JOURNAL OF APPLIED PHYSICS 114, 244701 (2013)
Automation of a high-speed imaging setup for differential viscosity measurements C. Hurth,a) B. Duane, D. Whitfield, S. Smith, A. Nordquist, and F. Zenhauserna) Center for Applied Nanobioscience and Medicine, The University of Arizona College of Medicine, 425 N 5th Street, Phoenix, Arizona 85004, USA
(Received 18 October 2013; accepted 17 November 2013; published online 26 December 2013) We present the automation of a setup previously used to assess the viscosity of pleural effusion samples and discriminate between transudates and exudates, an important first step in clinical diagnostics. The presented automation includes the design, testing, and characterization of a vacuumactuated loading station that handles the 2 mm glass spheres used as sensors, as well as the engineering of electronic Printed Circuit Board (PCB) incorporating a microcontroller and their synchronization with a commercial high-speed camera operating at 10 000 fps. The hereby work therefore focuses on the instrumentation-related automation efforts as the general method and clinical application have been reported earlier [Hurth et al., J. Appl. Phys. 110, 034701 (2011)]. In addition, we validate the performance of the automated setup with the calibration for viscosity measurements using water/glycerol standard solutions and the determination of the viscosity of an “unknown” solution of C 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4840855] hydroxyethyl cellulose. V I. INTRODUCTION
Historically, most studies have focused essentially on the fluid dynamics and mechanical forces in play when a liquid drop is launched onto a liquid or solid surface and the resulting impact sequence is acquired with a high-speed recording device. Pioneering work, reviewed by Yarin,1 for instance, justifies the interest of solid impacts on liquids by possible industrial applications in inkjet printing or surface treatments. In addition, the surface of a liquid drop is always smooth and free of defects, which makes the impact situation reproducible and easier to interpret experimentally. The interest in using a solid impactor resides, for us, in the future possibility of modifying its surface. Most of the studies done so far focused on the differences between a hydrophilic and hydrophobic treatment2,3 as the solid sphere enters the liquid4 and the subsequent formation of an air entrainment cavity and its dynamics5 and texture.6 Our approach innovates in several aspects: (i) we focus on the splashing event after the bead enters the liquid to be probed, and (ii) we can later coat the bead (impactor) with sensor molecules to detect specific biomolecules in the sample probed. Because we chose to focus on the splashing event rather than the entrance of the bead or the cavity formation, we can restrict our studies to hydrophilic beads with or without chemical coatings. We have previously shown that the formation timeline and the shape of the produced splash can be directly linked to the viscosity of the sample.7 The approach of modifying the impactor was preferred over dropping liquid on solid surfaces8–10 and modifying the surface11,12 because of the possibility to multiplex the detection by dropping several beads with different coatings shortly after each other without having to prepare and switch multiple surfaces. Another notable approach previously published is to add surfactants to a)
Authors to whom correspondence should be addressed. Electronic addresses:
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the liquid probed and study the influence on the splashing event,13 but this is not suitable for clinical settings where minimum sample processing and direct probing are expected by clinicians.7 The motivation to design and build an automated apparatus to probe the impact of solid spheres using high-speed imaging originates in several aspects. A first challenge we addressed is the launching of the glass spheres, as it has been known that the geometry of the impact is a major factor in how the liquid responds after impact since Worthington’s original work.14 The solid impactor ideally falls straight in the center (apex) of the liquid. Spinning spheres have recently been shown to modify the cavity formation due to a modified surface velocity distribution.15 It is therefore essential to build a launching system improved from the initial attempts.5,16 In addition, our approach offers a higherthroughput since up to 80 beads can be launched individually or in a sequence, which offers a higher statistical accuracy of the measurement. Potentially, several beads can be launched in a row to study the response of the sample to a series of perturbation or investigate hysteresis effects. We subsequently describe the mechanism and electronic controls and sensors used to load, position, launch, and reload 2 mm borosilicate glass spheres, then synchronize the recording of their impact on a liquid sample using a high-speed camera operated at 10 000 frames per second (fps). Finally, we characterize the fall trajectory of the bead and the effects of air resistance, and illustrate the performance of the system on water-glycerol mixtures of known tabulated viscosities and use this calibration curve to measure the viscosity of a 0.1% hydroxyethyl cellulose (HEC) solution. II. INSTRUMENTATION AND MATERIALS USED
The apparatus presented in this work is composed of several sub-elements that will be briefly described hereafter.
