1. Introduction

Ultrasound microbubbles (MB) have been intensively investigated due to their many applications, such as enhancing ultrasound imaging for diagnosis and potential drug/gene delivery for therapy [1, 2]. The size of ultrasound microbubbles is usually distributed between 1 and 10 μm to pass through human capillaries. A microbubble is filled with gas and shelled with a thin layer that may be made of lipids, proteins, or polymers. When injected into the bloodstream and illuminated with radio-frequency (RF, such as 1-10 MHz) ultrasonic radiation, microbubbles can oscillate and emit acoustic signals that can be detected for ultrasound imaging. When ultrasound exposure is strong enough, microbubbles can be broken, which enables delivery of drugs or genes [1, 2]. Recently, targeting microbubbles are attracting much attention because the potential applications for ultrasound molecular imaging and local drug/gene delivery [1, 2]. The idea is to attach specific ligands or peptides on the surface of microbubbles because these have a high affinity to specific molecules on the endothelial cells of blood vessels. By oscillating or breaking these microbubbles, molecular imaging or local drug/gene delivery can be conducted.

Studies have shown that the oscillation of a microbubble attached on a wall (known as a bound microbubble) is significantly different from that of a free microbubble [3–5]. Generally, if a free microbubble is approximately considered a sphere, the oscillation of the entire microbubble is symmetrical (independent of the sections of the bubble). In contrast, different parts of a bound microbubble oscillate asymmetrically in 3D due to the attached wall. For therapeutic purposes, microbubble oscillation might greatly affect the opening of cell membranes on the nearby surface, and the drug and gene uptake. Therefore, investigation of the oscillation of a single microbubble attached on a wall is attracting much attention as well [6]. Unfortunately, observation of microbubble oscillation in 3D is challenging and costly, primarily for two reasons: (1) micron-sized particles, which are challenging in the spatial domain, and (2) MHz (>10⁶ cycles/second) high-speed oscillations, which are challenging in the time domain. The small size leads to a weak signal, and the high speed results in a low gain of the detection system because of a large bandwidth.

Currently, observation of a free or bound microbubble oscillation in a 2D plane (or a 1D line) has been successfully developed by using an ultra-fast framing camera (20-200 million frames per second) or a streak camera combined with an optical microscope [3, 7]. This technique is especially useful for investigating bubble oscillation and characterizing microbubble properties because it can directly show the ultrasound-induced radius change of a microbubble. However, its extremely high cost (approximately $200,000-$400,000 for an ultra-fast framing camera and $150,000-$200,000 for a fast steak camera, depending on the performance of the system) prevents it from becoming a widely-used technique. Another optical method is based on the detection of the intensity variation of the scattered light caused by an ultrasound-oscillated microbubble [8]. While this technique is much simpler and less costly than the ultra-fast camera-based system, the detected signal represents an average of the oscillation of an entire microbubble. Thus, 3D and asymmetrical oscillation of a microbubble cannot be detected using this method.

To reduce the cost dramatically and detect 3D microbubble oscillation, in this study an optical confocal microscopic system combined with a gated and intensified charge-coupled device (ICCD) camera was developed. The capability of imaging microbubble high-speed oscillation (driven by MHz ultrasound pulses) with much lower costs than with an ultra-fast framing or streak camera system was demonstrated, as were microbubble oscillations along both lateral (x and y) and axial (z) directions. Therefore, the system is an excellent alternative for 3D investigation of microbubble high-speed oscillation, especially when budgets are limited.

2. Method

2.1 System setup and principles

A schematic diagram of the overall instrumental setup is shown in Fig. 1(a). The system was constructed based on an inverted microscope (Ti-U, Nikon). It consists of an ultrasonic system and two optical systems: a confocal optical system and a time-gated ICCD imaging system. Also, a water tank containing the contrast agent was designed and positioned on the microscope. The acoustic and optical systems were aligned so that both were focused on the contrast agent. When the acoustic pressure pulses were sent out to oscillate the bubble, one of the two optical systems was used to capture the fast bubble oscillation. The detection sequence of the two optical systems can be controlled by rotating a mirror inside the microscope. In addition, a cooled CCD camera (TCC-1.4HICE-II, Tucsen) was attached to the back port (the third port) of the microscope for optically focusing the sample (not shown in the Fig. 1).

