A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.
In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.
There is a growing recognition that cellular heterogeneity, arising from the stochastic expression of genes, proteins, and metabolites, exists within a large cell population and serves as a fundamental principle in biology to allow for cell adaptation and evolution1. Therefore, it is often inaccurate and unreliable to use population-based bulk measurements to understand the function of individual cells and their interactions. Developing new technologies for single-cell analysis is therefore of high interest in biological and pharmacological research, and can be used, for example, to better understand the key signaling pathways and processes in stem cell biology and cancer therapy2-4. In recent years, the emergence of microfluidic platforms has greatly facilitated single-cell analysis, where the positioning, treatment, and observation of the response from individual cells have been performed with novel analytical strategies5.
Cavitation plays an important role in a diverse range of biomedical applications, including the treatment of cancers by high-intensity focused ultrasound (HIFU)6, the non-invasive fragmentation of kidney stones by shock wave lithotripsy (SWL)7, drug or gene delivery by sonoporation8, and the recently reported destruction of cells or tissues by hydrodynamic bubble cavitation9,10. Despite this, the dynamic processes of cavitation bubble(s) interactions with biological tissue and cells have not been well understood. This is due to the randomness in cavitation initiation and bubble dynamics produced by ultrasound, shock waves, and local hydraulic pressure; furthermore, there is a lack of enabling techniques to resolve the inherently complex and fast responses of biological cells, especially at the single-cell level.
Because of these challenges, it is not surprising that very few studies have been reported to investigate bubble-cell interactions under well-controlled experimental conditions. For example, membrane poration of individual cells trapped in suspension11 and the impulsive large deformation of human red blood cells12 have been demonstrated using laser-generated single bubbles in microfluidic channels. The latter technique, however, can only produce very small deformation in eukaryotic cells because of the presence of the nucleus13. Moreover, it is difficult to monitor downstream bioeffects when treating cells in suspension. In other studies, ultrasound excitation of a cell-bound microbubble (or ultrasound contrast agent) for producing membrane poration and/or intracellular calcium responses in single adherent cells has been reported8. Membrane poration of single adherent cells can also be produced by using laser-generated tandem bubbles in a thin liquid layer containing light-absorbing Trypan blue solution14, or by an oscillating gas bubble generated by microsecond laser pulses irradiating through an optically absorbing substrate in microchambers15. When compared, the optically absorbing substrate has an advantage over the laser-absorbing Trypan blue solution because the latter is toxic to cells. More importantly, laser-generated bubbles are more controllable in terms of bubble size and location than acoustically excited bubbles. Nevertheless, in all these previous studies, the cell shape, orientation, and adhesion conditions were not controlled, which may substantially influence the cell response and bioeffects produced by mechanical stresses16.
To overcome these drawbacks in previous studies, we have recently developed an experimental system for bubble generation, cell patterning, bubble-bubble-cell interactions, and real-time bioassays of cell response in a microfluidic chip constructed by using a unique combination of microfabrication techniques. Three main features that distinguish our experimental system from others in the field are: 1) the patterning of micron-sized gold dots on the glass substrate to enable localized laser absorption for bubble generation17; 2) the patterning of micron-sized islands of extracellular matrix (ECM) for cell adhesion on the same substrate to control both the location and geometry of individual cells; and 3) the compression of the dimension of the bubble-bubble-cell interaction domain from 3D to a quasi-2D space to facilitate in-plane visualization of bubble-bubble interactions, jetting flow fields, cell deformation, and bioeffects, all captured in one streamlined imaging sequence (Figure 1d).
Figure 1: The microfluidic chip and schematics of different assays. a) An assembled microfluidic chip with channels filled with blue ink for visualization. b) A region inside the microfluidic chip with patterned cells and gold dots (the distance between the two gold dots in proximity is 40 µm). Many pairs of working units can be arranged in a channel. c) Close-up image of a single working unit consisting of a pair of gold dots and a HeLa cell adhered to the cell-patterning region. d) Schematic of the device operation. A single cell adheres to and spreads on the "H"-shaped island coated with fibronectin. A pair of cavitation bubbles (tandem bubble) with anti-phase oscillation are produced by illuminating pulsed laser beams on the gold dots (see Figure 4a), leading to the generation of a fast and localized jet moving towards the target cell nearby. The cell may be deformed, porated for macromolecular uptake, and/or stimulated with a calcium response, depending upon the standoff distance (Sd) of the cell to the tandem bubble. Please click here to view a larger version of this figure.
