Name: a tactile automated passive-finger stimulator (taps)
Uploaded: 2009-06-03T00:00:00
Duration: 19 min 44 s
Description: Watch this Scientific Journal Video about A Tactile Automated Passive-Finger Stimulator TAPS at JoVE.com

This work was supported by National Eye Institute Grant 1 R15 EY13649-01, and by an individual Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). Special thanks to Deda Gillespie for metal machining, and for consultation on device design and construction.

Introduction
The Tactile Automated Passive-Finger Stimulator (TAPS) is a computer-controlled system that presses structured surfaces against the skin in order to assess a person&#39;s tactile spatial acuity. We designed and constructed the device in Duquesne University, and modified it to its current form in McMaster University. Here, we provide an overview of the function of TAPS. We then describe the components of the device, in order to facilitate its reproduction by other researchers. Finally, we show how TAPS is used to conduct a psychophysical experiment.
1. Device Overview
TAPS uses gravity to press a stimulus surface against the skin (Fig. 1). The subject&#39;s arm rests comfortably in prone position on a tabletop. The fingertip (or other skin area to be tested) lies over a tunnel in the table. Beneath the table, a stepper motor rotates a disk that holds up to 40 stimulus pieces, positioning one of the pieces under the tunnel. Gravity exerts a downward force on a mass hanging from one end of a rotatable rod. A crossbar extending from the carriage of a linear actuator prevents the rod from moving. As the actuator motors forward, the rod pivots under the influence of gravity, pressing a stimulus piece upward through the tunnel and onto the skin. Simple physics relates the speed of the actuator to the speed with which the stimulus surface rises to contact the skin, and the weight of the hanging mass to the static force of the tactile stimulus. As the actuator reverses direction to return the rod to its starting position, the stimulus piece drops. Thus, the device applies tactile stimuli with controlled force, velocity, contact duration, and inter-stimulus interval. The subject selects a button with the non-tested hand to register a response. TAPS detects the response and proceeds to deliver the next stimulus.
<img alt="Figure 1" src="/files/ftp_upload/1374/Fig1.png" />
Figure 1. The Physics of TAPS. TAPS controls both the onset velocity and the steady-state force of the stimulus.
Onset velocity: As the crossbar from the linear actuator (red) moves to the right with velocity V1, the left end of the rotating rod drops under the influence of gravity; the right end of the rod therefore presses a stimulus piece upwards towards the finger with velocity V2 = V1 (L2 / L3). Since V1 is under computer control, V2 may be set to any value desired by the investigator.
Force: Gravity exerts a downward force, F1 = Mg, on the mass M (where g = 9.8 m/s2). This causes the stimulus piece to press against the finger with an upward force, F2. For a massless rod, F2 = F1 ( L1 / L2). Since the rod is not in fact massless, F2 is determined empirically, by measurement with a force sensor. To make fine adjustments to F2, the investigator can change the position of M along the rod. F2 increases slightly as M is moved to the left (increasing L1), and decreases slightly as M is moved to the right (decreasing L1). To make larger adjustments to F2, the mass can be changed.
2. Components and Details of Device Function
i. Moving Parts
TAPS uses two stepper motors, one to drive the carriage of the linear actuator, and another to rotate the disk containing the stimulus pieces. These two motors control all of TAPS&#39; moving parts. These parts lie on a lower table that is mechanically uncoupled from the upper table on which the subject&#39;s arm rests. Because the two tables are uncoupled, vibrations caused by the motors do not transfer to the skin. The tables are carefully aligned so that the tunnel through the upper table lies directly above the stimulus piece contacted by the rotating rod.
We machined 40 square slots around the circumference of the rotating disk, to accommodate the stimulus pieces. We constructed stimulus pieces from round plastic rods, machining the rods to have square shafts that fit into the slots of the disk. This ensures that the pieces do not rotate within their slots as the disk spins. We cut the stimulus surfaces into the round faces of the pieces.
When the actuator carriage motors forward, the end of the rotating rod opposite the hanging mass rises to contact the bottom of the selected stimulus piece. Because the rod describes an arc as it swings upwards, it is fit with a lightweight plastic bearing wheel to eliminate friction with the bottom of the stimulus piece. This plastic wheel rotates slightly as it pushes the stimulus piece upward; without the plastic wheel, the end of the rod would pull the base of the piece slightly laterally (towards the left in Fig. 1), causing the piece to jam within the tunnel through the upper table. To further ensure a smooth upward trajectory of the piece, the tunnel is fit with a plastic collar that is beveled to direct the piece towards the center of the tunnel as the piece rises.
ii. Sensors
TAPS makes use of six sensors to ensure proper functioning: two home sensors, three limit sensors, and a force sensor.
The actuator and disk home sensors signal to the computer that the actuator carriage and the disk are in their desired initial positions at the start of an experiment. The actuator home sensor is a Hall-effect sensor; the disk home sensor is a U-shaped photoelectric sensor, activated when a piece of stiff paper glued to the disk rim breaks the infrared sensor beam. When the disk is in home position, a particular slot is aligned directly under the tunnel in the upper table. All subsequent disk movement calculations are made relative to this known starting position.
The three limit sensors serve as safety features in case of malfunction. To prevent runaway movement of the actuator carriage in the unlikely event of a control malfunction, the actuator is fit with forward and reverse limit sensors (Hall-effect sensors). If activated, these cut power to the actuator. To prevent disk movement in the event that a stimulus piece becomes stuck in the tunnel, an infrared retro-reflective sensor is attached to the underside surface of the upper table. A piece that is elevated sufficiently to enter the tunnel breaks the infrared beam cast by this sensor, and blocks command voltages from reaching the disk stepper motor.
The sixth and final sensor is a force sensor that rests gently on the subject&#39;s fingernail (Fig. 2). This sensor detects downward (into the tunnel), backward or forward finger movement. The program discards trials in which movement occurred. Because the sensor is attached to a rigid arm, it additionally prevents the finger from moving passively upwards upon contact with the rising piece. Since we use TAPS to test passive (finger stationary) tactile spatial acuity, the force sensor is a crucial element. As a final precaution against finger movement, we place plastic barriers gently against the sides of the finger to prevent lateral movements.
<img alt="Figure 2a" src="/files/ftp_upload/1374/fig2a.png" /> 
<img alt="Figure 2b" src="/files/ftp_upload/1374/fig2b.png" /><img alt="Figure 2c" src="/files/ftp_upload/1374/fig2c.png" />
