This protocol was developed for research supported by the Natural Sciences and Engineering Research Council of Canada (grant RGPIN 401963).

The following protocol was approved by the Research Ethics Board at Holland Bloorview Kids Rehabilitation Hospital.
1. Motor Calibration
<ol>
	<li>Connect the microcontroller board to the computer using an USB port.</li>
	<li>Using the original microcontroller software, upload the custom script, &#34;Motor_and_AccelerometerTest.ino&#34; to the board using the USB connection by clicking the &#34;Upload&#34; icon, denoted by the circled right arrow.
	<ol>
		<li>Ensure that the vibration level is set to zero to put the motor in the off-position using the &#34;analogWrite&#34; command. The code should read &#34;analogWrite(vibe1,0);&#34;.</li>
		<li>In the microcontroller code, specify the pulse-width modulated (PWM) output pin corresponding to the motor of interest by initializing the &#34;vibe1&#34; variable. 
		Note: PWM signals generate approximate analog outputs from the digital signals generated by the microcontroller. The pins are labeled numerically on the physical microcontroller board. For example, if the motor is connected to PWM output pin &#39;3&#39;, then ensure that the &#34;int vibe1 = 3;&#34; is specified in the code.</li>
	</ol>
	</li>
	<li>Connect the z-axis output of the tri-axis accelerometer to one of the analog input ports of the microcontroller board, and connect the positive and ground leads from the accelerometer to the 5V and ground (GND) ports of the microcontroller board, respectively.</li>
	<li>Mount the accelerometer to the vibrating motor, ensuring that its z-axis is orthogonal to the flat surface of the motor, as shown in Figure 2, and place the motor on a hard surface.</li>
	<li>Open the &#34;Motor_Calibration.vi&#34; file in the data acquisition software and connect the microcontroller to the computer through an USB port.</li>
	<li>Using the fields provided, specify the serial port for the microcontroller input, using the dropdown menu, as well as the sampling rate, and number of samples to collect. Note: 500 Hz sampling rate is standard for these experiments to prevent aliasing of the acceleration data, and 1,000 samples are commonly recorded.</li>
	<li>Using the &#34;Motor_and_Accelerometer.ino&#34; code, specify the desired duty cycle of the PWM pulses supplied to the vibratory motor, once again using the &#34;analogWrite&#34; command, and re-download the program to the microcontroller board controlling the motors (see Step 1.1). For example, to set the number of pulses to 100, the code should read &#34;analogWrite(vibe1,100);&#34;. Table 1 lists the PWM values and their corresponding duty cycles.</li>
	<li>Using the Fast-Fourier Transform (FFT) display on the &#34;MotorCalibration.vi&#34; interface, identify the largest peak and record the corresponding vibration frequency value (from the horizontal axis).</li>
	<li>Repeat Steps 1.7-1.8, adjusting the PWM levels until the desired frequency is reached, recording each PWM-frequency pair. For example, if targeting a 100 Hz frequency, perform Steps 1.7-1.8 until the largest peak occurs over the 100 Hz mark on the horizontal axis. 
	Note: For the vibrational motors used in this protocol, the targeted vibrations should lie in the 60-400 Hz range to better match the response frequencies of the mechanoreceptors in the skin described in the literature5,10,13.</li>
	<li>Repeat Steps 1.2.2 to 1.8 for each motor, manually recording the PWM-frequency relationship for each motor with a spreadsheet or pencil and paper.</li>
	<li>Open the &#34;Experiment_1.vi&#34; file. For each motor, right-click on the dropdown frequency menu and select &#34;Property&#34;. Under the &#34;Edit Items&#34; tab, use the table to enter the desired frequencies and corresponding PWM levels determined in Steps 1.8-1.9. Select &#34;OK&#34; to exit.</li>
	<li>Repeat Step 1.11 for each virtual interface (VI) file of the system design software to be used during testing (e.g., &#34;Experiment_2.vi&#34;, &#34;Experiment_3.vi&#34;, etc.).</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54223/54223fig2.jpg" /> 
Figure 2. Accelerometer Mounted to Motor. The tri-axis accelerometer (green) is mounted to the coin motor with its z-axis orthogonal to the flat surface of the motor for calibration. Each motor was activated using different duty cycles, and the corresponding vibration frequencies were recorded by the accelerometer. <a href="https://www.jove.com/files/ftp_upload/54223/54223fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Placing the Motors
<ol>
	<li>Once all motors have been calibrated (section 2), mount them to the thigh.
	<ol>
		<li>To achieve the results described in this manuscript, place the one motor on each of the anterior, posterior, medial, and lateral surfaces of the thigh, approximately midway between the greater trochanter and lateral femoral condyle (or distal end of the limb for above-knee amputees). 