This platform can be further combined with fluorescence assays and functionalized beads attached to the cell surface for cavitation-induced bioeffects. In particular, this platform opens the way for reliable and quantifiable assays at the single-cell level. Up to now, we have used the device for the analysis of tandem bubble-induced cell membrane deformation, cell poration and intracellular uptake, viability, apoptosis, and intracellular calcium response. In the following protocol, we describe the process of chip fabrication and the procedure for analyzing the various bioeffects mentioned above. Moreover, the operations of the chip are also described.
1. Microfabrication
NOTE: All the microfabrication procedures are performed in a cleanroom. A chrome mask is designed prior to the microfabrication, see Figure 2.
Figure 2: Schematic of the channel design in the microfluidic chip and the dimensions of the working units. a) Mask design of the aligned PDMS microchannels (green) and the patterns on the glass substrate (blue and red). b) Enlarged image of the mask design in a representative section of the working unit arrays. c) Schematic of one working unit showing a cell pattern (blue) and a pair of gold dots (red). All the length scales are shown on the bottom right. Please click here to view a larger version of this figure.
Figure 3: Schematic diagrams for microfabrication and chip assembly. a) Pattern of gold dots on the glass substrate. b) Preparation of the glass substrate for cell patterning via MAPL. c) Assembly of the microfluidic channel with plasma bonding. See the Materials List for the definitions of the abbreviations. Please click here to view a larger version of this figure.
2. Bubble Generation and Flow Visualization
NOTE: A schematic of the experimental setup is shown in Figure 4a for tandem bubble generation and the resultant flow interaction with a single cell grown nearby in a microfluidic channel.
Figure 4: Schematic of the experimental setup and image recording. a) Experimental setup for tandem bubble generation. b) Time sequence used for different types of imaging acquisitions. FL: fluorescence microscopy, BF: bright field, IFT: interframe time. Please click here to view a larger version of this figure.
3. Single-cell Analysis and Bioeffects
NOTE: The tandem bubble is produced next to the target cells, and the resultant bioeffects are studied in a standoff distance-dependent manner. The time sequence for different types of image acquisitions is depicted in Figure 4b.
The microfluidic platform described in this work can be used to investigate bubble-bubble interactions and to analyze a variety of cavitation-induced bioeffects at the single-cell level. Here, we present several examples to demonstrate a variety of experimental studies and bioassays that can be performed in our experimental system. We will first illustrate the transient interactions of tandem bubbles with the jet formation, the visualization of the resultant flow field, and the calculation of the jet speed (Figure 5a). We will then present examples of tandem bubble-induced localized cell membrane deformation (Figure 5b), pinpointed membrane poration with PI uptake (Figure 5c), and intracellular calcium response (Figure 5d). Other examples, such as cell viability and apoptosis assays, can be found in Reference 22.
Figure 5a shows an example of the tandem bubble interaction with the jet formation, captured by high-speed imaging, and the resultant flow field revealed by PIV. Specifically, following its maximal expansion, the collapse of the first bubble B1 (produced at 0 µs) is distorted asymmetrically by the rapid expansion of the second bubble B2 (produced at 2.5 µs), leading to the formation of an "upward" jet (J1) at 3.4 µs and the subsequent jetting flow, shown at 5.4 µs. The average jet speed is determined by the slope of a fitted line for the position of the proximal end (or pole) of B1 before touchdown (i.e., contact with the distal end of B1) versus time. The average jet speed is found to increase from 20 to 58 m/s when the maximum diameter of B2 increases from 40 to 60 µm. Based on the results of PIV analysis, the directional jetting flow around the tandem bubble is on the order of 10 m/s and is confined within a width on the order of 10 µm. It is thus capable of producing an impulsive and localized shear stress and stress gradient onto a target cell grown nearby.