Figure 2. Force Sensor to detect finger movement. A. The force sensor presses at 90-deg to the surface of the fingernail, near the cuticle, with a force between 50 and 80 grams. B. Example force sensor trace showing one second preceding contact of the stimulus piece with the finger, followed by one second of maintained contact with the stationary finger. Note that, upon contact (time = 0), the force of the stimulus piece from below pushes the finger more firmly up against the sensor (rising slope in force trace). C. If the subject attempts to move the finger downward to better feel the stimulus surface, then the force between the fingernail and the sensor diminishes (arrow). The sensor also registers force fluctuations if the subject moves the finger backwards or forwards. TAPS is programmed to discard any trials in which the force of contact between the sensor and the fingernail fluctuates by more than a threshold amount (e.g., 20 grams) while the skin is in contact with the stimulus surface. Using a computer-generated voice, TAPS can optionally warn the subject that movement was detected.
iii. Vibration and Sound Damping
TAPS&#39; stepper motors produce vibration, and this in turn causes sound. Both vibration and sound are undesirable. Vibration, if transmitted to the skin, could interfere with the subject&#39;s ability to perform the tactile task. Over time, vibration could also cause mechanical instability in TAPS&#39; components. Sound could distract the subject. For these reasons, we have taken measures to reduce both vibration and sound, and to prevent any vibration from reaching the subject.
Most importantly, the lower and upper tables do not contact one another. They are connected only in the sense that they both contact the floor. Hence, vibration from the motors, which rest on the lower table, is not transmitted to the subject&#39;s arm, which rests on the upper table. Additionally, vibration and sound are reduced as follows: a fiberglass curtain enclosing the lower table muffles device sound; PVC/fiber reinforced anti-vibration pads sandwiched between the actuator and its base absorb some of the actuator-produced vibration; the actuator base sits on four rubber cylindrical sandwich mounts that serve to further dampen vibration; the disk stepper motor base also sits on rubber mounts (syringe plunger rubber); and silicone O-rings surround the shafts of the stimulus pieces, reducing clatter as the disk turns.
iv. Stimulus Pieces
TAPS uses modified half-inch diameter rods to press spatially structured surfaces against the skin (Fig. 3). The rods are machined to have square shafts. The round faces of the rods contain the stimulus surfaces; the square shafts of the rods fit into the 40 square slots around the circumference of the rotating disk, ensuring that the stimulus surfaces maintain their proper orientations as the disk spins.
Any stimulus surfaces may be machined into the rod faces. We have used TAPS mainly to apply square wave gratings (i.e., parallel ridges and grooves). To create these, we started with 0.5&#34; diameter Delrin plastic cylindrical rods, 3&#34; long. Using a Sherline milling machine, we cut the rods to 2.75&#34; long, with 2&#34; long square-cross-section shafts (8.7 x 8.7 mm), and a 0.75&#34;-long, 12.74 mm diameter round cylindrical head. We then milled grooves in the round face to create the stimulus surfaces. Each stimulus surface has equal ridge and groove widths. We made pieces with groove widths ranging from 0.25 to 3.10 mm in 0.15 mm increments. We made two identical stimulus pieces for each of these 20 groove widths, resulting in 40 pieces total. We then inserted the pieces into the square slots of the disk such that each groove width was represented in both the vertical (parallel to long axis of the finger) and horizontal (transverse to the long axis of the finger) orientation.
<img alt="Figure 3" src="/files/ftp_upload/1374/Fig3.png" />
Figure 3. Stimulus piece. Each stimulus surface is milled into the face of a 0.5-inch diameter Delrin rod. The base of the rod is cut to have a square cross section, allowing it to fit into the square cutouts in the rotating disk. The surface of the piece shown is a square-wave grating, with parallel grooves and ridges of equal width.
v. Computer Control
TAPS is a fully automated, computer-controlled system. Many computer hardware and software configurations could be used to control the device. We currently use a Macintosh G3 running LabVIEW 6.1 (National Instruments). The computer communicates via a motor controller board with a stepper motor driver to command the actuator and disk motors, and to read the home sensors and limit switches. A PCI data acquisition board reads the force sensor and subject response buttons.
vi. Force and Timing Limitations
Force range: We have used TAPS to deliver stimuli with forces ranging from 10 to 50 grams. We suspect that friction in the components would impede the delivery of stimuli at forces under about 5 grams, and that some mechanical instability would arise for forces in excess of about 100 grams.
Inter-stimulus interval (ISI): The distance between the stimulus surfaces and the finger, and the stable speeds of the linear actuator and disk stepper motor, together set a lower limit on the ISI that the device can achieve. We estimate this limit at about 1.5 seconds. Thus, the device is not appropriate for applications such as masking experiments, which require sub-second ISI.
3. Conducting an Experiment
Here we describe one protocol for which TAPS can be used: a two-interval forced-choice (2-IFC) grating orientation task (GOT) with Bayesian adaptive tracking. TAPS can be programmed for many other psychophysical protocols as well. We have used it previously to conduct 2-IFC experiments with staircase tracking1,2 and with the method of constant stimuli. Of course, it is able to conduct yes/no procedures as well as 2-IFC.
For the 2-IFC GOT task, we use square wave gratings that contact the distal pad of the finger with 4 cm/sec onset velocity, 50 gm contact force, and 1 sec contact duration. Each trial consists of two sequential stimulus presentations (ISI: 2 sec) with gratings of identical groove width, but differing 90-deg in orientation. In one presentation, the grooves are aligned vertically (parallel), and in the other, horizontally (transverse), to the long axis of the finger. Stimulus order is chosen randomly by the computer program. The subject indicates whether the horizontal orientation occurred in the first or second interval, by pressing one of two buttons with the non-tested hand. A Bayesian adaptive method adjusts groove width from trial to trial.
We programmed TAPS with a modified version of the psi (&#936;) method, a Bayesian adaptive algorithm3. This method has several advantages over conventional adaptive methods, such as staircase methods. First, the psi method estimates not just a single point on the psychometric function (e.g., the 71% threshold estimated by a 2-down 1-up staircase), but the entire psychometric function. Second, the method yields Bayesian posterior probability distributions (and thereby confidence intervals) for the parameters of the psychometric function. Finally, the method is efficient. It holds in memory many thousands of possible psychometric function shapes, and updates the posterior probability of each function after each response. It then uses an expected entropy minimization procedure to choose the next stimulus; that is, it chooses the stimulus on each trial that is expected to maximize the information gain.
Following Kontsevich and Tyler3, we modeled d-prime as a power function of stimulus level, and each subject&#39;s psychometric function &#8211; Pc(x), the probability of a correct response as a function of stimulus level, x &#8211; as a mixture of a cumulative normal (probit) function and a lapse rate term: 
<img alt="Equation 1" src="/files/ftp_upload/1374/eq1.png" /> 
<img alt="Equation 2" src="/files/ftp_upload/1374/eq2.png" /> 
Here, a is the threshold (stimulus level corresponding to 76% correct response probability, d-prime = 1), and b is the slope, of the subject&#39;s psychometric function.
We modified the &#936; algorithm by treating the lapse rate (delta) as a parameter of unknown value, and by calculating a &#34;guessing Bayes factor&#34; after each trial: 
<img alt="Equation 3" src="/files/ftp_upload/1374/eq3.png" /> 
 