		Note: The specific locations of each motor can vary, depending on the research questions and regions of interest, and can be affected by anatomical and physiological factors, such as the type and spatial distribution of mechanoreceptors in the skin.</li>
	</ol>
	</li>
	<li>Attach motors directly to the skin using double-sided tape. 
	Note: Shaving the region around each motor is not necessary, but may improve their adhesion to the skin (Figure 3). For applications where the effects of clothing, a prosthetic liner, or some other material on user perception are of interest, place the motors on top of said material, rather than against the skin.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/54223/54223fig3.jpg" /> 
Figure 3. Test Platform for Experiments. A custom test platform was built to house the microcontroller boards and push buttons. Motors can be attached directly to the skin (as shown), or with a prosthetic liner between the motor and skin. <a href="https://www.jove.com/files/ftp_upload/54223/54223fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Experiment 1: Applying Stimuli and Recording Reaction Time
<ol>
	<li>Re-flash the microcontroller board with the firmware to enable control of the board through the data acquisition software by opening the &#34;LVIFA_Base.pde&#34; file with the controller&#39;s accompanying software package, and repeating Step 1.1, replacing &#34;Motor_and_Accelerometer_Test.ino&#34; with the &#34;LVIFA_Base.pde&#34; script.</li>
	<li>Connect the push-button directly to one of the computer&#39;s USB ports using a serial-to-USB connector. Ensure that all required drivers are installed.</li>
	<li>Open the &#34;Experiment_1.vi&#34; interface.</li>
	<li>Specify the serial ports for the microcontroller board connected to the motors, and push-button by selecting the corresponding serial port identification numbers from the dropdown menus labeled &#34;Motor Input&#34; and &#34;Green Button&#34;, respectively. Identify the serial port identification numbers using the computer&#39;s operating system device manager utility.</li>
	<li>Select the file to record the results and start the program.</li>
	<li>Select the motors and frequencies to be activated by selecting from the dropdown menus for each motor in the &#34;Experiment_1.vi&#34; interface. Have the participant press the push-button with the leg on which the feedback is applied when a vibration is felt. After the button has been pressed, confirm the response in the data acquisition software interface by noting that the clock has stopped counting, and reset the motors for the next trial by selecting the new set of frequencies from the dropdown menus.</li>
	<li>Once the experiment is concluded, use the dropdown menus to return all motor frequencies to the zero position and select the &#34;Stop Program&#34; button to terminate the connection to the motor control board.</li>
</ol>
4. Experiment 2: Distinguishing between Stimuli
Note: This experiment can be conducted entirely independently from Experiment 1. A single motor or multiple motors can be used. The specific locations of the motors can vary depending on the application and research questions.
<ol>
	<li>Connect a second push-button to another USB port using a serial-USB connector.</li>
	<li>Open the &#34;Experiment_2.vi&#34; data acquisition interface.</li>
	<li>Place the motors in the locations and configurations required for the specific research application. For example, place single motors on the thigh, midway between the greater trochanter and lateral femoral condyle (or the distal end of the limb for amputees), on each of the anterior, posterior, lateral, and medial surfaces of the thigh to examine the frequency sensitivity at each of these portions of the leg10,11.</li>
	<li>Specify the serial ports for the microcontroller board and both push-buttons using the same procedure as Step 3.4. Be sure to note which push-button is assigned to each port.</li>
	<li>Select the specific motors to be activated and the desired sequence of frequencies by clicking on the &#34;Motor&#34; icons in the software interface. For example, suppose three frequencies are being tested, such as 140 Hz, 180 Hz, and 220 Hz. A series of tests might be (1) 180 Hz followed by 140 Hz, (2) 220 Hz followed by 140 Hz, and (3) 180 Hz followed by 220 Hz.</li>
	<li>Input the delayed start time and stimulation duration. 1.5 sec is typical for both the delay and stimulation times.</li>
	<li>Start the program. 
	Note: The after the delay specified in Step 4.6, the program will activate the motor(s) with the sequence of paired frequencies chosen in Step 4.5. For example, if 180 Hz vs. 220 Hz was selected in Step 4.5, the corresponding motor will first vibrate at 180 Hz for the specified duration before switching to 220 Hz.</li>
	<li>Have the participant press one of the two push-buttons to choose whether the second perceived frequency was higher or lower than the first. Responses will automatically be recorded by the program.</li>
</ol>

