The research and development leading to the above described vibration test was funded by the European Research Council (ERC, ADG 294678) and the German Research Council (grant, SFB665). Thanks go to the Max-Delbr&#252;ck-Center technical and support staff, Mr. R. Fischer, B. Neumann, and Mr. M. Pflaume, who provided invaluable assistance with the project.

The testing protocol was approved by the Charit&#233;-Universit&#228;tsmedizin Ethics Committee.
1. Vibration Detection Threshold (VDT)
<ol>
	<li>Device and Testing Protocol Assembly&#160;&#8211; Pretesting
	<ol>
		<li>Assemble the components of the device according to Figure 1A. Place a smooth-surfaced board (40 cm x 80 cm) on a table. Place the brass bar on the board.</li>
		<li>Connect the piezoelectric actuator (vibration stimulator) to the controller unit.</li>
		<li>Connect the response box and the monitor device to the data acquisition system (see supplemental code file).</li>
		<li>Connect the data acquisition system to a computer (or laptop), and to piezo actuator controller unit.</li>
		<li>Screw the custom-made stimulating probe to the moving part of the piezoelectric actuator (for specifics of the probe see Materials).</li>
		<li>Mount the piezoelectric actuator with the probe on the balanced brass bar.</li>
	</ol>
	</li>
	<li>Testing Protocol
	<ol>
		<li>Script a test protocol that implements the two interval forced-choice and transformed-rule up and down method. See supplemental code file for an outline of the script.</li>
		<li>Construct the waveform of the vibration stimulus as a sinusoidal wave and specify stimulus duration, rise and fall characteristics.
		<ol>
			<li>Open software (e.g., LabChart). Select Setup &#62; Stimulator.</li>
			<li>Choose a custom waveform and configure the stimulator options. Create 2 stimulus waveforms pertaining to each interval (stim1 and stim2). 
			NOTE: The waveform stim1 is composed of 3 parts: a delay of 4 sec, followed by a sinusoidal wave of 1.8 sec, and a delay of 1.8 sec (no stimulus). The waveform stim 2 is composed of 3 parts: a delay of 4 sec, followed a delay of 1.8 sec (no stimulus), and by a sinusoidal wave of 1.8 sec.</li>
			<li>For the sinusoidal waveform, create new variable parameters for frequency and amplitude. Modify the sinusoidal wave function by inputting the following functions to the rise and fall. 
			Rise waveform: (1-e-bt)&#8729;Amplitude&#8729;sine(frequency), b = 9.1 
			Fall waveform: (e-bt)&#8729;Amplitude&#8729;sine(frequency), b = 9.1</li>
			<li>In the data panel of the lab chart, create the set of 35 voltage outputs pertaining to the 35 amplitude intensities (or levels) of the vibration stimulus. See Table 1.</li>
		</ol>
		</li>
		<li>Set the starting/default amplitude of the vibration stimulus for the tested vibration frequency in the macro script for the testing procedure (see supplemental code file section).</li>
	</ol>
	</li>
	<li>Preparation and Training of Subjects &#8211; Testing Session
	<ol>
		<li>Inform test subjects about the testing procedure and have them sign a written consent form. To ensure anonymity and fulfill data protection requirements, assign each participant a number.</li>
		<li>Seat the subjects comfortably in a quiet room at temperatures between 20-30 &#176;C. Instruct them about the test in a simple and clear manner so that the subject knows what to expect during the testing.</li>
		<li>Place the arm of the subject on the board. Pad the little finger with medical dough to minimize movement. Place the brass bar on the board to position the probe on the little finger of the tested hand just below the nail bed. Ensure proper contact between the probe and the skin and adjust the position of the probe to a horizontal position using the water-level. Avoid skin contact with the edges of the circular flat probe. 
		NOTE: This ensures that the flat surface of the probe applies around 30 g (0.3 N) to the skin surface. Sharp edges can lead to decreased detection thresholds. The heavy mass of the brass bar prevents the transmission of distracting oscillations from the surroundings to the device and minimizes dissipation of the applied sine wave.</li>
		<li>Prior to testing, get the participants familiarized with the setup. Depending on the testing frequency, present both an easy (level 23) and a hard (level 7) to perceive vibration stimulus by varying the amplitude until the subject perceives a vibration to ensure that the experimental procedure is understood.</li>
		<li>If necessary, reset the starting (default) amplitude at the selected frequency so that the subject can easily detect the stimulus when the testing protocol starts (see 1.2.3).</li>
		<li>Minimize interactions between subject and examiner during the test.</li>
		<li>Start the test by running the script that uses the two-alternative forced-choice procedure with the up-down adaptive method22. 
		NOTE: Actions in 1.3.7-1.3.12 are automated by the program script.</li>
		<li>In each trial, randomly administer a vibration stimulus during one of the two intervals that are visually indicated to the test subject as &#34;1&#34; and &#34;2&#34; on the screen of the monitor (Figure 1B). Have the subject indicate if the first or second sequential intervals contained the vibration stimulus by pressing one of two buttons &#34;1&#34; or &#34;2&#34; on the response box. Let the subject make a guess if he or she is not sure when the stimulus is presented. 
		NOTE: The forced-choice technique requires that the subject responds even when the vibration is not perceived.</li>
		<li>In a trial series, which consists of 6 to a maximum of 9 single trials, repeat the same vibration stimulus at one amplitude level at least six times consecutively. If the responses are all correct, reduce the stimulus intensity level (down rule) for the subsequent trial series.</li>
		<li>Based on the decision rule of the adaptive method (logical operators in script), grant the subject more trials to the same stimulus intensity if the subject makes errors in a trial series. Reduce the stimulus intensity if the stimulus is correctly identified in at least 5 trials, and incorrectly in less than 2 trials.</li>
		<li>Increase the stimulus level if the subject makes the 2 incorrect responses in a trial series; or, more than one incorrect response and fewer than 5 correct responses.</li>
		<li>Document the change in the direction of stimulus intensity, as reversal point. Change the stimulus intensity according to reversal point number: prior to the third reversal point by 4 intensity levels; at the 3rd reversal point by 2 intensity levels; else by 1 level (for more detail see supplemental code file and Figure 4B).</li>
		<li>End the testing when the subject completes a total of 8 reversals.</li>
		<li>Calculate the VDT by taking the median of the stimulus amplitude value of the last 6 reversals.</li>
	</ol>
	</li>
</ol>
2. Tactile Spatial Acuity Test
<ol>
	<li>Determine tactile acuity with a two-alternative forced choice grating orientation test using the Tactile Acuity Cube (TAC). The TAC is comprised of 6 sides each containing a grating (bar and groove) whose widths are 0.75 mm, 1.25 mm, 1.75 mm, 3.0 mm, 4.5 mm, and 6.0 mm.</li>
	<li>Seat subjects in a quiet room at temperatures between 20-30 &#176;C and instruct on the task.</li>
	<li>During the experiment, blindfold the test subjects using shielded eyeglasses. Place the dominant hand on a table with the palmar surface facing up.</li>
	<li>On each trial, apply the TAC to the finger pad at one of two grating orientations: vertically (parallel direction) or horizontally (transverse direction) aligned to the long axis of the finger. Randomly choose the order of the grating orientation for each trial.</li>
	<li>Apply the gratings of the TAC for 2 sec to the finger pad of the index finger so that the cube exerts its whole weight on the finger (233 g). Avoid pressing the TAC on the finger pad.</li>
	<li>Ask the subjects to determine the orientation of the alignment before the cube is removed from their finger.</li>
	<li>Avoid movement of the participant&#39;s finger because it might provide a cue to the orientation. Discard the trial if the experimenter senses that the finger has moved. 
	NOTE: Beware that small finger movements might not be detected by the experimenter in the procedure.</li>
	<li>Employ a two-down and one-up adaptive method in the staircase algorithm.</li>
	<li>Start with the largest grating, 6.0 mm.</li>
	<li>Decrease the grating width after two correct identification of the orientation (correct response).</li>
	<li>Test the next, smaller width and continue with the stepping rule until the subject makes an incorrect response and document the grating width as a reversal point.</li>
	<li>Increase the grating width stepwise again until the two orientations of a width are determined correctly again.</li>
	<li>End the test after completion of thirteen reversals.</li>
	<li>Calculate the tactile grating orientation threshold by taking the median of the grating widths of the last 10 reversals.</li>
</ol>