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Most efforts were focused on the design, fabrication, and testing of the control electronic boards using an desktop PCB milling machine (LPKF Laser and Electronics, Tualatin, OR). A. System description
The system is composed of 3 main elements designed to pick up, position, and launch small solid glass spheres on top of a liquid sample and record the subsequent events at a high frame rate; a positioning stage, a bead pick-up assembly, and a high-speed imaging device. A system diagram is given in Figure 1 showing the interaction between the components controlled by electronic boards designed and built in-house. The subsections below describe each of the elements in detail. In addition, an annotated photograph of the setup is given as supporting information (Figure S1).17
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1. Glass spheres
The solid spheres used as impactor are 2 6 0.02 mm diameter surface polished borosilicate glass beads (Sigmund Lindner GmbH, Warmensteinach, Germany). They are cleaned for 2 min in a O2 plasma cleaner (Harrick Plasma, Ithaca, NY) to remove organic pollutants adsorbed on the surface and leave a hydrophilic surface behind. Silane-coated beads were obtained from MO-SCI Healthcare LLC (Rolla, MO) with either a “heavy” or “light” coating using 3-aminopropyltriethoxysilane (APTES). They were used as received. 2. Positioning stage
The experiment to be conducted requires the bead to be launched at a series of known heights from the sample, i.e., different impact velocities. For this purpose, we control a stepper-motor driven long travel (300 mm) stage (Thorlabs, Inc., LTS300) mounted vertically using the universal serial bus (USB) interface and a LabView routine. The stage has a specified maximum load of 4 kg when used vertically. The linear stage holds the components of the bead pick-up assembly described below. 3. Bead pick-up assembly
The bead pick-up assembly is mounted to the moving stage. It consists of a fixed nozzle, a machined notched polycarbonate (PC) disk, a stepper motor to rotate the disk, a linear actuator to tip the disk, a vacuum pump, sensor, and solenoid valve. The mechanical parts were machined out of PC to keep the assembly weight well below the maximum vertical load of the positioning stage. The miniature diaphragm vacuum pump (Parker BTC H022C-11) is located in the controller unit, away from the travel stage. It is powered by a switched 12 V DC power supply and offers vacuum down to 4 psi. a. Nozzle. The pick-up nozzle is made of a 0.3 mm I.D. (0.5 mm O.D.) quartz capillary embedded in a machined PC piece to which the vacuum tubing (1/4” polyurethane) is connected using a push-to-connect fitting (SMC Corporation KQ2H07-32). When vacuum is applied to the nozzle and the capillary end is placed about 0.5 mm above the glass bead, it will pick it up and hold it until the vacuum is released. The vacuum sensor can detect when the nozzle is clear and drawing air, or is blocked by a bead or clog. b. Sensor. The vacuum sensor (SMC Corporation ZSE10-N01-PG) measures the vacuum in the range of 0.0 to 101.0 kPa, to generate two signals. A digital signal used for bead drop timing. The vacuum threshold is programmed into the sensor itself. A 0–5 V analog signal is sent to the controller for conversion. The analog conversion result is used to generate a measurement similar to the reading displayed on the sensor itself, but it is used only for diagnostics. FIG. 1. Block system diagram of the setup. The main board provides I/O controls to the different system components such as the vacuum pump, the linear stage, a stepper motor to rotate the bead-containing disk, and a linear actuator to tip the disk.