 

Fig. 1 (a) A schematic diagram showing the imaging system setup and (b) a schematic diagram showing the timing relationship among the ultrasound pulse, bubble oscillation, strobe light illumination and ICCD camera acquisition.

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The acoustic system employed a focused 1 MHz single element ultrasound transducer (UST, V314-SU-F-1.00-IN-PTF, Olympus NDT) with a focal length of 25.4 mm, a lateral full-width-at-half-maximum (FWHM) of 1.3 mm, and an axial FWHM of 13 mm. It was mounted on a 3D translation stage so that its focus could be positioned on the microbubble. An arbitrary function generator (Agilent 33220A, Agilent Tech.) was used to generate an acoustic driving pulse. The driving pulse was amplified by a radio-frequency power amplifier (PA, 2100L, Electronics & Innovation Ltd.) and was then applied to the ultrasound transducer. The driving pulse consisted of a few cycles of a 1 MHz sinusoidal wave with a repetitious rate of 2 Hz (500 ms between every two pulses). We chose a short pulse length and a low repetition rate to minimize the potential of bubble damage or significant shrinkage.

The confocal optical system was designed to detect the bubble oscillation along the vertical axis (z direction or the optical axis direction that is perpendicular to the x-y image plane of the microscope), as shown in the right dashed rectangle in Fig. 1(a). In this system, a 532 nm laser (532GLM50, Dragon Lasers) was used as a light source. The laser beam was first coupled into a single mode fiber (P1-460A-FC-2, Thorlabs) and then collimated by a lens (L1, AC253-030-A, Thorlabs) that was positioned at a distance equal to its focal length away from the fiber output. A 50/50 beam splitter (21000, Chroma Technology Corp.) was used to deliver ~50% energy of the collimated laser beam into a 100X oil immersion objective lens (CFI Plan Fluor, N.A. = 1.3, W.D. = 0.16mm, Nikon). After the objective lens, the laser beam was tightly focused into a small spot and projected onto the surface of an immobilized microbubble. The backscattered light from the microbubble surface was collected by the same objective lens and then focused on the conjugate image plane by a 200 mm tube lens. Note that the microscope provided an additional 1.5X magnification lens inside the microscope, and it was used in this study. Thus, the total magnification is 150X. A pinhole with a diameter of 50 µm (P50S, Thorlabs) was placed on the image plane and overlapped with the light focus to block the majority of the out-of-focal-plane light [9]. The light passing through the pinhole was detected by a photomultiplier tube (PMT, H7422-20, Hamamatsu). The electronic signal from the PMT was further amplified by a broadband amplifier (SR445A, Stanford Research Systems) with a gain of 25. After that, a low pass filter (BLP-10.7 + , cutoff frequency = 11 MHz, Mini-circuits) was used to reduce the high frequency noise. The amplified and filtered signal was acquired and displayed by a high-speed digital oscilloscope (DPO7254, Tektronix, Inc.) triggered by the pulse delay generator.

To image the horizontal oscillation of the microbubble on x-y plane (parallel to the image plane of the microscope), a time-gated ICCD-based imaging system was designed, as shown in the left dashed rectangle in Fig. 1(a). In this system, an ultra-bright xenon strobe light (AC-4020-C, Electromatic Equip’t CO., Inc.) with a ~10 µs pulse width was employed as a light source. Considering the spectrum of the light source is centered at λ = ~550 nm, the horizontal resolution of the system can be calculated as λ/(2*N.A.) = 550 nm/(2*1.3) = 0.21 µm. The light was coupled into a 3.3 mm diameter fiber bundle (40-644, Edmund Optics). The other end of the fiber bundle was submerged into the water tank and used to illuminate the microbubbles from the top. The transmitted light through the microbubble was detected by the gated ICCD camera (Picostar HR, LaVision, Goettingen, Germany). The ICCD camera was triggered and gated on/off by a high rate imager (HRI) (Kentech Instruments Ltd., Oxfordshire, England). The camera capture window was determined by the “gate on” width, which was set to 20 ns in this study. A programmable delay unit (DEL 150/350 PC interface card) sent the trigger signal to the HRI with designed delay times using DaVis software (LaVision). The delay unit was programmed so that the 20 ns gating pulse was subsequently delayed by 100 ns relative to each ultrasonic pulse. A total of 44 images was acquired to reconstruct a bubble oscillation event with 4.4 µs duration time. Note that the number of frames could be selected by a user based on the specific experimental conditions, such as ultrasound frequency and pulse duration time. The acquired images were stored in a personal computer (PC) for processing.