This Bayes factor is a likelihood ratio that compares the probability of the data under hypothesis 1, that the subject is guessing, to the probability of the data under hypothesis 2, that the subject has a psychometric function. The numerator is the probability of the subject&#39;s data, D (correct and incorrect responses at each of the groove widths presented) given that the subject is simply guessing (50% probability correct) on all trials up to and including the current trial. The denominator is the probability of the data given the algorithm&#39;s best estimate of the subject&#39;s psychometric function. In our experience, for most young subjects the Bayes factor rapidly approaches zero as the testing block progresses, indicating that the subject&#39;s performance conforms to a psychometric function. For some older subjects, the Bayes factor rises above one, indicating that the subject is unable to perform the task. TAPS can be programmed to terminate the testing block if the guessing Bayes factor has exceeded a threshold value after a specified number of trials.
4. Results
Figure 4 shows an experimental block consisting of 40 trials from a 2-IFC GOT experiment. This 40-trial block consisted of 80 stimuli to the subject&#39;s right middle finger, with 1-second contact duration and 2-second ISI between contacts in a single trial. The subject&#39;s 76% correct threshold was 1.7 mm, as indicated by the mode of the posterior probability density function (PDF) for the threshold parameter (panel C). The 95% confidence interval for the subject&#39;s threshold parameter, indicated by the width of the posterior PDF, was 1.3 - 1.9 mm.
<img alt="Figure 4a" src="/files/ftp_upload/1374/fig4a.png" /><img alt="Figure 4b" src="/files/ftp_upload/1374/fig4b.png" /> 
<img alt="Figure 4c" src="/files/ftp_upload/1374/fig4c.jpg" />
Figure 4. Results from a 2-interval forced-choice grating orientation task. A. An experimental block, consisting of 40 trials applied in this case to the right middle finger. The subject&#39;s correct (plus symbols) and incorrect (open circles) responses at different groove widths are plotted against trial number. A Bayesian adaptive method (see text) determined the sequence of groove widths to apply, based on the subject&#39;s performance. B. The best-fit psychometric function for this subject. C. Posterior probability density function for the subject&#39;s 76% correct groove width (psychometric function a-parameter, corresponding to d-prime = 1).