c. Solenoid valve. A 3-way solenoid valve (Parker Series 3) switches the nozzle vacuum. The common port connects to the vacuum sensor and nozzle. When de-energized, the
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common port is vented to ambient pressure, thus blocking the vacuum. When energized, the common port is connected to the vacuum pump, applying vacuum to the nozzle and sensor. d. Bead holder. A 2.8 mm thick 130 mm diameter PC disk was machined using a computer-numerical-control (CNC) system (Roland MDX-540). The disk is segmented into 4 quarters of 22 slots aligned along a 120 mm diameter circumference. The 2 mm glass beads are placed inside machined spherical pockets (slots) that are 1.25 mm deep and 2 mm wide so that the beads are recessed by 0.5 mm into the slot and do not move while the disk is rotated. To avoid excessive contact with the bead surface, the bottom of the slots is opened over a 1.5 mm diameter circle. Four wider (Ø ¼ 6 mm) holes are placed on the disk to serve as launching holes. The disk is rotated using a stepper motor (Circuit Specialists 35BYG101, NEMA 14, 200 steps/revolution) to position a bead or a launching hole below the nozzle. Beads and drop openings on the disk are located every other motor step. Mechanical damping is provided by two Teflon friction blocks to the bottom surface of the disk. Without these blocks, the beads cannot sit stable in the slots due to the inherent jerks of the stepper motor actuation. A linear actuator (Firgelli PQ12-100-12-S) incorporating limit switches at both ends of the operating range tips the stepper motor and bead loader disk up to the nozzle, or down away from the nozzle. The actuator drives to one limit switch then stops when DC power is applied. Reversing the DC polarity causes the actuator to drive towards the other limit switch and stop. Tipping operations are timed without positional feedback. Raising the disk (and bead) to the nozzle drives the actuator to its limit switch, and places the bead loader in a known position. 4. Electronic controls a. Main board. The main board provides two independent serial communication channels (RS-232 and RS-485 (unused)), each on their own connector. Eight I/O pins, 12 V DC power and ground are offered in one connector— these connect directly to the driver board. One pin was modified to connect the analog vacuum signal to an analog input on the microcontroller (PIC18F8722-I/PT). An additional 8 I/O pins offer þ5 V and ground signals. Three CMOS outputs are used for camera triggering. Three more outputs control the stepper motor controller (Enable, Step, and Direction). b. Driver board. The main board outputs low power signals for driving power switches on the driver board. These switches in turn control the vacuum pump, vacuum solenoid, and actuator power and polarity. 12 V DC power is provided to the vacuum sensor, and analog and digital signals from the vacuum sensor are relayed back to the main board. c. Stepper motor driver. The stepper motor controller
(Circuit Specialists QJ-215) drives the stepper motor. Single
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stepping mode and motor current limiting (420 mA) are set using Dual In-line Package (DIP) switches. 3 control inputs (Enable, Direction, Step) are optically isolated from the motor drive circuits and asserted by grounding, otherwise they are allowed to flow high. A separate motor power source is used to drive the Aþ, A, Bþ, and B outputs. These are intended for use with a 2-phase bipolar (4-wire) stepper motor. The main 12 V supply that powers the pump and solenoids is also used to power the stepper motor outputs. d. Enclosure. The electronic control boards are contained in an enclosure containing the vacuum pump and the main 12 V power supply. The controller and driver boards operate all the components on the bead pick-up assembly, as well as the vacuum pump. They also generate camera triggers, and provide communications with the host computer. B. High-speed imaging 1. Imaging device
An Olympus iSpeed TR camera is used and is capable of capturing 1280 1024 pixels full frame 8-bit grayscale images up to 2 k FPS to its 4 GB buffer, providing up to 1.22 s of recording time. Using the “Economy” mode, various sized portions of the video frame may be captured at higher rates. At 5 k FPS, the image is 804 600 pixels (1.33 s); at 10 k FPS, 528 396 pixels (1.54 s). Configuration, control, and image downloading occur over a dedicated 1000-base-T Cat5e local area network (LAN) connection to the controlling computer. 2. Camera lens
A Nikkor Micro 200 mm F/4.0 internal focus (IF) lens and 27.5 mm extension tube are used to provide the desired magnification while maintaining a short focal distance. The iSpeed TR image size of 34.4 mm diagonal required a lens able to fully illuminate a 1.4 in. format frame. The extension tube decreased the lens minimum focusing distance from 710 mm to less than 375 mm. The light source (100 W ultra-high pressure (UHP) mercury lamp, Olympus ILP-2) allows well-contrasted images >10 000 fps. The camera/lens assembly weighs 5.9 kg. When equipped with the lens, the center of gravity of the assembly is about even with the lens mount face on the camera, well forward of the camera tripod mount. The lens and camera were mounted on a common plate to ease the load exerted on the lens mount, especially when the assembly is tipped to the 45 imaging position. 3. Triggering