A diagram displaying the timing relationship between the gated ICCD imaging system and the ultrasonic system is presented in Fig. 1(b). A multi-channel pulse-delay generator (PDG) (DG645, Stanford Research Systems, CA) was used as the master clock in the system. PDG sent a 2 Hz pulse signal from channel 1 to trigger the function generator and drive the transducer. The travelling time of the ultrasound pulse in water from the transducer to the microbubble was t0 (~22 µs). Meanwhile, PDG sent out the second pulse from channel 2 with a delay time of t1 (15 µs) to trigger the strobe light. The delay time t1 was predetermined through experiments to ensure that the bubble was illuminated before oscillation. Similarly, PDG sent out the third pulse from channel 3 with a delay time of t2 (20 µs) to trigger the delay unit. Once the delay unit received an external trigger, it generated a triggering signal shifted by a time of (n-1)*Δt (n indicates the Nth of the received trigger; Δt = 100ns that was selected by a user) to the HRI control unit. t1 and t2 were selected for appropriately synchronizing the ultrasound pulse, bubble oscillation, light illumination, and image acquisition.

2.2 Sample preparation

The microbubbles used in this study were purchased from Targeson, Inc., (Targestar-SA, CA) and have a diameter distribution between ~1 and ~10 µm. This type of microbubble contains a lipid shell encapsulating perfluorocarbon gas. Streptavidins are attached onto the lipid shell, which allows conjugation with biotinylated ligands. The experimental samples were prepared based on the following protocols. A large petri dish (0875710E, Thermo Fisher Scientific) was used as a water tank and filled with deionized (DI) water for coupling both optic and acoustic energies onto the bubble sample. Before being filled with water, the petri dish was opened with a hole (18 mm in diameter) at the bottom. The hole was then covered by a 22 × 22 × 0.12 mm³ coverslip whose surface was coated with biotins (Bio-01, Microsurfaces, Inc., TX). A ~5 µl microbubble solution was taken from the purchased stock solution (2.4~2.7 × 10⁹ particles/ml) and diluted with 400 µL PBS buffer (~3 × 10⁷ particles/ml after dilution). A small drop (~200 µL) was pipetted onto the biotinylated coverslip surface. Then the petri dish was carefully placed upside down (before being filled with water). The buoyancy generated by the microbubble solution forced the microbubbles to attach to the biotinylated coverslip surface. After the solution incubated for 20 minutes, the microbubbles were immobilized on the coverslip surface via biotin-streptavidin interaction. Thereafter, the petri dish was placed right-side up and filled with DI water. Unbounded bubbles floated away due to buoyancy.

The optical focus of the microscope objective and acoustic focus of the transducer were aligned before the experiments. A circular cone-shaped cap was designed, machined, and mounted onto the transducer. The height from the transducer surface to the cone apex was 25.4 mm, indicating that the apex was on the focus of the transducer. Then the apex was positioned on the focus of the microscope objective via a 3D translational stage. Finally, the cap was removed and the optical and acoustical focuses were aligned. Note that the size of the acoustic focus was much larger than the size of the optical focus. Therefore, the overlapping between the two foci was straightforward.

2.3 Image processing for diameter-time curves

In order to extract the diameter information of the bubble, the original images were processed with a code programmed in MATLAB (The MathWorks, Inc.). In general, the size of the object was determined by tracking the contour of the object. Specifically, each grayscale image was first transformed into a binary image by using a user-specified threshold. The threshold value was computed and adjusted based on the intensity and gradient of the image. The binary image showed lines of high contrast, which delineated the outline of the bubble. Then the interior gaps of the outline were filled to complete the segment of the bubble. Before the diameter was calculated, the area value $A$ of the bubble was obtained as the total pixel number of the consecutive area in the bubble-filled image. Thereafter, the equivalent diameter was calculated using equation $d=\sqrt{4A/\pi }$. The image pixel size was calibrated earlier using a micro-scaled ruler.

