitations (light leakage).
The recording of the effect of turning on a light switch was adapted from Peper, Gibney, Tylova, Harvey, and Combatalade (2008).
Caption:
Fluorescent light artifact recorded with a Triode™ electrode placed on the left forearm extensor muscles and a bandpass filter set to 400 wide. The artifact only occurred during an abrupt change in the electrical flow. Specifically, there was only a spike in the signal when the light was turned on or off.
A BioGraph ® Infiniti display of 60-Hz artifact is shown below.
Note the cyclical voltage fluctuations and the 60-Hz peak in the power spectral display.
ECG Artifact
ECG artifact results when the R-spike is
detected by sensors on the
upper limbs or trunk (a wide trapezius placement is particularly vulnerable).
Peper, Gibney, Tylova, Harvey, and Combatalade (2008) generously provided the
photograph below.
Caption: Wide upper trapezius electrode placement with two active electrodes placed on the center of the right and left upper trapezius muscles and the reference electrode placed on T1 of the spine.
The frequency range for this artifact is 0.05-80 Hz. ECG contamination is
seen in rhythmic meter fluctuations in the signal
(20-500 Hz) displayed below by a screen from Thought Technology Ltd.'s Basics of Surface Electromyography
Applied to Physical Rehabilitation and Biomechanics (2009).
Clinicians must visually
inspect the analog signal because digital values can conceal artifacts.
The recording below adapted from Peper, Gibney, Tylova, Harvey, and Combatalade (2008) shows
that a narrow bandpass (100-200 Hz) can minimize ECG artifact.
Caption: Effects of filter setting on ECG artifact recorded from the upper left trapezius Triode™ SEMG.
Below
is a BioGraph ® Infiniti display of ECG-artifact contamination of a trapezius SEMG recording. Note the
sharp upward deflection of the R-spike almost every second.
Radiofrequency Artifact
Radiofrequency (RF) artifact radiates outward like a cone from
the front of televisions and computer monitors (Montgomery, 2004).
The recording below adapted from Peper, Gibney, Tylova, Harvey, and Combatalade (2008) shows
the effect of a cell phone on the SEMG signal.
Caption: SEMG recording with Triode™ electrodes from the forearm extensors. There is a significant artifact from placing a cell phone near the Myoscan sensor and turning it on. The artifact disappears if the cell phone is further than 30 cm from the sensor.
Below
is a BioGraph ® Infiniti display of radiofrequency artifact contamination of a masseter SEMG recording. Note
the contamination at 60 Hz and its harmonics (120 Hz, 180 Hz, 240 Hz, 300 Hz, 360 Hz, 420 Hz, and 480 Hz) across
the EMG spectrum. Artifact amplitude fluctuates with the distance of the cell phone from the electrodes.
Electrostatic Artifact
Electrostatic artifact is produced by
static electricity, often encountered in low-humidity environments. This artifact can produce false voltages (see the center of the screenshot below).
Electrostatic discharges can also damage circuitry.
Movement Artifact
Movement artifact occurs when patient
movement displaces the electrode cable, shown below by a graphic adapted from Thought Technology Ltd.'s Basics of
Surface Electromyography Applied to Physical Rehabilitation and Biomechanics (2009).
You should suspect this
artifact
when you see unexpectedly elevated values or high-amplitude waveforms. Movement artifact occupies the region from 20 Hz and below (De Luca, 1997).
Note the four voltage spikes due
to electrode cable movement in the
BioGraph ® Infiniti display below.
Cross-Talk Artifact
Cross-talk artifact occurs when adjacent
SEMG activity contaminates your
readings. You can detect cross-talk by monitoring an adjacent muscle with
a second SEMG channel.
The
BioGraph ® Infiniti display below shows periodic masseter interference with recording from a frontales
placement.
Clinicians performing relaxation training may intentionally place electrodes across muscle fibers (frontalis
placement). They may place active electrodes on the belly of two separate muscles (bilateral upper trapezius placement).
Also, they may position electrodes on non-specific sites away from any muscle belly (wrist-to-wrist placement) (Cram,
1998). In these cases, they invite cross-talk to help clients recognize and reduce patterns of muscle
contraction that involve several muscle groups (Shaffer & Neblett, 2010).
Removing Artifacts
Data acquisition software has become increasingly sophisticated and can
now assist clinicians in artifact detection and removal. For example,
BioGraph ® Infiniti software
automatically detects artifacts and highlights them for visual
inspection and removal. After clinicians have removed contaminated data
segments, they can calculate accurate session statistics from the
remaining data.