Outputs from the microcontroller are 5 V digital signals, timed with 12.8 ls resolution. Several outputs are made available. A. Trigger A is asserted when the vacuum detected drops as part of a normal bead drop and timing begins. The trigger is released as the timer runs out. This trigger output was
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used to determine operational delays experimentally, and to verify that bead releases occurred consistently. B. Timing for trigger B also begins when vacuum is detected as “lost.” The trigger is asserted when the first timer runs out (when a bead enters the camera field of view). The timer is reloaded to run out as the bead exits the field of view. As the second timer period runs out, the trigger is released. This output is used for normal camera triggering when dropping beads for gathering data. These trigger functions are not intended to be used with cameras using mechanical switches for triggering. These cameras usually contain a debounce filter with a ResistorCapacitor Low Pass Filter (RC LPF) to clean up the trigger signal from a manually operated mechanical switch. This would introduce an unpredictable, variable 10–30 ms delay, which makes precise trigger timing impossible. The camera used in this setup does not have such a low pass filter. 4. Timing model
The general assumption for the timing model assumes beads drop from 0 to 100 mm into a camera field of view 8 mm high. Recording is to start when a bead enters the field of view and stop approximately 15 ms after the initial impact with the liquid sample. All system timing is based on a 12.8 ls timer tick. At 10 000 fps, this allows for 7.8 timer ticks per frame, with reasonable computational requirements. A bead dropped from 150 mm requires 13 481 (0x34A9) timer ticks (172.6 ms) to reach the cameras field of view, and an additional 364 (0x016C) timer ticks (4.7 ms, 47 frames) to exit the cameras view. All timer tick values can be represented using an unsigned 16-bit integer (0-65 535, 0-0xFFFF) up to 1000 mm. The validity of this timing model based on the contribution of air resistance and the deviation of the falling conditions from those of free fall is discussed later. III. RESULTS AND DISCUSSION
Two levels of characterization and testing will be exposed in Sections III A and III B. The first one simply
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concerns the reliability of picking up, positioning, and launching the bead using vacuum generated by a miniature pump. Then, we will assess the bead release and fall conditions using the high-speed camera and discuss the timing model. Finally, an application using viscosity standards made of mixtures of water and glycerol will be given to illustrate the data processing and the intended use of the presented apparatus. A. Reliability of the bead pick-up assembly
The success of the experiment for which the apparatus is designed is the reliable pick-up and launching of multiple glass beads in a row. Figure 2 (multimedia view) gives the timeline of the pick and launch sequence from a movie illustrating the functioning of the bead pick-up assembly. The whole movie (7.7 MB) is available. In Figure 2 (multimedia view), the disk is first moved to a location determined by stepper motor clicks to align a bead location with the capillary nozzle as the vacuum pump has been turned on (a). The disk is then tipped up towards the nozzle and the bead sucked onto the capillary (b). The disk then tips down away from the nozzle as the disk is rotated again to align the nozzle with the nearest larger diameter drop hole (c). At this point, the vacuum is switched off and the bead is launched (d). This starts the delay function of the trigger function. The next bead slot is treated similarly. The system can detect if a bead is on the nozzle. If a strong vacuum is measured by the sensor, this means a bead is loaded (or the nozzle is clogged). If a weak vacuum is detected, the bead was not picked up or there was no bead present in this particular slot. In this case, the stepper motor moves the disk to the next slot. The full movie illustrates such cases, as well as a proper pick-up and launching sequence. Once the position of the nozzle is manually adjusted by the user using the set screws provided, the success rate of picking up and dropping a bead is very high. We report >96% success out of hundreds of attempts. Similarly, the discrimination rate between a successful pick-up and an empty slot is >97%. The few failures originated from of misalignment between the nozzle and the bead in areas of the disc where there is deformation from
FIG. 2. Simplified sequence of the bead pick-up process. The vacuum is turned on and the disk is rotated to position a bead slot under the nozzle (a), the disk is tipped away from the nozzle after the pressure sensor has detected a strong vacuum (b), the disk is moved to place a launching hole under the nozzle (c), and the vacuum is turned off to release the bead (d). When a weak vacuum is detected, the delay timer in the trigger signal for recording starts. The entire movie sequence illustrates cases where no bead is present and the disk moves to the next available slot (Multimedia view [URL: http://dx.doi.org/10.1063/1.4840855.1], 7.7 MB).