3 Results

3.1 Microbubble static and dynamic measurement principles using the confocal system

The axial resolution of the confocal setup needs to be characterized before the experiments. Figure 2(a) shows the reflection signal created by translating a mirror through the focus of the objective peak. From this signal, an FWHM axial resolution of 2 µm was measured. In all the following experiments conducted with this system, bubbles with diameter >4 µm were chosen to avoid significant interference between the upper and lower bubble boundaries. Figure 2(b) shows a photo of a microbubble on the horizontal x-y plan with a diameter of 7.45 µm acquired by the cooled CCD camera.

 

Fig. 2 (a) A plot of the reflected signal from a mirror versus the depth of the objective focus along the z direction. The zero depth means the mirror is on the objective focus. The data were measured and normalized from the confocal setup by scanning the mirror along z direction around the focus of the objective lens. The inset schematically shows the principle of detecting the oscillation of a microbubble’s floating top surface using the confocal setup. (b) A photograph of a microbubble captured with a cooled CCD camera on the horizontal x-y plane. (c) Plots of the backscattered signals from the microbubble (shown in [b]) versus the depth of the objective focus along z direction. The zero depth means the bubble bottom surface is on the objective focus. The solid and dashed lines are measured using the confocal and non-confocal setups, respectively, by scanning objective lens along z direction around its focus. The inset schematically shows the measurement geometry.

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The capability of optical sectioning with a resolution of 2 µm along the z direction provides an opportunity to measure the static surface profile (without ultrasound pulses) and dynamic oscillation (driven by ultrasound pulses) of a microbubble along the optical axis of the microscope. The static diameter (along z direction) of the microbubble was measured and plotted in Fig. 2(c). The data were acquired by scanning the objective focus along the optical axis at the bubble’s center (see the inset). The measured backscattered optical signal was plotted as a function of the depth of the objective focus. The solid and dashed lines represent the measured results with and without the pinhole, respectively. Two peaks were observed in the data acquired by the confocal setup (with the pinhole). Because the boundary between the bubble shell and bubble gas has a large reflection coefficient, the two signal peaks should be generated by the bubble’s bottom (shell-to-gas) and top (gas-to-shell) boundaries. The distance between the two peaks was measured as ~7.5 µm, which is considered the vertical diameter of the microbubble. This vertical (z) diameter agrees well with the horizontal (x-y) diameter measured by the cooled CCD camera. In contrast, when the pinhole was removed, the resolution in the axial direction is low so that the system lost the capability to resolve the fine structure of the bubble along z direction. The central peaks between the two boundaries observed in Fig. 2(c) were called pseudo peaks (not noise) that were caused by the interference of the reflected optical signals from the two boundaries. The central peaks were usually weaker compared to the peaks generated from the boundaries.

After the characterization of the confocal system and measurements of the static properties of the microbubble, the dynamic oscillation of the microbubble during acoustic insonation was detected using the confocal setup. As one example, Fig. 2(a) schematically illustrates the principle of detecting the oscillation of the bubble’s floating top surface. (The top represents the central top in the following sections, unless otherwise noted.) The falling edge of the signal-vs.-depth curve showed a nearly linear relationship between the received signal and the sample depth. The middle point of the falling edge of the curve was selected as the operating point, which meant that the sample (or the top surface of the bubble) was positioned at this point before applying ultrasound pulses. When ultrasound pulses were sent out, the bubble contracted and expanded in response to the positive and negative pressure cycles, respectively. Accordingly, its top surface moved inward and outward with respect to the operating point. This motion therefore led to the correspondingly and nearly linear variation of the backscattered optical signal. In Fig. 2(a), the three circles schematically indicate bubble oscillation, and the line with double arrows indicates the optical signal variation.