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The authors have nothing to disclose.

The techniques used to evaluate VDT vary as to device specifications, hardware, and testing protocols. The International Organization for Standardization specifies the methods and procedures to analyze and interpret vibrotactile thresholds including recommendations for the various components of a vibrometer (ISO 13091-1 and 226,27) The described testing system abides to the relevant ISO recommendations for testing frequency range (4-125 Hz), method (variant of up-down staircase and forced choice), probe size (smooth edged flat and circular), and testing at room temperature. The setup can be equipped with a sensor determining the probe&#39;s position and displacement and optionally can be equipped with a firm surround isolating the vibration stimulus.
The benefits of the described device lie in the following characteristics: availability and easy assembly of the components; adjustable frequency range 1-500 Hz; wide amplitude range (5 nm-90 &#181;m displacement); multiple probe sizes; programmability of the vibration stimulus size and duration; and versatility to psychophysical testing protocol with various adaptive method. Although portable systems are available28, the setup is transportable and has been used in various settings such as schools, research institutes, and hospitals. Research centers, however, should have a well-equipped workshop to fabricate additional tools and devices needed for the experimental setup. There are commercially assembled and ready for use systems e.g. case IV or Medoc, but these systems are configured to deliver only a limited set of stimuli. In contrast, our system can be used to implement a very wide range of stimulation protocols that are only limited by the specifications of the piezo element used. Due to the brass bar size, it is not feasible to test other areas than the fingers and toes. Our device does not use a surround to limit the stimulation to the tested skin area and it is likely that a wider area larger than the actual skin to probe contact is stimulated. Lastly, the procedure requires attention from the subject for a longer period of time, 15 min on average. Usually the first 3 reversals have a larger change in intensity levels (4 levels) and once completed the threshold most likely lies within this range of 4 intensity levels. With progress of the test, fine determination of the threshold is made. There are other stopping criterion which could be implemented and integrated in the adaptive procedure described elsewhere29.
Several device parameters that can affect vibration detection threshold are adjustable in the setup17. These include device parameters such as the contact area of the probe to skin, use of surround encircling the probe to limit the area of stimulation, the selected vibration frequency, wrist posture, and psychophysical testing algorithm. Psychophysical testing algorithms incorporating the method of limits and the staircase method have been used to determine the VDT and there is no consensus favoring a particular method. Vibrotactile thresholds do vary depending on the psychophysical testing method used20. Estimates of VDT for 125 Hz are reliably obtained in the range of 50 to 600 nm using the outlined adaptive method in the psychophysical protocol and are in accord with VDT from other studies5,23. Furthermore; scripts for different adaptive methods can be easily developed and integrated to execute the psychophysical test for vibration detection threshold25. The adaptive procedure we described for the determination of the vibration threshold assures that for each stimulus strength level the subject&#39;s correct responses are over 76% correct in order to move down the stimulus strength staircase22. Since there are only 2 possible responses, a series of lucky guesses could erroneously alter threshold measurements, especially at low stimulus levels. For this reason we added a modification which is a series of trials at each vibration level in order to minimize such errors. Subjects who show no consistency during trial series &#8211; deviations of more than 4 levels between reversal points &#8211; are typically excluded from the study. The skin to probe contact and how the probe behaves while stimulating the skin are very important to any psychophysical experiment on touch sensation30,31. The piezo actuator is equipped with a compact strain gauge sensor (SGS) has a relatively high bandwidth (up to 3 KHz) and a very good resolution with good repeatability (0.1% of nominal displacement). Therefore, the piezoelectric device has high reliability characteristics especially when it comes to fine indentation even at high static loads. The motion is straight because the piezo (PICMA stack piezo linear actuator) we use is embedded in a guide and this ensures no lateral motion. In addition, the servo-controller can automatically compensate for varying loads or forces.
The assessment of tactile acuity we described here relies on manual delivery of tactile stimuli. The test requires careful application of the stimulus to produce perpendicular deformation of the skin and no shearing distortions that might provide cues to the subject. We choose a slightly different procedure for the determination of tactile spatial acuity and vibrotactile threshold. We did not elect to use a larger step size change initially using the TAC because it is equipped with few levels for grating width (6 levels) and these are not of constant or fixed size but vary from larger to smaller step size change. The change in size of the grating widths between the first 3 levels is 1.5 mm, between the 3rd and 4th level 1.25 mm, and 4th to 6th 0.5 mm. The subject&#39;s performance in the grating orientation task is affected by the depth of the indentation caused by the tactile stimulus, the force applied, and finger the size of the finger32,33. There are other alternatives to the tactile acuity cube: JVP domes, and the two-point discrimination task. The JVP domes are other alternatives to the tactile cube. The advantages are that the JVP domes have 8 grating widths ranging from 0.35 mm to 3.0 mm. JVP domes can be utilized to assess tactile spatial acuity of the tongue and lips8, whereas 2 point discrimination task does not rely on identifying grating orientation and is not a valid measure for tactile spatial acuity34. Recently, difficulties associated with manual testing have been improved through the introduction of an automated tactile acuity system that applies a forced-choice paradigm to determine tactile threshold for grating orientation35.
The methods for obtaining the vibrotactile and tactile acuity thresholds we described have been employed to screen individuals for touch-related traits3,7,9. In a study conducted by our group, we showed that touch traits are heritable, and that certain genetic lesions causing hearing impairment also influenced touch sensitivity7. Moreover, touch sensitivity can be assessed under different experimental condition such as water-induced fingertip wrinkling9. It will be important to make quantitative measurements of VDT and tactile acuity in patients with possible function altering mutations in genes involved in regulating mechanoreceptor sensitivity3,7. Indeed there has been a recent surge in the identification of genes directly involved in this process like Piezo2 and STOML31,13,14,36,37, and rapid progress in this area is sure to identify new genes that regulate touch. The influence of genetic variants in such &#34;touch&#34; genes will be ideally tested in genotyped patients with quantitative psychophysical methods like those described here.