Another example shown in Figure 5b illustrates the cell membrane deformation induced by the directional and localized jetting flow produced by the tandem bubbles at standoff distance Sd = 40 µm. The membrane deformation and recovery are highlighted by the displacement of a functionalized bead (indicated by the yellow dotted line) attached to the leading edge of the cell membrane. The local area strain can be calculated from the coordinates of a triad of adjacent beads. A schematic of the calculated maximum area change at different locations on the cell surface is shown in the middle. The leading edge is primarily stretched (see the green circles), while the trailing edge or lateral sides of the cell are compressed (as indicated by the red circles), indicating heterogeneity in cell deformation produced by the tandem bubble induced jetting flow. The temporal variation of the area strain at the cell leading edge is illustrated on the right. It is comprised of a few rapid oscillations in the beginning, followed by a large and sustained stretch for about 100 µs (FWHM) and a subsequent gradual recovery on a time scale of several ms.
Furthermore, the large area strain and strain integral at the cell leading edge may be responsible for the cell membrane poration observed at small Sd, as shown in Figure 5c. At small Sd (i.e., 10 µm, or in some cases, 20 µm) and intermediate Sd (i.e., the majority of 20 and 30 µm), a localized membrane disruption is induced, leading to a pinpoint uptake of extracellular PI into the cytosol. If the PI intensity inside the cell keeps increasing without saturation, necrosis occurs. In comparison, if the PI intensity is an order of magnitude lower and reaches a plateau within 10 s following the tandem bubble treatment, repairable poration with likely cell survival will occur, which is further supported by the minimal change in cell morphology. At large Sd (i.e., 40 µm), negligible PI uptake is found following tandem bubble treatment, indicating negligible or non-poration. The cell can survive with regular growth and proliferation22. Overall, the results show a clear transition in cell response from necrosis, through repairable membrane poration, to non-poration in the range of Sd ≈ 20-40 µm.
Finally, we show the results of ratiometric imaging of the intracellular calcium response elicited by tandem bubbles in individual cells at different Sd, especially in the sublethal range of Sd = 30-50 µm (Figure 5d). The intensity ratio is proportional to the amount of intracellular calcium. At small Sd (i.e., 30 µm or, in some cases, 40 µm), a calcium wave travelling from the leading edge to the trailing edge is clearly observed within a few seconds following the tandem bubble treatment. After the intracellular calcium reaches a peak concentration, it decays slowly back to the resting level within a few minutes. In contrast, at large Sd (i.e., a majority of 50 µm, or in some cases, 40 µm), the increase of intracellular calcium is much milder, and no clearly visible travelling calcium wave can be identified. These two different calcium responses can also be distinguished from their intensity ratio versus time profiles, with significant differences in the peak amplitude of the intensity ratio and the rise time from resting level to peak value. At an even larger Sd (i.e., above 60 µm), no calcium response can be observed. The percentage of HeLa cells displaying calcium responses at various Sd is summarized in Figure 5d (right).
All in all, our results have demonstrated that, by altering the strength of the jetting flow (or changing Sd), a variety of bioeffects can be produced in individual HeLa cells under similar culture conditions, which is likely correlated with the amplitude and duration of the mechanical deformation imposed on the cell membrane22.
Figure 5: Results from tandem bubble interaction, cell membrane deformation, membrane poration, and calcium response assays. a) Fluid motion and the jet induced by tandem bubble interaction. Left: Selected frames showing tandem bubble interactions with the jet formation and flow field revealed by PIV. The maximum diameters of the two bubbles are about 50 ± 2 µm. Middle: Schematic of the jetting tandem bubbles, illustrating the center axis, origin of the coordinates (o), and proximal and distal ends (or poles) of the first bubble B1. Right: Measured pole positions (with error = ±1 µm) at the proximal and distal end of B1. b) Cell membrane deformation. Left: The yellow dotted line labels the displacement of a PS bead attached to the leading edge of the cell membrane, indicating the local membrane deformation and recovery. Middle: a schematic showing the peak area strains at different locations on the cell surface shown on the left. Right: The time courses of the area strains in the middle of the cell leading edge. Δε is the area strain calculated based on principal strains and ΔΔ is the area strain calculated based on the triad geometry change. More details about the area strain calculation and error analysis are documented in Reference 22. c) Cell membrane poration. Left: bright field images before and after tandem bubble treatment and time-lapse fluorescence images of PI uptake (0 and 120 s). The white arrows indicate the jetting flow direction. Middle: the typical change of average PI intensity versus time inside the cells. Right: Statistical results of the percentage of cells undergoing necrosis (red), repairable poration (blue), and non-poration (green) at different Sd. Number of cells treated: N = 9, 14, 14, and 8 for Sd = 10, 20, 30, and 40, respectively. The statistic error is ±1/√N. d) Intracellular calcium response. Left: sequences of ratiometric images showing the intracellular calcium responses of individual cells at Sd of 30 µm and 50 µm. The red arrows indicate the jet direction. Middle: typical response profiles (ratio-time curves) at different Sd. Right: the probability of a calcium response at different Sd. Please click here to view a larger version of this figure.