<ol>
	<li>Tate, J. J., Milner, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+kinematic,+temporospatial,+and+kinetic+biofeedback+during+gait+retraining+in+patients:+a+systematic+review.">Real-time kinematic, temporospatial, and kinetic biofeedback during gait retraining in patients: a systematic review.</a> Phys. Ther. 90 (8), 1123-1134 (2010).</li><li>Onate, J. A., Guskiewicz, K. M., Sullivan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmented+feedback+reduces+jump+landing+forces.">Augmented feedback reduces jump landing forces.</a> J. Orthop. Sports Phys. Ther. 31 (9), 511-517 (2001).</li><li>Cholewiak, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+perception+of+tactile+distance:+Influences+of+body+site,+space,+and+time.">The perception of tactile distance: Influences of body site, space, and time.</a> Perception. 28 (7), 851-875 (1999).</li><li>Zhang, Z., Wu, H., Wang, W., Wang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+smartphone+based+respiratory+biofeedback+system.">A smartphone based respiratory biofeedback system.</a> Proc. 2010 3rd Int. Conf. Biomed. Eng. Informatics. 2, 717-720 (2010).</li><li>Wentink, E. C., Mulder, A., Rietman, J. S., Veltink, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrotactile+stimulation+of+the+upper+leg:+Effects+of+location,+stimulation+method+and+habituation.">Vibrotactile stimulation of the upper leg: Effects of location, stimulation method and habituation.</a> Proc. Annu. Int. Conf. IEEE Eng. Med. Biol. Soc. , 1668-1671 (2011).</li><li>Rusaw, D., Hagberg, K., Nolan, L., Ramstrand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+vibratory+feedback+be+used+to+improve+postural+stability+in+persons+with+transtibial+limb+loss?.">Can vibratory feedback be used to improve postural stability in persons with transtibial limb loss?.</a> J. Rehabil. Res. Dev. 49 (8), 1239-1254 (2012).</li><li>Goodworth, A. D., Wall, C., Peterka, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+feedback+parameters+on+performance+of+a+vibrotactile+balance+prosthesis.">Influence of feedback parameters on performance of a vibrotactile balance prosthesis.</a> IEEE Trans. Neural Syst. Rehabil. Eng. 17 (4), 397-408 (2009).</li><li>Asseman, F., Bronstein, A. M., Gresty, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+vibrotactile+feedback+of+instability+to+trigger+a+forward+compensatory+stepping+response.">Using vibrotactile feedback of instability to trigger a forward compensatory stepping response.</a> J. Neurol. 254 (11), 1555-1561 (2007).</li><li>Fan, R. E., Culjat, M. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+haptic+feedback+system+for+lower-limb+prostheses.">A haptic feedback system for lower-limb prostheses.</a> IEEE Trans. Neural Syst. Rehabil. Eng. 16 (3), 270-277 (2008).</li><li>Sharma, A., Torres-moreno, R., Zabjek, K., Andrysek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+an+artificial+sensory+feedback+system+for+prosthetic+mobility+rehabilitation:+Examination+of+sensorimotor+responses.">Toward an artificial sensory feedback system for prosthetic mobility rehabilitation: Examination of sensorimotor responses.</a> J. Rehabil. Res. Dev. 51 (6), 416-425 (2014).</li><li>Sharma, A., Leineweber, M. J., Andrysek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+cognitive+load+and+prosthetic+liner+on+volitional+response+times+to+vibrotactile+feedback.">The effects of cognitive load and prosthetic liner on volitional response times to vibrotactile feedback.</a> J. Rehabil. Res. Dev. , (2016).</li><li>Crea, S., Cipriani, C., Donati, M., Carrozza, M. C., Vitiello, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Providing+Time-Discrete+Gait+Information+by+Wearable+Feedback+Apparatus+for+Lower-Limb+Amputees:+Usability+and+Functional+Validation.">Providing Time-Discrete Gait Information by Wearable Feedback Apparatus for Lower-Limb Amputees: Usability and Functional Validation.</a> IEEE Trans. Neural Syst. Rehabil. Eng. 23 (2), 250-257 (2015).</li><li>Bolanowski, S. J., Gescheider, G. A., Verrillo, R. T., Checkosky, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+channels+mediate+the+mechanical+aspects+of+touch.">Four channels mediate the mechanical aspects of touch.</a> J. Acoust. Soc. Am. 84 (5), 1680-1694 (1988).</li><li>Giggins, O. M., Persson, U. M., Caulfield, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofeedback+in+rehabilitation.">Biofeedback in rehabilitation.</a> J. Neuroeng. Rehabil. 10 (1), 60 (2013).</li><li>Shull, P. B., Jirattigalachote, W., Hunt, M. A., Cutkosky, M. R., Delp, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantified+self+and+human+movement:+A+review+on+the+clinical+impact+of+wearable+sensing+and+feedback+for+gait+analysis+and+intervention.">Quantified self and human movement: A review on the clinical impact of wearable sensing and feedback for gait analysis and intervention.</a> Gait Posture. 40 (1), 11-19 (2014).</li><li>Goodworth, A. D., Peterka, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+integration+for+multisegmental+frontal+plane+balance+control+in+humans.">Sensorimotor integration for multisegmental frontal plane balance control in humans.</a> J. Neurophysiol. 107 (1), 12-28 (2012).</li></ol>