3.2. Microbubble oscillation measurements using the gated ICCD system and the confocal system

A typical microbubble with a diameter of 5.6 µm was insonified by an ultrasound pulse generated by a 1 MHz electronic wave with two sinusoidal cycles. The peak pressure was measured as 180 kPa by using a calibrated needle hydrophone (HNP-0200, Onda Corp., CA) and a preamplifier (AH2012, Onda Corp., CA). In the horizontal plane, the time-gated ICCD system captured a sequence of 44 consecutive images of the oscillating bubble, as shown in Fig. 3(a). In each image, the diameter was determined by tracking the contour of the bubble (as discussed in section 2.3), which was outlined by the green line. The horizontal diameter-vs.-time curve, $d(t)$, of the bubble was plotted as the red dashed line in Fig. 3(c). It can be seen that the measured microbubble oscillation curve on the x-y plane is very close to a two-cycle sinusoidal wave, except that a small oscillation occurs after the first two cycles. This indicates that the ultrasound driving pressure wave has an extra small oscillation after the two main cycles. This is normal and mainly due to the dynamic response of the ultrasound transducer to the electronic driving signal. This is further confirmed in Fig. 4 by directly measuring the acoustic pressure wave using the needle hydrophone.

 

Fig. 3 (a) A sequence of 44 images shows the ultrasound-driven oscillation of a microbubble (the static diameter = 5.6 µm), recorded by the gated ICCD camera. The number indicates the absolute acquisition time in µs. The time interval between two adjacent images is 100 ns. (b) The backscattered signal as a function of the time from the central top surface of the same oscillating microbubble, acquired by the confocal optical system. (c) Microbubble diameter oscillation (with a unit of micron) along the vertical and horizontal directions.

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Fig. 4 (a) A schematic diagram shows the four selected locations on the bubble surface. (b) the measured ultrasound pressure wave excited by a 3-cycle sinusoidal wave. (c) AC/DC data acquired at the four different locations (as shown in Fig. 4(a)) on the surface of a microbubble with a diameter of 8.5 µm. The AC/DC data represent the oscillation strength of the microbubble surface.

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The vertical oscillation of the same bubble was measured using the confocal optical system. Before oscillation, a signal-vs.-depth profile as in Fig. 2(c) was obtained and used for converting the optical signal into the bubble diameter after experiments. The bubble’s top surface was then positioned on the operating point, as described previously. When the bubble was oscillated, the backscattered optical signal was acquired (with a unit of millivolts, mV) as a function of time, as shown in Fig. 3(b). The acquired voltage signal was converted into the depth or bubble vertical diameter via the previously measured signal-vs.-depth profile. The converted data of the vertical oscillation versus time are shown in Fig. 3(c) as a solid blue line. The oscillation amplitude in vertical direction is slightly greater than that in horizontal plane by ~0.3 µm. This can be explained by the asymmetrical oscillation of an attached microbubble [2, 3].

3.3. Oscillations at different locations on a microbubble surface using the confocal optical system

Because the confocal system has the capability to selectively focus the laser beam on different locations on the bubble surface by translating the microbubble sample (adopted in this work) or scanning the laser beam, we compared the oscillation behaviors at four different locations on a microbubble surface based on the backscattered signal. Figure 4(a) schematically shows the relative locations of the four measurements. When the beam focus was on the central axis of the microbubble [central top and central bottom positions in Fig. 4(a)], the oscillation direction was parallel to the optical axis. Thus, the detected signal can be converted into the distance change based on the prior measured signal-vs.-depth. However, when the locations are a certain distance off from the bubble central axis [see the right top and right bottom in Fig. 4(a)], the bubble oscillation direction has a certain angle relative to the optical axis, which may affect the received backscattered photons. Thus, the absolute distance oscillation may be difficult to quantify based on the above-mentioned signal-vs.-depth method. In order to compare the oscillations at different locations, the detected oscillation signal when ultrasound was on [so-called alternating-current (AC) signal] was normalized by the static signal when ultrasound was off [so-called direct-current (DC) signal]. Thus, a relative ratio AC/DC was used for the comparison among different locations.