Tracking Tests
You can determine whether a SEMG display
mirrors your client's muscle contraction by performing a tracking test, during which you instruct your client to contract and then relax the monitored muscles briefly.
For example, for a frontal placement over the
forehead, you might instruct your client, "Please gently tighten the muscles
in your forehead for a few seconds and then allow them to relax." The
integrated SEMG signal should increase during the contraction phase
and decrease during the relaxation phase.
Below is a BioGraph ®
Infiniti SEMG tracking test display in which the client briefly contracts and relaxes the frontales muscles.
A tracking test checks the entire signal chain's integrity from the three individual sensors to the encoder and the correct software selection of input channels.
Normal Values
SEMG values depend on muscle size, sensor placement, narrow (1.5-2 centimeters) or wide (6 centimeters), bandpass, narrow (100-200 Hz) or wide (20-500 Hz), degree of adipose tissue, client position, and muscle activity (Shaffer & Neblett, 2010). Graphic retrieved from mTrigger.com.
During a resting baseline, typical values lie below 3 μ V for small-to-moderate-sized muscles and below 5 μV for large muscles (Khazan, 2019).
Clinicians may attempt to down-train SEMG activity to below 1 μV (Khazan, 2013, pp. 45, 143).
Computers in Biofeedback
Inegrating computers into biofeedback has allowed more powerful
data analysis, displays, and recordkeeping. Fast Fourier Transformation provides power
spectral analysis of complex EEG, SEMG, and heart rate variability (HRV) signals. Z-score calculations allow neurofeedback training that compares performance to a normative database. Finally, computers also
enable clinicians to administer and score psychological tests like the
Minnesota Multiphasic Personality Inventory-2 (MMPI-2) and Tests of Variables of Attention (TOVA).
The downside of computer use is that they are sources of 50/60Hz artifact
and radiofrequency artifact, and can present a shock hazard without
proper electrical isolation.
Drug Effects
Since over-the-counter, prescription, and social drugs can affect SEMG values, identify all medications that clients are taking during theinitial assessment and use this list to interpret readings. Muscle relaxation and reduced muscle spasms may reduce EMG.
Glossary
active electrode: the electrode placed over a target muscle like the
masseter.
adhesive collars: self-adheringrings that
secure SEMG electrodes to the skin.
aliasing: an artifact created during analog-to-digital conversion of a continuous
signal by a slow sampling rate.
amplitude: the strength of the EMG signal measured in microvolts.
analog display: a displaythat continuously represents
biofeedback signal amplitude over time.
analog-to-digital (A/D) converter: an electronic device that converts continuous signals to discrete
digital values.
average voltage: 0.637 of the peak voltage.
bandpass: a filter thatcombines a low-pass
filter and high-pass filter.
bandpass filter: an electronicdevice that
combines a low-pass filter and high-pass filter to transmit frequencies within a specific range and attenuate
those outside that range, such as a 100-200 Hz bandpass filter.
bandwidth: the difference between a filter's lower and upper cutoff frequencies. The bandwidth
of a 100-200 Hz bandpass filter is 100 Hz.
beat frequency: a falsesignal produced during analog-to-digital
conversion by frequencies near the sampling rate.
binary display: displays that show whether the signal is above or below a threshold.
bipolar recording: a recording method that uses two active electrodes and a
common reference.
bit depth: the number of voltage levels that an A/D converter can discern. A resolution of 16 bits
means that the converter can discriminate among 65,536 voltage levels.
center frequency: the frequency located at the middle of a bandpass filter. For example, 150 Hz is the
center frequency for a 100-200 Hz bandpass filter.
common-mode rejection ratio (CMRR): the degree by which a differential amplifier boosts signal
(differential gain) and artifact (common-mode gain).
contraction phase: the segment of a SEMG tracking test where the patient is instructed to contract a
monitored muscle.
corner or cutoff frequencies: the frequencies at which voltage is reduced to .707 of its center
frequency strength. The corner frequencies are 100 Hz and 200 Hz for a 100-200 Hz bandpass filter.
cross-talk artifact: in SEMG biofeedback, interference with recording at one site by muscle action
potentials generated by another muscle.
decibel: the logarithmic ratio of two measurements using the same unit of intensity or power. A ratio of 60
dB is 1000 to 1, and 100 dB is 100,000 to 1.
differential amplifier: an electronic amplifier containing two single-ended amplifiers that are 180 degrees out of phase that amplifies the difference between two input voltages.
differential input impedance: the opposition to an AC signal entering a differential amplifier as it is
dropped across a resistor network.
digital display: the numeric display of signal amplitude, for example, a temperature display of 92° F
(33.3° C).
disposable electrodes: surface electrodes that are discarded after use to
prevent transmission of infection.
dummy subject: deviceconsisting of two resistors that simulates
the skin-electrode impedance of a human subject used to measure noise generated by the environment and
instrument. For example, a SEMG dummy electrode.