Specialized mechanosensory receptors in the skin mediate the perception of vibration and grating orientation. Each mechanosensory receptor type is tuned to detect distinct features of tactile stimuli1,2. This property provides the psychophysical basis for a differentiated assessment of mechanosensory function by using tests that deliver simple sinusoidal oscillations (vibration) or fine gratings. It is known that psychophysical thresholds for vibration perception are lower for high than low frequency vibration3,4. Two types of rapidly adapting mechanoreceptors associated with the Meissner and Pacinian corpuscles primarily detect low (10-40 Hz) and high (100-200 Hz) frequency vibration stimuli, respectively5. It is thought that the psychophysical thresholds for vibration perception rely to a considerable extent on the activation of these two sets of mechanoreceptors at their best frequencies6,7. Tactile spatial acuity is tested by the grating orientation task to determine the finest grating whose orientation can be discriminated by a subject7-9. Merkel cell-neurite complex afferents are essential for detecting grating orientation8,10. Interestingly, there has been considerable recent progress in our understanding of the molecular basis of how mechanoreceptors detect extremely small tactile stimuli11. Mechanosensitive ion channels like Piezo2 and modulators like STOML3 have been directly implicated in the detection of fine tactile stimuli in touch receptors12-15. There are already human patients identified with function altering mutations in the Piezo2 gene and it will be important to test whether such patients have touch deficits16. 
The determination of the vibrotactile threshold relies on the delivery of a vibrating stimulus to the skin. Vibration detection thresholds vary with the vibration frequency. Vibration frequency to detection threshold curves were described by von Bekesey, Verillo, and Bolanowski, among many others, from the 1930s up to the 1990s5,17. The devices in the early days were based on shakers and power amplifiers and several devices have been commercialized and there are a lot of variation as to choice of stimulator (testing frequency), the diameter of the probe in contact with skin, the use of a surround to limit the stimulation to one area of the skin, and the testing protocol that determines the threshold (see the following references for more detailed insight on the device and testing features)18-20. Most devices usually test one site; however, there are new devices that use vertical displacement stimulators equipped with 2 probes to deliver vibratory stimuli with separation ranges that can be varied21. Also detection thresholds are measured based on vibration frequency or intensity discrimination with continuous stimulation; or with intermittent vibration stimuli with or without a masking stimulus. Therefore, we recommend that the reader be aware of the plethora of developments in this field. 
Here, we describe the components of the device and a step-by-step guide on how to conduct psychophysical tests to estimate vibration detection threshold in human subjects. We then show how to assess tactile acuity manually by using the tactile acuity cube. We use a two-interval, forced-choice paradigm: the stimulus is always presented during one of two intervals and the subject has to indicate which interval has the vibration stimulus. We employ a transformed-rule up and down as the adaptive method that executes a threshold search by changing the stimulus intensity based on the performance during the test. Both psychophysical protocols can be used by investigators as a screening tool for evaluation of alterations in touch sensitivity.