<ol>
	<li>Goldreich, D. &. a. m. p. ;. a. m. p., Kanics, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+acuity+is+enhanced+in+blindness.">Tactile acuity is enhanced in blindness.</a> J. Neurosci. 23, 3439-3445 (2003).</li><li>Goldreich, D. &. a. m. p. ;. a. m. p., Kanics, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+blind+and+sighted+humans+on+a+tactile+grating+detection+task.">Performance of blind and sighted humans on a tactile grating detection task.</a> Percept. Psychophys. 68, 1363-1371 (2006).</li><li>Kontsevich, L. L. &. a. m. p. ;. a. m. p., Tyler, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+adaptive+estimation+of+psychometric+slope+and+threshold.">Bayesian adaptive estimation of psychometric slope and threshold.</a> Vision. Res. 39, 2729-2737 (1999).</li><li>Bleyenheuft, Y., Cols, C., Arnould, C. &. a. m. p. ;. a. m. p., Thonnard, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+tactile+spatial+resolution+from+6+to+16+years.">Age-related changes in tactile spatial resolution from 6 to 16 years.</a> Somatosens. Mot. Res. 23, 83-87 (2006).</li><li>Johnson, K. O. &. a. m. p. ;. a. m. p., Phillips, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+spatial+resolution.+I.+Two-point+discrimination,+gap+detection,+grating+resolution,+and+letter+recognition.">Tactile spatial resolution. I. Two-point discrimination, gap detection, grating resolution, and letter recognition.</a> J. Neurophysiol. 46, 1177-1192 (1981).</li><li>Gibson, G. O. &. a. m. p. ;. a. m. p., Craig, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+force+and+conformance+on+tactile+intensive+and+spatial+sensitivity.">The effect of force and conformance on tactile intensive and spatial sensitivity.</a> Exp. Brain Res. 170, 172-181 (2006).</li><li>Johnson, K. O. &. a. m. p. ;. a. m. p., Phillips, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rotating+drum+stimulator+for+scanning+embossed+patterns+and+textures+across+the+skin.">A rotating drum stimulator for scanning embossed patterns and textures across the skin.</a> J. Neurosci. Methods. 22, 221-231 (1988).</li><li>Craig, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grating+orientation+as+a+measure+of+tactile+spatial+acuity.">Grating orientation as a measure of tactile spatial acuity.</a> Somatosens. Mot. Res. 16, 197-206 (1999).</li><li>Killebrew, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dense+array+stimulator+to+generate+arbitrary+spatio-temporal+tactile+stimuli.">A dense array stimulator to generate arbitrary spatio-temporal tactile stimuli.</a> J. Neurosci. Methods. 161, 62-74 (2007).</li></ol>