standoff distance (Sd) (μm) | Reynolds number (Re) | averaged shear stress (Pa) in yz plane |
20 | 206.96 | 618.64 |
30 | 354.8 | 709.69 |
40 | 378.78 | 657.22 |
50 | 163.85 | 323.61 |
60 | 105.08 | 42.21 |
Table 1: A list of parameters for flow physics from the PIV results. Estimated Reynolds number and averaged shear stress with respect to different standoff distances (Sd). The YZ plane refers to the channel width x height plane. The averaged shear stress is estimated from a height of 0 to 7.7 µm from the channel bottom because the cell height in the channel is around 6-7 µm.
Single-cell analysis, in combination with live-cell imaging, has greatly enhanced our understanding of the dynamic and often variable processes in individual cells, such as phenotype development and immune response23. In contrast to the conventional cell culture in dishes or flasks, microfluidic systems enable precise control of the microenvironment, down to the single-cell level, in real time. Consequently, advances in microfluidic technology and techniques have largely improved the throughput and reproducibility of single-cell analysis. By integrating soft lithography and surface patterning, microfluidic systems can be further designed to facilitate the in-depth analysis of a single cell's response to complex patterns of spatiotemporally variable stimuli. For example, transient chemical or mechanical cues have been successfully delivered to single cells while their biological or mechanical responses were automatically recorded and analyzed24. Despite this general trend, the application of microfluidics to analyzing cavitation-induced bioeffects at the cellular level has been very limited. Most notably, it has only been applied to bubble-induced membrane poration of individual cells in suspension trapped by micropillars11. In the majority of studies on adherent cells, the benefit of controlling cell morphology to maintain consistency in dynamic bubble-cell interactions is either unavailable or largely neglected.
We have developed a microfluidic system to precisely control the initiation, subsequent expansion, and collapse dynamics of cavitation bubbles; bubble-bubble interactions with jet formation; and cell shape, orientation, adhesion pattern, and standoff distance from the bubbles, thus allowing for reproducible jetting flow to be applied to individual cells under well-controlled experimental conditions. This novel microfluidic system is ideal for carrying out fundamental studies on cavitation bubble(s)-cell interactions that are relevant to a diverse range of therapeutic ultrasound applications, such as SWL, HIFU, and sonoporation. We have demonstrated that our microfluidic system can be used to investigate various cavitation-induced bioeffects, including cell membrane deformation, membrane poration, viability and calcium response, in a more controllable and reproducible manner. Most notably, the directional jetting flow produced by tandem bubbles allows us to probe the mechanical property and calcium response in individual cells under high strain-rates that have not been well characterized. The localized, impulsive mechanical stress produced by such a jetting flow cannot be achieved by conventional stretching methods, such as micropipette aspiration25 and the use of optical26 and magnetic tweezers27. Furthermore, in contrast to previous studies using ultrasound contrast agents8,28-30 or laser-induced single bubbles31,32, our microfluidic system offers better control of cell geometry and adhesion conditions, thus reducing their substantial effect on cellular response and bioeffects16.