<table border="0" cellpadding="0" cellspacing="0" width="1381">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="145">
			<td height="145" style="height:145px;width:115px;">Piezo actuator</td>
			<td style="width:83px;">Physik Instrument, Germany</td>
			<td style="width:221px;">P-602.1L</td>
			<td style="width:411px;">The linear piezoelectric actuator, with integrated position sensor and motion amplifier, contains a piezoceramic material that elongates and contracts when voltage is applied. The piezoelectric actuator travels up to100 &#181;m. The actuator is equipped with a flexure guide that ensures straight motion without tilting or lateral offset. The displacement is linear and calibration is done and checked by the manufacturer. It is recommended that on-axis movement of the probe be checked under the microscope. According to the manufacturer, the stimulus amplitude dampens by less than 20% at oscillating frequencies of 1000 Hz. This can be checked by using a force or displacement measuring device (e.g. force transducer from Kleindiek).</td>
			<td style="width:555px;">The linear piezoelectric actuator, with integrated position sensor and motion amplifier, contains a piezoceramic material that elongates and contracts when voltage is applied. The piezoelectric actuator travels up to100 &#181;m. The actuator is equipped with a flexure guide that ensures straight motion without tilting or lateral offset. The displacement is linear and calibration is done and checked by the manufacturer. It is recommended that on-axis movement of the probe be checked under the microscope. According to the manufacturer, the stimulus amplitude dampens by less than 20% at oscillating frequencies of 1000 Hz. This can be checked by using a force or displacement measuring device (e.g. force transducer from Kleindiek).</td>
		</tr>
		<tr height="547">
			<td height="547" style="height:546px;width:115px;">Piezo Amplifier / Servo Controller</td>
			<td style="width:83px;">Physik Instrument, Germany</td>
			<td style="width:221px;">E-665</td>
			<td style="width:411px;">E-665 amplifier/controller drives and controls the displacement of a low-voltage piezoelectric actuator in a system with sensor position feedback (SGS sensors). The servo-controller provides the option for closed loop operation. When applying sinusoidal and oscillating stimuli the amplitude signal deviates from the set amplitude starting from 500 Hz and reaches a maximum decrease of 20% at 1000Hz.&#160;&#160;</td>
			<td style="width:555px;">E-665 amplifier/controller drives and controls the displacement of a low-voltage piezoelectric actuator in a system with sensor position feedback (SGS sensors). The servo-controller provides the option for closed loop operation. When applying sinusoidal and oscillating stimuli the amplitude signal deviates from the set amplitude starting from 500 Hz and reaches a maximum decrease of 20% at 1000Hz. &#160;</td>
		</tr>
		<tr height="39">
			<td height="57" rowspan="2" style="height:57px;width:115px;">LabChart Software</td>
			<td rowspan="2" style="width:83px;">ADInstruments, USA</td>
			<td style="width:221px;">LabChart 7, MLU60/8</td>
			<td style="width:411px;">Can create, store and run macro of the psychophysical testing algorithm.&#160;</td>
			<td rowspan="2" style="width:555px;">Can create, store and run macro of the psychophysical testing algorithm.&#160;&#160;</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:221px;"></td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:115px;">PowerLab</td>
			<td rowspan="2" style="width:83px;">ADInstruments, USA</td>
			<td style="width:221px;">PowerLab 4/35 PL3504</td>
			<td rowspan="2" style="width:411px;">Data Acquisition Hardware. Used with LabChart software.</td>
			<td rowspan="2" style="width:555px;">Data Acquisition Hardware. Used with LabChart software.</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:221px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:115px;">Brass bar</td>
			<td style="width:83px;">Custom-made</td>
			<td style="width:221px;"></td>
			<td colspan="2" style="width:965px;">Bar made of pure brass, weighs 15.5Kg. When the peizoelectric actuator is mounted on the brass bar it should exert a force of 30 g weight on skin surface.</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:115px;">Monitor</td>
			<td style="width:83px;">Custom-made</td>
			<td style="width:221px;"></td>
			<td colspan="2" style="width:965px;">To mark the 1st and the 2nd interval. The monitor indicates to the subject the time intervals during which the stimulus may be presented.</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:115px;">Response box</td>
			<td style="width:83px;">Custom-made</td>
			<td style="width:221px;"></td>
			<td colspan="2" style="width:965px;">The subject indicates the interval at which stimulus occurred.&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:115px;">Board&#160;</td>
			<td style="width:83px;">Custom-made</td>
			<td style="width:221px;"></td>
			<td colspan="2" style="width:965px;">Upper surface should be smooth (Plastic), lower surface made of foam to prevent stray vibration ot be transmitted to the stimulating pobe.&#160;</td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:115px;">Probe</td>
			<td style="width:83px;">Custom-made</td>
			<td style="width:221px;"></td>
			<td colspan="2" style="width:965px;">A flat circular probe with smoothed edges (thermoplastic material) attached to a screw head. The screw should be of appropriate size to be tightened directly to the moving part of piezoelectric actuator. Size of the probe can be according to preference; in our case, diameter 8.21mm and surface area 52.9mm2.</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:115px;">Labchart Script</td>
			<td style="width:83px;"></td>
			<td style="width:221px;"></td>
			<td colspan="2" style="width:965px;">Can be sent on request. See supplementary code file.&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:115px;">Tactile Acuity Cube</td>
			<td style="width:83px;">MedCore</td>
			<td style="width:221px;"></td>
			<td colspan="2" style="width:965px;">The cube is comprised of 6 sides each containing a grating (bar and groove) whose widths are 0.75, 1.25, 1.75, 3.0, 4.5, and 6.0 mm.&#160;</td>
		</tr>
	</tbody>
</table>

measurement of vibration detection threshold and tactile spatial acuity in human subjects

Tests that allow the precise determination of psychophysical thresholds for vibration and grating orientation provide valuable information about mechanosensory function that are relevant for clinical diagnosis as well as for basic research. Here, we describe two psychophysical tests designed to determine the vibration detection threshold (automated system) and tactile spatial acuity (handheld device). Both procedures implement a two-interval forced-choice and a transformed-rule up and down experimental paradigm. These tests have been used to obtain mechanosensory profiles for individuals from distinct human cohorts such as twins or people with sensorineural deafness.