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planarity over time. This could be fixed by machining a thicker disk and adding mechanical support to the disk. Another cause of failure observed is damages or deposits on the glass capillary. A timeout counter is built into the system. After N successive occurrences of weak vacuum readings on the sensor, the system reports an error and stops. The number N is set by the user and can be changed from the default value of 2. B. Influence of air resistance on bead drop conditions
In order to approximate the starting parameters of the timing function for camera triggering, we first assessed the fall conditions of the glass bead. A series of clean beads were dropped and the time needed for the beads to reach the top of the camera field of view was measured as a function of the increasing drop distance at which the bed was released into the sample (pure water). For this set of experiments, the trigger timer is asserted as soon as the vacuum is sensed as “lost” by the vacuum gauge. There is no delay and the timer starts ticking as soon as the vacuum is lost. This corresponds to situation A in Sec. II B 3. The time to the impact with the sample can be affected by air resistance on the falling glass bead. Two models linking the drop height z to the time t were considered. First, a simple free fall model without air resistance where the fall time t is linked to the drop height z by pffiffiffiffiffiffiffiffiffiffi t ¼ 2z=g; (1) where g is the gravitational constant (g¼ 9.81 m s2). Additionally, a model where air resistance is taken into account by a velocity-dependent Rayleigh drag force is considered 2
Fdrag ¼ 1=2 qv Cd A;
(2a)
t ¼ vt =g acoshðexpðgz=v2t ÞÞ; pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi vt ¼ mg=qCd A ¼ 0:02616=Cd ;
(2b) (2c)
where vt is the terminal velocity for a 2-mm-diameter glass sphere of mass m and surface area A. q is the density of air at 25 C (q ¼ 1.1839 kg m3). The fit of the experimental data to both models (Figure 3) yields a terminal velocity vt ¼ 18.4 m s1. Given the experimental conditions (Eq. (2c)), this would correspond to Cd ¼ 2.7 106 1, indicating a negligible Rayleigh drag force and contribution from air resistance. This is also shown in Figure 3 where both fitting curves overlap. In both cases, there is however a constant offset t0 ¼ 37 ms. This delay is independent of the drop distance and originates from various delays accumulated in the system such as electronics delays or even bead stickiness on the capillary before release. The contribution of the bead stickiness on the quartz capillary can be put into evidence by using identical beads coated with a heavy aminosilane layer. When plasmacleaned bare borosilicate beads are dropped from a distance of 80 mm, they reach the camera field of view after, on average, 157.04 ms with a standard deviation of 0.072 ms.
FIG. 3. Evolution of the time need for the bead to reach the camera field of view with the distance from the camera the bead was dropped from. For this set of experimental data (-•-), the camera is triggered as soon as the vacuum sensor senses a pressure change. The red solid line is the fit to free fall conditions, whereas the dotted blue line represents the fit of the data when an additional Rayleigh drag force is taken into account. There is very little difference between these cases.
However, the aminosilane-coated beads travel the same distance in, on average, 140.33 ms with a standard deviation of 1.048 ms. The much higher standard deviation indicates a much higher variability of the measured fall time that is due to the delay needed for the bead to fall from the quartz capillary once the vacuum is released, due to the additional adhesion created by the heavy silane coating. The increase is however still acceptable since it still only represents 1) to a jet (W/H < 1) occurs for a higher distance.
evolution of the time to the maximum extension of the air/liquid interface and the width to height ratio at the maximum as a function of the launch distance will typically show a very small amplitude as there is much less variation recorded
over the probed launch distances. The corresponding data is made available as supporting information (Figure S2).17 The difference with uncoated beads originates from several factors. The surface of the bead, as observed under the
FIG. 6. Comparison of the evolution of the maximum vertical extension between a 0.1% (HEC, 250 kDa) solution (red dots and trace) and the calibration glycerol-water solutions presented in Figure 5. The solid lines are 2nd order polynomial fits. The 0.1% HEC solution evolves between the 10% and 15% glycerol-water solutions. This can be used to perform a linear interpolation of the viscosity: gHEC ¼ 1.38 6 0.06 cP at 25 C.