Figure 4(b) shows the measured ultrasound pressure wave excited by a 3-cycle sinusoidal electronic wave using the calibrated needle hydrophone. The measured pressure has an oscillation a little more than 3 cycles, which is commonly observed in the literature and due mainly to the convolution between the 3-cycle driving signal and the impulse response functions of the ultrasound transducer and the hydrophone [10]. The oscillation signal ratio AC/DC at the four locations are shown in Fig. 4(c). The results show that the oscillation strength depends greatly on the locations. For example, the solid blue and the dotted black lines represent the oscillations at the central top and the central bottom surface of the bubble, respectively. The oscillation at the central bottom is the weakest. In contrast, the oscillation at the central top is the strongest among all the oscillations at the four locations. This is expected because the central bottom surface of the bubble is attached onto the coverslip and has little freedom to move, but the central top location has the most freedom to oscillate. The dashed red and the dash-dotted green curves show the oscillations at the right top and right bottom locations of the bubble, respectively. The oscillation on the right top is slightly weaker than that at the central top but stronger than that at the right bottom. The oscillation at the right bottom is stronger than that at the central bottom. These observations imply that the oscillations become relatively weaker toward the bound site of the bubble (such as the bottom and side locations compared with the central and top locations). This conclusion is true for all the bubbles studied in this work.

3.4. Microbubble oscillation as a function of acoustic pressure measured by the gated ICCD system

Microbubble oscillation behaviors under different ultrasound pressures were studied using the gated ICCD imaging system. In the applied pressure range, we did not observe microbubble pushing away caused by the acoustic force. Three bubble parameters, static diameter before oscillation, maximum diameter during bubble expansion, and minimum diameter during bubble contraction, were plotted against acoustic pressure, as shown in Fig. 5. With the increase of ultrasound pressure, the microbubble was gradually shrunk. At 1350 kPa, the static diameter of the bubble was reduced below 30% of its initial static diameter. This may result from the diffusion of the gas from the bubble core and/or the fragmentation of the bubbles during oscillation [2, 11]. In majority of the experiments, we did not observe microbubble break or cavitation at certain pressure point. Instead, we observed microbubbles’ shrinkage and deformation at high pressures. The oscillation amplitude was calculated as the difference between the maximum and the minimum oscillating diameters ($d_{\max }-d_{\min }$) at each pressure, which reached a maximum value of ~2.9 µm at 630 kPa. Additionally, when the pressure was between 270 and 810 kPa, the bubble was relatively easier to compress than to expand. This asymmetrical oscillation can be found in the literature and explained based on the nonlinear oscillation behavior [12–14]. Many factors may affect this phenomenon, such as microbubble materials (shell material, gas materials, surface phospholipid concentration, and extra coating materials), microbubble size, immobilization characterization of the microbubble (if immobilized), the properties of the substrate where the microbubble is attached (if immobilized), and the ultrasound frequency. It has been studied that the resonance frequency of 1~10 µm microbubbles lies between 1~10 MHz, and usually the larger sized microbubbles have a lower resonance frequency. Matching the insonification frequency with the resonant frequency of the microbubble may provide optimized conditions for microbubble oscillation. It may be helpful to investigate that how different sized bounded microbubble respond to different ultrasound frequency in the future studies.

 

Fig. 5 Microbubble’s static diameter before oscillation, maximum diameter during bubble expansion, and minimum diameter during bubble contraction were plotted as a function of applied ultrasound pressure.

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4. Discussion

Detection of microbubble oscillation was demonstrated using this combined confocal optical system and gated ICCD imaging system. Compared with a framing or streak camera-based system, the major advantages and limitations of the current system are discussed.