The piezoelectric actuator provides the vibration stimulus to the subject. The vibration stimulus has a total duration of 1.8 sec and is presented only once during a trial during the first or second interval (Figure 2A). The rise and fall time at the onset and offset of the stimulus is determined by the functions (1-e-bt)&#8729;Amplitude&#8729;sine(frequency), and (e-bt)&#8729;Amplitude&#8729;sine(frequency), respectively, where b is set at 9.1. The rise and fall time at onset and offset are 500 and 600 msec, respectively, and are independent of the testing frequency and amplitude. The duration of the stimulus between the onset and offset phases is 700 msec. The gradual rise and fall ensures a smooth stimulus delivery.
Vibration detection thresholds are dependent on stimulation frequency because they are mediated by different sensory receptors. According to the human psychophysical tuning curve, thresholds lie between ~20 nm to ~45 &#181;m5. Therefore, a set of 35 levels of stimulation (range from 18 nm to 45 &#181;m) of the vibration waveform is constructed (Figure 2B) whose amplitude values are arranged logarithmically (base 10; stimulusn+1 = 100.1&#8729;stimulusn). This range of amplitudes is designed to allow to test at frequencies ranging from 1-250 Hz. The starting stimulus amplitude is usually set above the average vibration detection threshold for a particular vibration frequency test. Previous observations on frequency tuning curves obtained from both psychophysical studies measured average detection threshold around 300 nm for high frequency (&#62;100 Hz) stimuli, and ~3 &#181;m for lower frequencies (&#60;40 Hz)5,23,24.
The relationship of the driving input voltage to the output displacement of the piezoelectric actuator measured by the strain gauge sensor (SGS) at 10 and 125 Hz are illustrated in Figures 3A and 3B. The relationship is linear (correlation coefficient, r2 = 0.9992) for the set of vibration intensities used (Figure 3C). There was a nearly identical relationship between input voltage and output displacement at sinusoidal waveforms of 20 nm amplitude (Figure 3D).
In Figure 4, a typical testing session where the vibration detection threshold at the right little finger is determined at 125 Hz. The threshold search starts with a stimulus that is above threshold of amplitude level 23 (676 nm displacement). The experimental session consists of multiple series of single trials. A series consists of up to 9 single trials applied at one vibration stimulus level. A decrease in stimulus intensity (level) in general requires at least 6 sequential correct responses (Figure 4A). A change in the direction of stimulus intensity marks a reversal point in trial series track and requires at least two incorrect responses. The magnitude of change in stimulus intensity depends on the reversal number. To rapidly search for the threshold, the stimulus intensity is changed in steps of 4 levels when the number of reversals is less than 3, followed by 2 levels at the 3rd reversal (upward direction). Otherwise, the stimulus intensity changes in 1-level steps to finely determine the threshold (Figure 4B). The subject&#39;s threshold is calculated by converting the median stimulus intensity of the last 6 reversal points; in this case 401 nm. The transformed-rule up and down procedure converges to the threshold value at which 75% of the responses are correct. This was calculated based on the response sequences leading to a down rule using the following formula: p6 + 6p6.(1-p)+6p6.(1-p)2, where p is the probability of a correct response.
In the same subject, the tactile cube experiment was done (Figure 5). Each trial consists of 2 stimulations. A two-down and one-up rule is employed and the experiment ends after 13 reversal points. The tactile acuity threshold is 1.6 mm is the median value of the grating widths of the last 10 reversal points. The 2-down and 1-up staircase rule converges to a 71% correct threshold25.
<img alt="Figure 1" src="/files/ftp_upload/52966/52966fig1.jpg" /> 
Figure 1: Schematic diagram illustrating the setup components for measurement of vibrotactile thresholds (A) and a typical test trial (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52966/52966fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52966/52966fig2.jpg" /> 
Figure 2: Physical characteristics of the vibration stimulus. (A) schematic presentation of the stimulus waveform. (B) amplitudes for vibration stimuli (the x-axis has a logarithmic scale). The black circle designates the starting amplitude strength for vibration testing at 125 Hz. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52966/52966fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52966/52966fig3.jpg" /> 
Figure 3: Relationship of the driving input voltage to output displacement of the piezoelectric actuator at 10 Hz and 125Hz sinusoidal input waveform, (A and B), measured by the SGS integrated sensor. (C) the relationship between the 125 Hz driving input voltage and measured output displacements for the set of vibration intensity levels used in testing. (D) Nearly identical input voltage to output displacement at the lowest intensity level, 20 nm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52966/52966fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52966/52966fig4.jpg" /> 
Figure 4: A typical performance of a subject is shown on the two-interval forced-choice vibration detection test. (A) the responses of the subject (correct, open circles; incorrect, black circles) are plotted against the trial series number. (B) a diagram depicting the adaptive method that searches for the threshold by changing both step sizes and direction (i.e., increasing and decreasing level) across a set of trials. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52966/52966fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52966/52966fig5.jpg" /> 
Figure 5: Typical performance of a subject on the two-alternative forced-choice algorithm for the grating orientation task using the tactile acuity cube. The responses of the subject (correct, open circles; incorrect, black circles) are plotted against the trial number. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52966/52966fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="6">Stimulus levels and the corresponding output voltages (V) for stimulus amplitudes</td>
		</tr>
		<tr>
			<td colspan="6">(1 V = 10 &#181;m displacement amplitude)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.00179</td>
			<td>13</td>
			<td>0.02839</td>
			<td>25</td>
			<td>0.45</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.00226</td>
			<td>14</td>
			<td>0.03574</td>
			<td>26</td>
			<td>0.56652</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.00284</td>
			<td>15</td>
			<td>0.045</td>
			<td>27</td>
			<td>0.7132</td>
		</tr>
		<tr>
			<td>4</td>
			<td>0.00357</td>
			<td>16</td>
			<td>0.05665</td>
			<td>28</td>
			<td>0.89787</td>
		</tr>
		<tr>
			<td>5</td>
			<td>0.0045</td>
			<td>17</td>
			<td>0.07132</td>
			<td>29</td>
			<td>1.13035</td>
		</tr>
		<tr>
			<td>6</td>
			<td>0.00567</td>
			<td>18</td>
			<td>0.08979</td>
			<td>30</td>
			<td>1.42302</td>
		</tr>
		<tr>
			<td>7</td>
			<td>0.00713</td>
			<td>19</td>
			<td>0.11303</td>
			<td>31</td>
			<td>1.79148</td>
		</tr>
		<tr>
			<td>8</td>
			<td>0.00898</td>
			<td>20</td>
			<td>0.1423</td>
			<td>32</td>
			<td>2.25534</td>
		</tr>
		<tr>
			<td>9</td>
			<td>0.0113</td>
			<td>21</td>
			<td>0.17915</td>
			<td>33</td>
			<td>2.83931</td>
		</tr>
		<tr>
			<td>10</td>
			<td>0.01423</td>
			<td>22</td>
			<td>0.22553</td>
			<td>34</td>
			<td>3.57448</td>
		</tr>
		<tr>
			<td>11</td>
			<td>0.01791</td>
			<td>23</td>
			<td>0.28393</td>
			<td>35</td>
			<td>4.5</td>
		</tr>
		<tr>
			<td>12</td>
			<td>0.02255</td>
			<td>24</td>
			<td>0.35745</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Stimulus levels and the corresponding output voltages (V) for stimulus amplitudes.