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microscope, is much smoother with the heavy coating (data not shown) and the aminosilane layer is known to make the bead surface more hydrophobic. This is attested by slightly larger entrainment cavities being formed after the bead enters the liquid, especially the 20% glycerol solution. The purpose of the aminosilane coating was to immobilize fluorescent streptavidin on the bead and investigate the effect of the strong interaction with biotin when a 1.2 mM solution of biotin in 10 mM phosphate buffer at pH ¼ 7 is probed instead of just the buffer solution. Although the coverage density of the streptavidin on the surface of the APTES-coated bead is high, as judged from the strong fluorescent signal on the surface of the bead (data not shown), no large difference in the evolution after impact by the coated beads could be observed. This is due to the limitation by the slower diffusion of the ligand to its receptor than the impact timescale, as well as the low interaction force compared to the mechanical forces of the fluid. Such an interaction between a coated bead and the solution could be detected if the interaction is coupled to a large change in viscosity, such as, for instance, in the case of collagen I formation.21 3. Viscosity interpolation
The splashing event is a secondary manifestation of the bead entry into the liquid, it is therefore very dependent on many external factors that are sometimes hard to control such as the presence of bubbles on the surface from the previous experiment. In addition, an analytical formulation of the problem is extremely complex, which makes quantitative measurements of parameters such as the viscosity difficult, all the more so as contributions from viscosity and surface tension are difficult to separate. However, the results obtained from glycerol-water solutions can be used as a calibration curve for the system because in the range of glycerol concentrations chosen, from 0% to 50% v/v, the surface tension decreases by 5.7% at 20 C while the viscosity increases by a factor 7.7.20 This also means they can be used to interpolate the viscosity of an “unknown” solution of similar surface tension. To illustrate this, we prepared a 0.1% w/w solution of 2-hydroxyethyl cellulose (HEC, 250 kDa, SigmaAldrich 308633, surface tension c ¼ 57 mN/m measured using the pendant drop method) in water and applied the method described here (Figure 6). The evolution of the time needed to reach the maximum extension of the splash with the bead launch distance D was measured and plotted towards that of the 0% (water), 5%, 10%, 15%, and 20% v/v glycerol solutions. The glycerol curves (Figure 5(a)) were used for each distance to linearly interpolate the viscosity of the 0.1% solution using the value in Table I. The average value obtained for the 0.1% HEC solution is 1.38 6 0.06 cP. This value is in good agreement with previously published measurements.22,23 The presented system could provide a novel automated metrology tool for the characterization of physic-chemical properties of several biological fluids of relevance in biomedical and clinical applications.
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IV. CONCLUSION
We show the successful automation of a setup able to reproducibly pick-up, position, and launch 2 mm solid glass spheres into a liquid sample, adjust the launch distance, and synchronize the recording of the impact with the liquid by a high-speed camera. We expose an empiric method to study the behavior of the probed liquid depending on its viscosity based on plotting two quantities, i.e., the time to reach the maximum extension of the air liquid interface and the ratio of width over height (aspect ratio) at this maximum. These two quantities are useful in determining the transition between a blob and jet after impact by the sphere. We were able to determine the viscosity on an “unknown” liquid, e.g., 0.1% v/v 2-HEC, using a calibration by water-glycerol standard solutions with good agreement with previously published measurements. ACKNOWLEDGMENTS
The technical assistance of Glen McCarty and Matthew Barrett throughout this work is highly appreciated. The authors acknowledge funding from the Arizona Biomedical Research Commission, a division of the Arizona Department of Health Services, under Contract No. ADHS-13-031272.
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