ECG artifact: contamination of EEG or EMG signals by the 0.05-80-Hz components of the ECG signal generated by the heart.
electrode: a sensor that detects biological signals like the EMG by converting
an ionic potential into an electrical potential.
electrode gel: an electrically-conductance solution used to detect biological potentials (e.g., ECG, EEG, and EMG) from the skin surface and standardize electrodermal measurement.
electrolyte: an electrically-conductive medium that contains free ions.
electrostatic artifact: artifactual voltages in EEG and EMG recording produced by static
electricity.
epoch: a signal sampling period, for example, a 30-second sample of EMG
activity.
epoch number: a location in a session's record. For example, epoch 52 is the
52nd 1-second epoch.
Fast Fourier Transform (FFT) analysis: an algorithm thatdecomposesa complex waveform into its component frequencies.
filter: an electronic circuit that removes or enhances signal components.
floating skin electrode: an SEMG electrode that contains a recessed reservoir for electrode gel.
frequency (Hz): the number of complete cycles that an AC signal
completes in a second, usually expressed in hertz.
frontales placement: a horizontal placement of SEMG over the two frontales muscles. The actives are
centered over each eye with the reference above the nose.
full-wave rectifier: an electronic device that changes an AC signal's upper and lower halves into a positive DC signal.
gel-bridge artifact: a short circuit created when electrode gel smears and creates a bridge between
closely-spaced active and reference electrodes, resulting in abnormally low readings.
half-wave rectifier: an electronic device that changes an AC signal's upper or lower half into a positive DC signal..
high-pass filter: an electronic device that selects frequencies above a cutoff, for example, a 100 Hz
high-pass filter.
impedance (Z): the complex opposition to an AC signal measured in Kohms.
impedance meter: a device that measures skin-electrode impedance.
impedance test: the automated ormanual measurement of skin-electrode
impedance.
integrator: a circuit that calculates rectified signal voltage.
interstitial fluid: the fluid between cells through which biological signals travel via volume
conduction.
level detection: a device that decides whether the signal voltage matches the threshold setting to
activate a feedback display.
line current artifact: the frequencies at 50/60Hz and their harmonics produced
by AC devices can contaminate EEG, ECG, or SEMG recordings. A fluorescent light can create false SEMG
voltages.
logarithmic display: the display of the logarithm of a biofeedback signal to feature greater resolution
at the lower end of the scale.
low-pass filter: an electronic device that selects frequencies below a cutoff, for example, a 200 Hz
low-pass filter.
microvolt: a unit of amplitude (signal strength) that is one-millionth of a volt.
millivolt: a unit of amplitude (signal strength) that is one-thousandth of a volt.
monopolar recording: recording method that uses one active and one reference electrode.
movement artifact: the high-amplitude signals produced by client movement that displaces the electrode
cable that contaminates EEG and SEMG recordings.
notch filter: a circuit that suppresses a narrow band of frequencies, such as
contamination produced by line current (50/60Hz artifact).
Nyquist-Shannon sampling theorem: position that the perfect reconstruction of an analog signal requires sampling at two
times its highest frequency. A signal whose highest frequency is 1000 Hz should be sampled 2000 times per
second.
octave: a 2:1 ratio between two frequencies. Filter slope is specified in dB per octave.
operational amplifier (op-amp): a high-gain DC amplifier that uses external feedback to perform
computations like addition, subtraction, and averaging on biological signals.
passband: the frequencies transmitted by a bandpass filter, for example,
100-200 Hz.
peak frequency: the highest amplitude frequency in a signal.
peak voltage: 0.5 of the peak-to-peak voltage.
peak-to-peak voltage: the energy contained between the positive and negative peaks of the original AC
waveform, which is 2 times peak voltage.
polarization: charge segregation produced by chemical reactions producing separate regions of positive and negative charge where an
electrode and electrolyte make contact, reducing ion exchange.
power spectral analysis: the measurement ofsignal amplitude across
its frequency range using a Fourier Transform algorithm.
preamplifier: the first amplification stage that prepares an electronic signal for additional
processing. A preamplifier can be built into an EEG or SEMG electrode housing to reduce signal loss.
radiofrequency (RF) artifact: the frequencies from 3 Hz to 300 GHz that contaminate EEG and SEMG recordings.