Watch this Scientific Journal Video about Measurement of Vibration Detection Threshold and Tactile Spatial Acuity in Human Subjects at JoVE.com

Measurement of Vibration Detection Threshold and Tactile Spatial Acuity in Human Subjects

Here, we present protocols to determine vibration detection thresholds and tactile acuity using psychophysical methods in man.

Tests that allow the precise determination of psychophysical thresholds for vibration and grating orientation provide valuable information about ...

measurement-vibration-detection-threshold-tactile-spatial-acuity

Nanyang Technological University

Research

JoVE Journal

Behavior

12.6K Views.  Charite Universitätsmedzin, Campus Virchow Klinikum und Campus Charite Mitte. Here, we present protocols to determine vibration detection thresholds and tactile acuity using psychophysical methods in man.

Video: Measurement of Vibration Detection Threshold and Tactile Spatial Acuity in Human Subjects

Quantitative Assessment of Cortical Auditory-tactile Processing in Children with Disabilities

Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology to quantitatively assess children&#39;s cortical processing of light touch, speech sounds and the multisensory processing of the 2 stimuli, without requiring active subject participation or causing children discomfort. To accomplish this we developed a dual channel, time and strength calibrated air puff stimulator that allows both tactile stimulation and sham control. We combined this with the use of event-related potential methodology to allow for high temporal resolution of signals from the primary and secondary somatosensory cortices as well as higher order processing. This methodology also allowed us to measure a multisensory response to auditory-tactile stimulation.

Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology to quantitatively assess infants and children&#39;s cortical processing of light touch, speech sounds, and the multisensory processing of the 2 stimuli, without requiring active subject participation or causing discomfort in vulnerable patients.

Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology ...

Performing Behavioral Tasks in Subjects with Intracranial Electrodes

Patients having stereo-electroencephalography (SEEG) electrode, subdural grid or depth electrode implants have a multitude of electrodes implanted in different areas of their brain for the localization of their seizure focus and eloquent areas. After implantation, the patient must remain in the hospital until the pathological area of brain is found and possibly resected. During this time, these patients offer a unique opportunity to the research community because any number of behavioral paradigms can be performed to uncover the neural correlates that guide behavior. Here we present a method for recording brain activity from intracranial implants as subjects perform a behavioral task designed to assess decision-making and reward encoding. All electrophysiological data from the intracranial electrodes are recorded during the behavioral task, allowing for the examination of the many brain areas involved in a single function at time scales relevant to behavior. Moreover, and unlike animal studies, human patients can learn a wide variety of behavioral tasks quickly, allowing for the ability to perform more than one task in the same subject or for performing controls. Despite the many advantages of this technique for understanding human brain function, there are also methodological limitations that we discuss, including environmental factors, analgesic effects, time constraints and recordings from diseased tissue. This method may be easily implemented by any institution that performs intracranial assessments; providing the opportunity to directly examine human brain function during behavior.

Patients implanted with intracranial electrodes provide a unique opportunity to record neurological data from multiple areas of the brain while the patient performs behavioral tasks. Here, we present a method of recording from implanted patients that can be reproducible at other institutions with access to this patient population.

Patients having stereo-electroencephalography (SEEG) electrode, subdural grid or depth electrode implants have a multitude of electrodes implanted in ...

Testing Tactile Masking between the Forearms

Masking, in which one stimulus affects the detection of another, is a classic technique that has been used in visual, auditory, and tactile research, usually using stimuli that are close together to reveal local interactions. Masking effects have also been demonstrated in which a tactile stimulus alters the perception of a touch at a distant location. Such effects can provide insight into how components of the body's representations in the brain may be linked. Occasional reports have indicated that touches on one hand or forearm can affect tactile sensitivity at corresponding contralateral locations. To explore the matching of corresponding points across the body, we can measure the spatial tuning and effect of posture on contralateral masking. Careful controls are required to rule out direct effects of the remote stimulus, for example by mechanical transmission, and also attention effects in which thresholds may be altered by the participant's attention being drawn away from the stimulus of interest. The use of this technique is beneficial as a behavioural measure for exploring which parts of the body are functionally connected and whether the two sides of the body interact in a somatotopic representation. This manuscript describes a behavioural protocol that can be used for studying contralateral tactile masking.

Here we explore contralateral tactile masking between the forearms in which tactile detection thresholds are modulated by vibration applied to a remote site. The details of which remote sites have an effect can tell us about how the body is represented in the brain.

Masking, in which one stimulus affects the detection of another, is a classic technique that has been used in visual, auditory, and tactile research, ...