While our technique is reliable and reproducible, one must follow the protocol cautiously to ensure that the experimental system is faithfully reproduced. The quality of surface patterning in the microchannel is primarily responsible for the reproducibility of the bubble-bubble-cell interactions and the cascade of downstream bioeffects. For example, for HeLa cells, the area of each gold dot must be around 25-30 µm2 to ensure stable bubble generation while preventing the cells from adhering. On the other hand, the area of each fibronectin-coated island has to be around 700-900 µm2 to facilitate the adequate spreading of a single cell in a square while reducing the chance of multiple cells forming aggregates on the island. Alignment between the gold dots and the fibronectin-coated islands must be precisely performed with the aid of the mask aligner to keep the angular error within 1° and the axial error within 0.5 µm. Moreover, this experimental system provides versatility in monitoring a series of events, with their time scale varied by orders of magnitude (e.g., bubble dynamics: µs, cell deformation: ms, membrane poration: s, apoptosis: h). Therefore, it is important to accurately control the timing of the image acquisition and recording of each sequence by fine-tuning the trigger signals (controlled by a digital delay generator). The single-cell culture must be well maintained in the microchannel to minimize cell-to-cell variation in phenotype (mostly referred to as morphology). Therefore, a minimum 2-h incubation is required for cell attachment and spreading on the islands before proceeding with downstream cell treatment. It is recommended to always keep the flow rate in the microchannel under 3 µL/min, so that the flow shear stress produced by the circulating culture medium on the cell is within the physiological range.
One current limitation of our technique is that tandem bubble interaction and the resultant jetting flow can only be applied to a target cell once, because the gold dots will be ablated by the laser irradiation. This drawback limits the ability of our experimental system to mimic the bioeffects produced by therapeutic ultrasound, where cells are often exposed to cavitation events. However, this temporal limitation may be alleviated by applying another thin layer of silicon dioxide to protect the gold dots from direct exposure in the liquid15. Overall, our microfluidic system offers well-controlled experimental conditions to investigate the bioeffects produced from cavitation bubble(s)-cell interactions and has several unique features. First, both the size and the location of the cavitation bubbles in our experimental system can be precisely controlled by adjusting the incident laser energy absorbed by the gold dots. Second, the geometry and adhesion of individual cells are standardized by cell patterning to minimize their effects on cell responses and bioeffects, leading to more reproducible results. Third, the parallel working units in the microfluidic chip design make it feasible to study downstream bioeffects, such as cell growth, proliferation, and potentially gene expression, after cavitation exposure, since each cell can be addressed and monitored individually. In summary, our microfluidic system and methodology create reliable bubble-bubble interactions to study the bioeffects of individual cells with improved precision.
Our current chip is designed for the analysis of individual HeLa cells. However, a modification of the pattern islands for other mammalian cell lines are also possible. Several improvements can be made to further broaden the applications of the chip. For example, substitutes of the PLL-g-PEG molecules could be explored to stop individual patterned cells from migrating even after 24 h. In addition, new chip designs with microfluidic valves may be explored to control the local microenvironment of the cells, so that chemicals such as fluorescence markers or toxic reagents will only be applied to a specific location, without affecting the cells in other regions of the chip. With these improvements, the microfluidic system presented here may provide opportunities to study the long-term bioeffects of single cells, such as metabolism, segregation, differentiation, and gene expression, following cavitation exposure or mechanical stimuli produced by an impulsive flow.
The authors have nothing to disclose.
We would like to acknowledge the use of the clean room facility SMIF at Duke University. We also want to thank Hao Qiang for his assistance in measuring the jet velocity. The authors thank Todd Rumbaugh of Hadland Imaging for providing the Shimadzu HPV-X camera used in this study.The work was funded in part by NIH through grants 5R03EB017886-02 and 4R37DK052985-20.