The controlled delivery of spatially structured mechanical stimuli poses challenges not faced in the delivery of visual or auditory stimuli, for which commercially available equipment (computer screens, audio speakers) may be used. For this reason, many tactile psychophysics experiments are still done using manual stimulus delivery.
The manual delivery of tactile stimuli is time consuming, and requires great care and concentration on the part of the investigator. For instance, Bleyenheuft et al.4 report that "Manual application of approximately 1-2 mm of skin perpendicular deformation was used... the examiner was particularly attentive to avoid any shearing stress between the skin and the grating which could distort the measure..." Perhaps because manual stimulus delivery is relatively slow and concentration-intensive, many studies using this method have employed yes/no (just one stimulus per trial) rather than two-interval forced choice protocols.
Unfortunately, even when great care is taken, manual stimulus delivery leaves many stimulus parameters uncontrolled. These parameters include stimulus force, onset velocity, duration, and the stability of the stimulus surface on the skin. Of these parameters, perhaps the best studied with respect to its influence on performance is stimulus force. Humans show progressive improvement in grating orientation discrimination on the fingertip as indentation depth changes from 500 to 1200 microns5; performance is better at 10 gram force than at 50 gram force1, although no further improvement is observed between 50 grams and 200 grams6. For other applications, such as grating detection (distinguishing a smooth from a grooved surface), increases in force significantly improve performance throughout the range of 10 to 200 grams2,6.
To overcome these challenges associated with manual testing, we developed the automated system, TAPS. Our aim in building TAPS was to create a simple, safe, versatile, efficient and affordable device for controlled tactile stimulus application. The controlled use of gravity is a simple and safe way to apply a tactile stimulus. Safety is ensured, because the force against the skin cannot exceed that due to gravity. The powerful linear actuator serves only to transfer the force of gravity onto the skin, and then to withdraw the contact. The device is versatile, as it will accommodate any stimulus surfaces machined into (or attached onto) the ends of half-inch-diameter rods, and can be programmed to run a variety of psychophysical protocols. TAPS is efficient, as it rapidly deploys stimuli, and can run timesaving adaptive algorithms. Finally, the components of TAPS are affordable to small grant holders. The motors cost approximately $1,500; the electrical components (excluding the computer, but including the PCI boards and stepper motor driver), approximately $6,000. A mini-milling machine and accessories may be purchased for approximately $1,000, or a professional machinist can be hired to mill the stimulus pieces and make other small parts.
We hope this system will be replicated by others, and serve to promote the move towards controlled tactile testing, which in recent years has seen promising progress5,7,8,9.