Determining Pain Detection and Tolerance Thresholds Using an Integrated, Multi-Modal Pain Task Battery

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify potentially suitable therapeutic populations. The need to use a comprehensive battery of pain models is highlighted by studies whereby only a single pain model, thought to relate to the clinical situation, demonstrates lack of efficacy. No single experimental model can mimic the complex nature of clinical pain. The integrated, multi-modal pain task&#160;battery presented here encompasses the electrical stimulation task, pressure stimulation task, cold pressor task, the UVB inflammatory model which includes a thermal task and a paradigm for inhibitory conditioned pain modulation. These human pain models have been tested for predicative validity and reliability both in their own right and in combination, and can be used repeatedly, quickly, in short succession, with minimum burden for the subject and with a modest quantity of equipment. This allows a drug to be fully characterized and profiled for analgesic effect which is especially useful for drugs with a novel or untested mechanism of action.

Human pain models are valuable tools used to assess the analgesic potential of novel compounds and predict their clinical efficacy, especially when used in an integrated manner. Although implementation of these models is complex, with proper execution, the pain models described in this protocol can provide predictive and reliable results.

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify ...

The (Spatial) Memory Game: Testing the Relationship Between Spatial Language, Object Knowledge, and Spatial Cognition

The memory game paradigm is a behavioral procedure to explore the relationship between language, spatial memory, and object knowledge. Using two different versions of the paradigm, spatial language use and memory for object location are tested under different, experimentally manipulated conditions. This allows us to tease apart proposed models explaining the influence of object knowledge on spatial language (e.g., spatial demonstratives), and spatial memory, as well as understanding the parameters that affect demonstrative choice and spatial memory more broadly. Key to the development of the method was the need to collect data on language use (e.g., spatial demonstratives: &#34;this/that&#34;) and spatial memory data under strictly controlled conditions, while retaining a degree of ecological validity. The language version (section 3.1) of the memory game tests how conditions affect language use. Participants refer verbally to objects placed at different locations (e.g., using spatial demonstratives: &#34;this/that red circle&#34;). Different parameters can be experimentally manipulated: the distance from the participant, the position of a conspecific, and for example whether the participant owns, knows, or sees the object while referring to it. The same parameters can be manipulated in the memory version of the memory game (section 3.2). This version tests the effects of the different conditions on object-location memory. Following object placement, participants get 10 seconds to memorize the object&#39;s location. After the object and location cues are removed, participants verbally direct the experimenter to move a stick to indicate where the object was. The difference between the memorized and the actual location shows the direction and strength of the memory error, allowing comparisons between the influences of the respective parameters.

The Spatial Memory Game: Testing the Relationship Between Spatial Language, Object Knowledge, and Spatial Cognition

We present a protocol to explore the relationship between spatial language production, spatial memory, and object knowledge. The procedure allows experimental manipulation of, and control over, conditions of object knowledge, language at instruction, and physical location, thus teasing apart cognitive and linguistic models describing interactions between these variables.

The memory game paradigm is a behavioral procedure to explore the relationship between language, spatial memory, and object knowledge. Using two different ...

Whole Body Vibration Methods with Survivors of Polio

The purpose of the original study was to examine the use of whole body vibration (WBV) on polio survivors with and without post-polio syndrome as a form of weight bearing exercise. The goal of this article is to highlight the strengths, limitations, and applications of the method used. Fifteen participants completed two intervention blocks with a wash-out period in between the blocks. Each block consisted of twice a week (four weeks) WBV interventions, progressing from 10 to 20 min per session. Low intensity (peak to peak displacement 4.53 mm, frequency 24 Hz, g force 2.21) and higher intensity (peak to peak displacement 8.82 mm, frequency 35 Hz, g force 2.76) WBV blocks were used. Pain severity significantly improved in both groups following higher intensity vibration. Walking speed significantly improved in the group who participated in higher intensity intervention first. No study-related adverse events occurred. Even though this population can be at risk of developing overuse-related muscle weakness, fatigue, or pain from excessive physical activity or exercise, the vibration intensity levels utilized did not cause significant muscle weakness, pain, fatigue, or sleep disturbances. Therefore, WBV appears to provide a safe method of weight bearing exercise for this population. Limitations included the lack of measurement of reflexes, muscular activity, or circulation, the difficulty in participant recruitment, and insufficient strength of some participants to stand in recommended position. Strengths included a standard, safe protocol with intentional monitoring of symptoms and the heterogeneity of the participants in their physical abilities. An application of the methods is the home use of WBV to reduce the barriers associated with going to a facility for weight bearing exercise for longer term interventions, and benefits for conditions such as osteoporosis, particularly for aging adults with mobility difficulties due to paralysis or weakness. Presented method may serve as a starting point in future studies.

The goal of this article is to highlight the strengths, limitations, and applications of the method used with whole body vibration on polio survivors with and without post-polio syndrome as a feasible and safe form of weight bearing exercise.

The purpose of the original study was to examine the use of whole body vibration (WBV) on polio survivors with and without post-polio syndrome as a form ...

Applying Incongruent Visual-Tactile Stimuli during Object Transfer with Vibro-Tactile Feedback

The application of incongruent sensory signals that involves disrupted tactile feedback is rarely explored, specifically with the presence of vibrotactile feedback (VTF). This protocol aims to test the effect of VTF on the response to incongruent visual-tactile stimuli. The tactile feedback is acquired by grasping a block and moving it across a partition. The visual feedback is a real-time virtual presentation of the moving block, acquired using a motion capture system. The congruent feedback is the reliable presentation of the movement of the block, so that the subject feels that the block is grasped and see it move along with the path of the hand. The incongruent feedback appears as the movement of the block diverts from the actual movement path, so that it seems to drop from the hand when it is actually still held by the subject, thereby contradicting the tactile feedback. Twenty subjects (age 30.2 &#177; 16.3) repeated 16 block transfers, while their hand was hidden. These were repeated with VTF and without VTF (total of 32 block transfers). Incongruent stimuli were presented randomly twice within the 16 repetitions in each condition (with and without VTF). Each subject was asked to rate the difficulty level of performing the task with and without the VTF. There were no statistically significant differences in the length of the hand paths and durations between transfers recorded with congruent and incongruent visual-tactile signals &#8211; with and without the VTF. The perceived difficulty level of performing the task with the VTF significantly correlated with the normalized path length of the block with VTF (r = 0.675, p = 0.002). This setup is used to quantify the additive or reductive value of VTF during motor function that involves incongruent visual-tactile stimuli. Possible applications are prosthetics design, smart sport-wear, or any other garments that incorporate VTF.