Reagent/Materials | |||
75x38mm Plain Microscope Slides | Corning | 2947-75X38 | |
Acetone | Sigma Aldrich, Co. | 320110 | ACS reagent, ≥99.5% |
Isopropyl alcohol | Sigma Aldrich, Co. | W292907 | ≥99.7%, FCC, FG |
Sulfuric acid | Sigma Aldrich, Co. | 320501 | ACS reagent, 95.0-98.0% |
Hydrogen peroxide | Sigma Aldrich, Co. | 216763 | 30 wt.% in H2O |
Primer P-20 | Microchem | MCC Primer 80/20 | |
NFR photoresist | JSR | NFR016D2 | |
Photomask | Photoplotstore | N/A | 4×4 Direct write mask |
MF-319 Developer | Shipley (Rohm and Haas) | Microposit MF-319 | |
1165 Photoresist Remover | Dow Chemical, Co. | DEM-10018073 | 1-methyl-2-pyrrolidinone based |
S1813 photoresist | Shipley (Rohm and Haas) | S1813 | |
PLL-g-PEG | SuSoS | PLL(20)-g[3.5]- PEG(2) | |
HEPES | ThermoFisher Scientific | 15630080 | |
Paraffin film | HACH | 251764 | |
SU-2025 photoresist | Microchem | SU-2025 | |
PDMS | Dow Corning | 184 SIL ELAST KIT 0.5KG | |
Microbore Tubing | Saint-Gobain PPL Corp. | S-54-HL | |
Metal pins | New England Small Tube | NE-1300-01 | Cut Tube (straight), 0.025” OD x 0.017” ID x 0.50” Long |
HeLa cells | Duke Cell Culture Facility | (307-CCL-2) HeLa, p.148 | |
DPBS(1X) buffer | ThermoFisher Scientific | 14190144 | |
DMEM culture medium | ThermoFisher Scientific | 11995065 | |
Fibronectin Bovine Protein, Plasma | ThermoFisher Scientific | 33010018 | |
0.25% Trypsin-EDTA (1X) | ThermoFisher Scientific | 25200056 | |
Propidium Iodide | ThermoFisher Scientific | P21493 | |
Carboxylate Microspheres 1.00μm | Polysciences, Inc | 08226-15 | |
Carboxylate Microspheres 2.00μm | Polysciences, Inc | 18327-10 | |
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) | ThermoFisher Scientific | 22980 | |
Sulfo-NHS | Sulfo-NHS (N-hydroxysulfosuccinimide) | 24510 | |
Peptite-2000 | Advanced BioMatrix | 5020-5MG | |
FITC Annexin V | ThermoFisher Scientific | A13199 | |
Fura-2, AM | ThermoFisher Scientific | F1221 | |
DMSO | Sigma Aldrich, Co. | D2650 | |
F-127 | invitrogen | P6866 | 0.2 µm filtered (10% Solution in Water) |
Reduced serum media | ThermoFisher Scientific | 11058021 | |
Name | Company | Catalog Number | Comments |
Equipment | |||
Plasma asher | Emitech | K-1050X | O2 / Ar plasma ashing of photoresist and other organic materials |
Mask aligner | SUSS MicroTec | Karl Suss MA6/BA6 | |
E-beam evaporator | CHA Industries | CHA Industries Solution E-Beam | |
RIE | Trion Technology | Trion Technology Phantom II | (oxide/ nitride/ polymer) etching |
Stereoscope | AmScope | American Scope SM-4TZ-FRL | Stereo Microscope |
Syringe pump | Chemyx Inc | NanoJet | |
Cell culture incubator | NuAire | AutoFlow NU-8500 Water Jacket CO2 Incubator | |
Biological Safety Cabinets | NuAire | NU-425-400 | |
Water bath | VWR | 1122s | |
Centrifuge | IEC | Centra CL2 | |
Microscope | Zeiss | Axio Observer Z1 | |
Nd:YAG laser (laser 1) | New Wave Research | Tempest | |
Nd:YAG laser (laser 2) | New Wave Research | Orion | |
Delay generator | Berkeley Nucleonics | BNC 565-8c | |
Flash lamp | Dyna-Lite | ML1000 fiber-coupled flashtube | |
high speed camera | DRS Hadland | Imacon 200 | |
high speed camera | Shimadzu | HPV-X | |
high speed camera | Vision Research | Phantom V7.3 | |
PIV software | LaVision | DaVis 7.2 | |
camera | Zeiss | AxioCam MRc 5 | |
software | Zeiss | AxioVision | |
PTI system | Horiba | S/N: 1705 RAM-X | |
EasyRatio software | Horiba | Easy Ratio Pro 2 | version 2.3.125.86 |
63× objective | Zeiss | LD Plan Neofluar |