Cell phone transmissions and computer monitors can produce spuriously raise SEMG amplitudes.
R-spike: an initial upward deflection in the QRS complex of the ECG.
rectifier: an electronic device that changes an AC signal into a positive DC signal.
reference electrode: the electrode placed over a less electrically active site like the mastoid
process behind the ear or the spine.
relaxation phase: the segment of a SEMG tracking test where the patient is instructed to relax a monitored
muscle.
resolution: the degree of detail in a biofeedback display (0.1 μV) or the number of voltage levels
that an A/D converter can discriminate (16 bits or discrimination among 65,536 voltage levels).
root mean square (RMS) voltage: 0.707 of the peak voltage.
scale: the range of displayed values, for example, a SEMG scale of 0-5 microvolts.
sampling rate: the number of times per second that the voltage is measured.
SEMG scanning: bipolar recording technique that involves the sequential monitoring of a series of
muscle sites using two active post electrodes and a common reference.
sensitivity: the ability of a biofeedback device to detect weak signals that depends on its
signal-to-noise ratio. The ability of an electromyograph to discriminate a 0.1 μV voltage from environmental
and instrument noise.
shaping: an operant procedure where a clinician rewards successive approximations of a target behavior.
Praise and auditory and visual feedbackfor progressively lower trapezius SEMG
levels.
silver-silver chloride electrode: an electrode fabricated
from a combination of silver and silver-chloride to reduce electrode noise.
signal-to-noise ratio: the ratio betweensignal and artifact voltages
that determines a biofeedback instrumen's sensitivity.
single-ended amplifier: an electronic amplifier that amplifies an input voltage.
slope: the rate by which voltage is reduced as frequency changes that is expressed as a ratio of
decibels per octave, such as a 20 dB/octave slope.
stratum corneum: the outermost epidermal layer that is abraded during skin preparation for EEG and SEMG
biofeedback.
telemetry: the remote monitoring and transmission of information. A biofeedback encoder measures
physiological activity and transmits these data to a computer for analysis.
threshold: the signal value that is a training goal, for example, 2 μv.
time constant: the periodduring which a biological signal is
averaged before it is displayed, for example, 5 seconds.
tracking test: a check whether a biofeedback display mirrors client behavior. SEMG amplitude should
rise when your client contracts a monitored muscle.
transducers: devices that transform energy from one form to another. Electrodes convert ionic
potentials into electrical potentials.
volume conduction: the movement of a biological signal through the interstitial fluid to the skin
surface.
zeroing clip: a device that shorts a sensor's inputs to allow a data acquisition system to adjust for
offset errors, for example, EEG or SEMG zeroing clips.
Test Yourself
Click on the ClassMarker logo to take 10-question tests over this unit without an exam password.
REVIEW FLASHCARDS ON QUIZLET
Click on the Quizlet logo to review our chapter flashcards.
Visit the BioSource Software Website
BioSource Software offers Human Physiology, which satisfies BCIA's Human Anatomy and Physiology requirement, and Biofeedback100, which provides extensive multiple-choice testing over BCIA's Biofeedback Blueprint.
Essential Skills
1. Explain the EMG and biofeedback to a client.
2. Explain skin preparation and electrode placement to a client, and obtain permission to monitor her.
3. Explain how to protect the client from infection transmitted by the sensor.
4. Identify active- and reference-electrode placements using a marking pencil for bilateral cervical paraspinal,
frontalis, masseter, sternocleidomastoid, and trapezius sites.
5. Demonstrate skin preparation and electrode placement.
6. Measure electrode impedance for each active-reference electrode pair and ensure that impedance is sufficiently
low and balanced.
7. Perform a tracking test for your placement, instructing the client to contract and then relax the monitored
muscle.
8. Identify common artifacts in the raw EMG signal, including 50/60Hz, bridging, ECG, loose electrode, movement,
and radiofrequency, and explain how to control for them and remove them from the raw data.
9. Demonstrate how to instruct a client to utilize a feedback display.
10. Demonstrate a surface EMG biofeedback training session, including record keeping, goal setting, site
selection, bilateral and unilateral recording, bandpass selection, baseline measurement, display and threshold
setting, coaching, and debriefing at the end of the session.
11. Demonstrate how to select and assign a practice assignment based on training session results.
12. Evaluate and summarize client progress during a training session.
Assignment
Now that you have completed this module, find the manual for your
electromyograph, electroencephalograph, or data acquisition system. Find
the following information:
Bandpass:
Common-mode rejection ratio:
Input impedance:
Integration method:
Signal-to-noise ratio:
Perform the behavioral test discussed in this module. Place a set of SEMG
electrodes on the palmar surface (underside) of your arm, and then watch
the display as you contract and relax these muscles.