We present a protocol to apply incongruent visual-tactile stimuli during an object transfer task. Specifically, during block transfers, performed while the hand is hidden, a virtual presentation of the block shows random occurrences of false block drops. The protocol also describes adding vibrotactile feedback while performing the motor task.

The application of incongruent sensory signals that involves disrupted tactile feedback is rarely explored, specifically with the presence of vibrotactile ...

Measurement of Spatial Stability in Precision Grip

The purpose of the protocol is to indirectly evaluate the direction of the finger force during manipulation of a handheld object based on the biomechanical relationships in which deviated force direction causes center of pressure (COP) replacement. To evaluate this, a thin, flexible, and high spatial resolution pressure sensor sheet is used. The system allows measurement of the COP trajectory in addition to the force amplitude and its temporal regulation. A series of experiments found that increased trajectory length reflected a sensorimotor deficit in stroke patients, and that decreased COP trajectory reflects a compensatory strategy to avoid an object slipping from the hand grip in the elderly. Moreover, the COP trajectory could also be decreased by dual task interference. This article describes the experimental procedure and discusses how finger COP contributes to an understanding of the physiology and pathophysiology of grasping.

The goal of this protocol is to measure the center of pressure (COP) replacement using a high spatial resolution sensor sheet to reflect the spatial stability in a precision grip. The use of this protocol could contribute to greater understanding of the physiology and pathophysiology of grasping.

The purpose of the protocol is to indirectly evaluate the direction of the finger force during manipulation of a handheld object based on the ...

Impact of High-intensity Interval Exercise and Moderate-Intensity Continuous Exercise on the Cardiac Troponin T Level at an Early Stage of Training

An elevation in cardiac troponin T (cTnT), as a highly specific biomarker of cardiomyocyte damage, after moderate-intensity continuous exercise (MCE) has been described. The exercise-induced cTnT response distorts the diagnostic role of the cTnT assay. Although high-intensity interval exercise (HIE) is growing in popularity and concerns remain about its safety, available data related to cTnT release after HIE is limited, which hampers the use of HIE as a health intervention. Here, we present three representative HIE protocols [traditional HIE (repeated 4 min cycling at 90% V&#775;O2max interspersed with 3 min rest, 200 kJ/session); sprint interval exercise (SIE, repeated 1 min cycling at 120% V&#775;O2max interspersed with 1.5 min rest, 200 kJ/session); and repeated sprint exercise (RSE, 40 x 6 s all-out sprints interspersed with 9 s rest)] and one representative MCE protocol (continuous cycling exercise at an intensity of 60% V&#775;O2max, 200 kJ/session). Forty-seven sedentary, overweight young women were randomly assigned to one of four groups (HIE, SIE, RSE, and MCE). Six bouts of respective exercise were performed by every single group, with each being 48 h apart. Meanwhile, for four groups, the duration of the entire testing period was identical, being 10 days. Before and after the first and final exercise bouts, an assessment was conducted of cTnT. The current study provides a frame of reference giving a clear picture of how a specific exercise session affects the circulating cTnT concentration at the early stage of training. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise, especially for HIE.

Here, we present protocols of high-intensity interval and moderate-intensity continuous exercise to observe the response of circulating cardiac troponin T (cTnT) concentration to acute exercise over 10 days. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise.

An elevation in cardiac troponin T (cTnT), as a highly specific biomarker of cardiomyocyte damage, after moderate-intensity continuous exercise (MCE) has ...

An Inertial Measurement Unit Based Method to Estimate Hip and Knee Joint Kinematics in Team Sport Athletes on the Field

Current athlete monitoring practice in team sports is mainly based on positional data measured by global positioning or local positioning systems. The disadvantage of these measurement systems is that they do not register lower extremity kinematics, which could be a useful measure for identifying injury-risk factors. Rapid development in sensor technology may overcome the limitations of the current measurement systems. With inertial measurement units (IMUs) securely fixed to body segments, sensor fusion algorithms and a biomechanical model, joint kinematics could be estimated. The main purpose of this article is to demonstrate a sensor setup for estimating hip and knee joint kinematics of team sport athletes in the field. Five male subjects (age 22.5 &#177; 2.1 years; body mass 77.0 &#177; 3.8 kg; height 184.3 &#177; 5.2 cm; training experience 15.3 &#177; 4.8 years) performed a maximal 30-meter linear sprint. Hip and knee joint angles and angular velocities were obtained by five IMUs placed on the pelvis, both thighs and both shanks. Hip angles ranged from 195&#176; (&#177; 8&#176;) extension to 100.5&#176; (&#177; 8&#176;) flexion and knee angles ranged from 168.6&#176; (&#177; 12&#176;) minimal flexion and 62.8&#176; (&#177; 12&#176;) maximal flexion. Furthermore, hip angular velocity ranged between 802.6 &#176;&#183;s-1 (&#177; 192 &#176;&#183;s-1) and -674.9 &#176;&#183;s-1 (&#177; 130 &#176;&#183;s-1). Knee angular velocity ranged between 1155.9 &#176;&#183;s-1 (&#177; 200 &#176;&#183;s-1) and -1208.2 &#176;&#183;s-1 (&#177; 264 &#176;&#183;s-1). The sensor setup has been validated and could provide additional information with regard to athlete monitoring in the field.&#160;This may help professionals in a daily sports setting to evaluate their training programs, aiming to reduce injury and optimize performance.

Monitoring athletes is essential for improving performance and reducing injury risk in team sports. Current methods to monitor athletes do not include the lower extremities. Attaching multiple inertial measurement units to the lower extremities could improve monitoring athletes in the field.

Current athlete monitoring practice in team sports is mainly based on positional data measured by global positioning or local positioning systems. The ...