The authors have nothing to disclose.

The purpose of this protocol is to provide the framework for evaluating stimulation parameters in vibrotactile ASF applications. Specifically, it examines the effects of vibration frequency, amplitude, location, and sequence on user sensorimotor response. This framework can be built upon and expanded to incorporate additional or alternative types of user response that may be more clinically relevant, such as bending a joint or shifting weight from one leg to another. These types of changes would require slightly differen...

Artificial sensory feedback (ASF) can be defined as the practice of providing real-time biological information to individuals, often compensating for compromised proprioception or other sensory mechanism. ASF has been long used in the realm of rehabilitation of injured or disabled persons to assist in recovering of aspects of physical function and movement1-3, allowing individuals to control physical processes that once were an involuntary response of the autonomic nervous system4. A subcategory of ASF, biomechanical biofeedback, uses external sensors to measure parameters relating to balance or gait kinematics, and communicate this information to the individual through some sort of applied stimulus. An increasingly popular approach to biomechanical feedback employs small vibrating motors, or tactors, placed at different parts of the body to provide spatial as well as temporal feedback. Previous literature has showed promising results supporting the use of vibrotactile feedback in applications to individuals with lower-limb amputations, vestibular impairments, and aging-related loss of balance5-9.
A thorough understanding of the mechanisms controlling an individual&#39;s perception and response to specific stimuli is necessary for informing effective implementation of ASF systems for different applications. For vibrotactile feedback, chief among these mechanisms are proprioception and the sensorimotor response, specifically the user sensitivity to the applied vibrations and the time required to execute the desired reaction. Any sensory information communicated through vibration stimuli must be encoded as specific combinations of vibration frequency, amplitude, location, and sequence. Therefore, design of vibrotactile ASF systems should select combinations of parameters to maximize user&#39;s perception and interpretation of the stimuli, as well as the timeliness and accuracy of the resulting motor response. The goal of this protocol is to provide a platform from which to evaluate response times and response accuracy to various vibrational stimuli to inform the design of ASF systems for use with different sensory-impaired populations.
The methods described here builds on prior research exploring human perception of tactile and vibrotactile feedback3,5,6, and was developed for use in two previous studies10,11. The latter two studies employed this protocol to examine the effects of vibration frequency and location on the accuracy and timeliness of user responses in lower-limb amputees, showing that both parameters significantly affect the outcome measures, and that a high degree of response accuracy can be achieved. These results can be used to inform the ideal placement of tactors in future studies and clinical applications of vibrotactile ASF systems. Other recent work by Crea et al.12 examined user sensitivity to changes in vibration patterns applied to the thigh during walking, using verbal responses to signify perceived changes to the vibration patterns, rather than a motor response. While these verbal responses can be used to measure detection accuracy, they do not account for errors and delays that may be present in the motor control process.