If you have a portable electromyograph, insert an electrode and move it
about the room to detect the lowest and highest zones for 50/60Hz noise.
References
Alleyne, J., & Dopico, A. M. (2021). Alcohol use disorders and their harmful effects on the contractility of skeletal, cardiac and smooth Muscles. Advances in Drug and Alcohol Research, 1, 10011. https://doi.org/10.3389/ADAR.2021.10011
Amar, P. B., McKee, M. G., Peavey, B. S., Schneider, C. J., Sherman, R.
A., & Sterman, M. B., (1992). Standards and guidelines for biofeedback
applications in psychophysiological self-regulation. Association for Applied Psychophysiology and Biofeedback.
Andreassi, J. L. (2007). Psychophysiology: Human behavior and physiological
response (5th ed.). Lawrence Erlbaum and Associates, Inc.
Basmajian, J. V. (Ed.). (1989). Biofeedback: Principles and practice for
clinicians. Williams & Wilkins.
Bolek, J. E., Rosenthal, R. L., & Sherman, R. A. (2016), Advanced topics in surface electromyography. In M. S. Schwartz and F. Andrasik (Eds.). Biofeedback: A
practitioner’s guide (4th ed.). The Guilford Press.
Cram, J. (1991). Clinical EMG for surface recordings: Vol. 2. Clinical Resources.
Griffin, C. E., 3rd, Kaye, A. M., Bueno, F. R., & Kaye, A. D. (2013). Benzodiazepine pharmacology and central nervous system-mediated effects. The Ochsner Journal, 13(2), 214–223. PMID: 23789008
Gruenthal, M., Mueller, M., Olson, W. L., Priebe, M. M., Sherwood, A. M., & Olson, W. H. (1997). Gabapentin for the treatment of spasticity in patients with spinal cord injury. Spinal Cord, 35(10), 686–689. https://doi.org/10.1038/sj.sc.3100481
Katzung, B. G., & Trevor, A. J. (2020). Basic and clinical pharmacology (15th ed.). McGraw-Hill Professional.
Khazan, I. (2013). The clinical handbook of biofeedback: A step-by-step guide for training and practice with mindfulness. John Wiley & Sons, Ltd.
Kubala, T. (2013). Electricity 1: Devices, circuits, and materials (10th ed.). Cengage Learning.
Lubar, J., & Gunkelman, J. (2003). Neurometrics, neurotherapy, and
clinical practice. Workshop presented at the 34th annual Association for
Applied Psychophysiology and Biofeedback convention, Jacksonville,
Florida.
Montgomery, D. (2004). Introduction to biofeedback. Module 3:
Psychophysiological recording. Association for Applied Psychophysiology and Biofeedback.
Nilsson, J. W., & Riedel, S. A. (2019). Electric circuits (11th ed.). Pearson
Prentice-Hall.
Peek, C. J. (2016). A primer of traditional biofeedback instrumentation.
In M. S.
Schwartz, & F. Andrasik (Eds.). (2016). Biofeedback: A practitioner's guide (4th ed.).
The Guilford Press.
Peper, E., Gibney, K. H., Tylova, H., Harvey, R., & Combatalade, D. (2008). Biofeedback mastery: An
experiential teaching and self-training manual. Association for Applied Psychophysiology and Biofeedback.
Stern, R. M., Ray, W. J., & Quigley, K. S. (2001). Psychophysiological
recording (2nd ed.). Oxford University Press.
Tassinary, L. G., Cacioppo, J. T., & Vanman, E. J. (2007). The skeletomotor system: Surface electromyography.
In
J. T. Cacioppo, L. G. Tassinary, & G. G. Berntson, (Eds.). Handbook of
psychophysiology (3rd ed.). Cambridge University Press.
Zajicek, J., Fox, P., Sanders, H., Wright, D., Vickery, J., Nunn, A., Thompson, A., & UK MS Research Group (2003). Cannabinoids for treatment of spasticity and other symptoms related to multiple sclerosis (CAMS study): Multicentre randomised placebo-controlled trial. Lancet,362(9395), 1517–1526. https://doi.org/10.1016/S0140-6736(03)14738-1
 300dpi_Premium Package_Best Quality indoboy fiverr.jpg)
 300dpi_Premium Package_Best Quality indoboy fiverr.jpg)
.jpg)
 Artifact.jpg)