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		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Delrin plastic rods 0.5&#x201D; diameter 3&#x201D; long </td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>ZRD-08</td>
<td>Rods from which the stimulus pieces are made</td>
</tr>
<tr>
<td>Chrome-steel bearing, 0.5" bore diameter</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>BR-08-01</td>
<td>Pivot for the rotating rod</td>
</tr>
<tr>
<td>Plastic bearing, 0.5&#x201D; bore, 1.125&#x201D; outer diameter, 0.25&#x201D; thick</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>BRP-08-01</td>
<td>Wheel at end of rotating rod</td>
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<tr>
<td>Fiberglass curtain, 0.75&#x201D; thick</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>9781T83</td>
<td>Sound absorbing curtain around lower table</td>
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<tr>
<td>Silicone O-rings 5/16" inside diameter</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>AS568A- 011</td>
<td>To reduce vibration of stimulus pieces</td>
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<tr>
<td>Anti-Vibration Pads 2"x 2", 93 PSI max </td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>60105K61</td>
<td>Put under the linear actuator</td>
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<tr>
<td>Rubber sandwich mounts 1/2" H x 3/8" W </td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>9378K11</td>
<td>Put under linear actuator base</td>
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<tr>
<td>Stepper motor Nema 23, 3-stack </td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>S23</td>
<td>For rotating the stimulus disk</td>
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<tr>
<td>Linear rodless actuator</td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>R2S23N-105A-18-l-M35M</td>
<td>To control rotating bar movement</td>
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<td>1 Hall effect switch &#x201C;normally open&#x201D; config. </td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>RP1</td>
<td>Actuator home sensor</td>
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<td>2 Hall effect switches &#x201C;normally closed&#x201D; config. </td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>RP2</td>
<td>Actuator limit sensors</td>
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<td>Micro switch</td>
<td>&nbsp;</td>
<td>Honeywell</td>
<td>FSG15N1A</td>
<td>Finger force sensor</td>
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<tr>
<td>Retro-reflective sensor</td>
<td>&nbsp;</td>
<td>Honeywell</td>
<td>FE7B-RB6VG-M</td>
<td>Piece position sensor</td>
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<tr>
<td>Motor controller board, closed-loop control</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI-step-4CX</td>
<td>Mediates communication between computer and Nudrive</td>
</tr>
<tr>
<td>Nudrive stepper motor driver</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>Nudrive 4SX-211</td>
<td>Sends command voltages to the linear actuator and disk stepper motors</td>
</tr>
<tr>
<td>Data acquisition board</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI-MIO-16E-1</td>
<td>Reads finger force sensor</td>
</tr>
<tr>
<td>LabVIEW</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>&nbsp;</td>
<td>Programming language</td>
</tr>
<tr>
<td>Valuemotion library</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>&nbsp;</td>
<td>Motor control routines</td>
</tr>
<tr>
<td>Photoelectric sensor</td>
<td>&nbsp;</td>
<td>Panasonic</td>
<td>PM-K53-C1</td>
<td>Disk home sensor</td>
</tr>
<tr>
<td>Mini milling machine</td>
<td>&nbsp;</td>
<td>Sherline</td>
<td>2010-DRO</td>
<td>For machining the stimulus surfaces</td>
</tr>
<tr>
<td>Ohaus precision mass set</td>
<td>&nbsp;</td>
<td>Edmund Scientific</td>
<td>&nbsp;</td>
<td>Mass on rotating bar</td>
</tr>
<tr>
<td>Parrish Magic Line aluminum cake pan bottom, 9&#x201D; diam.</td>
<td>&nbsp;</td>
<td>Sur La Table</td>
<td>&nbsp;</td>
<td>Rotating disk machined with square cut-outs to hold the stimulus pieces</td>
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a tactile automated passive-finger stimulator (taps)

Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering controlled spatially structured stimuli to the skin. Consequently, investigators often apply tactile stimuli manually. Manual stimulus application is time consuming, requires great care and concentration on the part of the investigator, and leaves many stimulus parameters uncontrolled. We describe here a computer-controlled tactile stimulus system, the Tactile Automated Passive-finger Stimulator (TAPS), that applies spatially structured stimuli to the skin, controlling for onset velocity, contact force, and contact duration. TAPS is a versatile, programmable system, capable of efficiently conducting a variety of psychophysical procedures. We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.
Corresponding Author: Daniel Goldreich

Watch this Scientific Journal Video about A Tactile Automated Passive-Finger Stimulator TAPS at JoVE.com

A Tactile Automated Passive-Finger Stimulator TAPS

We describe a computer-controlled device for investigating the sense of touch: the Tactile Automated Passive-finger Stimulator (TAPS). We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.

A Tactile Automated Passive-Finger Stimulator (TAPS)

Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering ...

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Research

JoVE Journal

Biology

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.