The primary setup for the following experiments consists of a number of vibrating motors connected to pulse-width-modulated output pins of a microcontroller board. The board is, in turn, controlled through a Universal Serial Bus (USB) connection to a computer running commercially available system design software. The motors require an additional amplifying circuit to ensure sufficient voltage and current is supplied over a wide range of vibration frequencies. An example amplifier circuit is shown in Figure 1. The bipolar junction transistor (BJR) in the figure can be replaced with smaller metal-oxide-semiconductor field-effect transistor (MOSFET) for more efficient operation and smaller size. Similarly, the entire amplifying circuit can be replaced by an off-the-shelf haptic motor driver to provide additional control and reduced size. Each motor requires its own circuit, and using the equipment listed in this paper, up to ten motors can be controlled by a single microcontroller board.
<img alt="Figure 1" src="/files/ftp_upload/54223/54223fig1.jpg" /> 
Figure 1. Motor Wiring. (A) The amplification circuit for a single vibration motor is shown. Each motor requires a separate circuit and must be connected to a unique PWM output port on the microcontroller. The VDD here represents the 3.3 V power supplied by the microcontroller board, and the resistor R2 serves as a pull-down resister to ensure the transistor switch remains open when zero voltage is applied. (B) An example of the physical wiring of two motors. Although eight individual amplification circuits are shown, only two are connected to vibration motors. In this protocol R1 = 4.7 k&#937; and R2 = 100 k&#937;. <a href="https://www.jove.com/files/ftp_upload/54223/54223fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="849">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vibrating Pager Motors</td>
			<td style="width:107px;">Precision Microdrives</td>
			<td style="width:119px;">Model 310-101</td>
			<td style="width:407px;">Coin eccentric rotating mass motors.&#160; As many as necessary to test all locations and interactions of interest</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Tri-axis Accelerometer</td>
			<td style="width:107px;">Dimension Engineering</td>
			<td style="width:119px;">ADXL 335</td>
			<td style="width:407px;">Advanced analog accelerometer. 500Hz bandwidth, 3.5-15V input.&#160; Designed for motion, tilt, and slope measurement, as well as vibration and shock sensing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Arduino Uno</td>
			<td style="width:107px;">Arduino</td>
			<td style="width:119px;">DEV-11021</td>
			<td style="width:407px;">Microcontroller board for communicating with the tri-axis accelerometer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Arduion Mega 2560</td>
			<td style="width:107px;">Arduino</td>
			<td style="width:119px;">DEV-11061</td>
			<td style="width:407px;">Microcontroller board for interfacing with the vibration motors.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LabVIEW</td>
			<td style="width:107px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:407px;">Data acquisition software used to control motors and display accelerometer signals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Arduino IDE Software</td>
			<td style="width:107px;">Arduino</td>
			<td style="width:119px;">v. 1.6.5</td>
			<td style="width:407px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Push-Button</td>
			<td style="width:107px;">Bridges</td>
			<td style="width:119px;">Buddy Button</td>
			<td style="width:407px;">Wired switch featuring a 2.5in/6.35cm activation surface that provides an auditory click and tactile feedback.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optional:</td>
			<td style="width:107px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dedicated haptic motor driver</td>
			<td style="width:107px;">Texas Instruments</td>
			<td>DRV2605L</td>
			<td style="width:407px;">Can be used to replace the entire amplification circuit described in Step 1.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Flexible wearable goniometer</td>
			<td style="width:107px;">Biometrics Ltd.</td>
			<td style="width:119px;">SG110</td>
			<td style="width:407px;">Twin axis flexible goniometers to measure angles in up to two planes of movement that can be used in lieu of the push button to measure joint movement in response to stimuli. 
			www.biometricsltd.com/gonio.htm</td>
		</tr>
	</tbody>
</table>

a method for evaluating timeliness and accuracy of volitional motor responses to vibrotactile stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

Figure 4 illustrates the calibration curves identifying the PWM value for a 180 Hz vibration frequency of a single motor. Starting at a 50% duty cycle, the PWM values are iterated until the primary frequency spike occurs at 180 Hz. Successful calibration trials should show a clear spike at the primary vibration frequency. Poor fixation of the accelerometer to the motor, or of the motor to a support surface may result in a more diffuse FFT without a clear spike. In this situation, the calibration trial sh...

Watch this Scientific Journal Video about A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli at JoVE.com

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

a-method-for-evaluating-timeliness-accuracy-volitional-motor

Research

JoVE Journal

Bioengineering

7.2K Views.  Holland Bloorview Kids Rehabilitation Hospital. This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant's volitional response for various combinations of stimulation location and frequency.

Video: A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Haptic/Graphic Rehabilitation: Integrating a Robot into a Virtual Environment Library and Applying it to Stroke Therapy

Recent research that tests interactive devices for prolonged therapy practice has revealed new prospects for robotics combined with graphical and other forms of biofeedback. Previous human-robot interactive systems have required different software commands to be implemented for each robot leading to unnecessary developmental overhead time each time a new system becomes available. For example, when a haptic/graphic virtual reality environment has been coded for one specific robot to provide haptic feedback, that specific robot would not be able to be traded for another robot without recoding the program. However, recent efforts in the open source community have proposed a wrapper class approach that can elicit nearly identical responses regardless of the robot used. The result can lead researchers across the globe to perform similar experiments using shared code. Therefore modular "switching out"of one robot for another would not affect development time. In this paper, we outline the successful creation and implementation of a wrapper class for one robot into the open-source H3DAPI, which integrates the software commands most commonly used by all robots.

Recently, a vast amount of prospects have come available for human-robot interactive systems. In this paper we outline the integration of a new robotic device with open source software that can rapidly make possible a library of interactive functionality. We then outline a clinical application for a neurorehabilitation application.

Recent research that tests interactive devices for prolonged therapy practice has revealed new prospects for robotics combined with graphical and other ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Evaluating Primary Blast Effects In Vitro

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such blast-induced injuries is of great importance considering the recent trend towards the use of explosives in modern warfare and terrorist-related incidents. To fully understand blast-induced injury, we must first be able to replicate such blast events in a controlled environment using a reproducible method. In this technique using shock tube equipment, shock waves at a range of pressures can be propagated over live cells grown in 2D, and markers of cell viability can be immediately analyzed using a redox indicator assay and the fluorescent imaging of live and dead cells. This method demonstrated that increasing the peak blast overpressure to 127 kPa can stimulate a significant drop in cell viability when compared to untreated controls. Test samples are not limited to adherent cells, but can include cell suspensions,&#160;whole-body and tissue samples, through minor modifications to the shock tube setup. Replicating the exact conditions that tissues and cells experience when exposed to a genuine blast event is difficult. Techniques such as the one presented in this article can help to define damage thresholds and identify the transcriptional and epigenetic changes within cells that arise from shock wave exposure.

Evaluating Primary Blast Effects In Vitro

Understanding how cells are modulated by exposure to shock waves can help identify the mechanisms behind injuries triggered from blast events. This protocol uses custom-built shock tube equipment to apply shock waves at a range of pressures to cell monolayers and to identify the subsequent effects on cell viability.

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...