First, the cost of the current system is much lower than that of a system based on an ultra-fast framing or streak camera. The major cost reduction of the current system is due to the adoption of a gated ICCD camera. An ICCD camera imaging system (including both hardware and software) with a gating width of tens of nanoseconds usually costs ~10 and ~5 times less than an ultra-fast framing camera or a streak camera, respectively. Therefore, the total cost of the system is dramatically reduced, and the system may be affordable for many research laboratories. (Note that the camera system is the most costly component.) The major components used in the current method include: (1) a gated ICCD camera imaging system with a gating with of tens of nanoseconds (~$30,000-$40,000), (2) an optical microscope and its illumination light source (such as a strobe light) (~$10,000-$20,000), (3) major electronic components (such as a pulse and delay generator, a function generator and a power amplifier, ~$10,000), and (4) a data acquisition device (such as an oscilloscope, <$3,000). Compared with the above-mentioned devices, the costs of other components used in this study can either be ignored (such as a laser, an ultrasound transducer, an electronic filter, optical lenses and a beam splitter, and a pinhole with a few microns in diameter) or be replaced by an affordable substitute (such as a PMT and a broadband amplifier). All the above-mentioned major components are similar to the major devices used in a system based on an ultra-fast framing or streak camera, except for the camera system and the data acquisition device. Thus, the total cost of a system similar to the one in this study can be estimated at between ~$50,000 and ~$70,000, depending primarily on the types of the adopted ICCD camera system and the optical microscopic imaging system. Note that the above estimate regarding the system’s cost is based on the general requirements of the performance of each device, not the specific cost of each device used in the current system. This is because several devices in the current system have higher performances (designed for other applications) than are needed for this application. For example, the gated ICCD camera has the narrowest gating width of 200 pico-seconds, which is much narrower than the used gating width of 20 ns in this study. Also, the oscilloscope can reach a GHz bandwidth, which is much wider than the required bandwidth of <100 MHz for current application.

Second, by combining the ICCD camera system with a confocal microscopic optical system, the high-speed microbubble oscillation along both the horizontal (via the ICCD camera) and vertical (via the confocal system) directions can be investigated on the same microbubble. This feature is especially useful for investigating asymmetrical bubble oscillation. Although a system based on a framing or streak camera is successful in imaging microbubble oscillation on the horizontal plane, it is difficult to image microbubble oscillation along both the axial and lateral directions on the same microbubble because the camera is usually lacking the imaging capability along the third axis (z), and its imaging plane is generally parallel to the horizontal plane (x-y). Studies have been conducted by using two orthogonally positioned microscopes equipped with an ultra-fast framing camera to observe microbubble oscillation along the three directions (x, y and z) [4]. While the system is highly successful, several disadvantages have been realized. (1) Only objective lenses with low magnification (such as 10 × or 20 × ) can be orthogonally positioned to focus on the same microbubble, which degrades the spatial resolution of microbubble oscillation measurement. When using two 40 × lenses, part of the protective housing of the objective lenses has to be machined away for this purpose. (2) Both system and operation complexities increase due to the use of two orthogonally positioned microscopes. (3) Cost is correspondingly increased. Besides the feature of 3-axis detection capability, the confocal system also provides the capability to investigate microbubble oscillation behaviors at various locations on the bubble surface, which is extremely difficult, if not impossible, for other systems. The above-mentioned features of the current system enable one to investigate and characterize the unique oscillation behaviors of a bound microbubble. For example, temporally and spatially (along 3 directions) asymmetrical oscillations, the effect of the attaching wall (such as rigid coverslip or soft blood vessel wall), the attaching methods and the distance between the wall and the microbubble can be investigated by using the current system. These topics are interesting and important in the field of microbubble-based ultrasound molecular imaging [15–17].

Several major limitations of the current system are discussed below. (1) The confocal system’s application is limited to a bound microbubble. (2) When using the current system, microbubbles with a diameter >4 µm should be selected to avoid any interference effect. This bubble size limit can be reduced by improving the axial resolution of the confocal system. This might be achieved by employing a shorter laser wavelength and/or using a smaller pinhole. (3) A signal-vs.-depth profile needs to be acquired for calibrating the vertical bubble size oscillation. (4) For the gated ICCD camera system, the total data acquisition time may be longer than that for a framing or streak camera system. This is because a bubble needs to be repeatedly insonified until the completed oscillation is sampled adequately. In this study, the total acquisition time of 22 s was required to capture 44 images with an ultrasound repetition rate of 2 Hz. The acquisition time can be decreased by selecting a higher ultrasound repetition rate if microbubble damage or shrinkage does not occur during the ultrasound insonation period.

5. Conclusions

In this study, to detect and investigate 3D high-speed oscillation of a bound ultrasound microbubble and significantly reduce the cost, a confocal microscopic optical system combined with a gated ICCD camera imaging system was developed. The confocal optical system provides a point-to-point measurement of the bubble dynamics along the vertical direction (z direction). The gated ICCD system measures the bubble oscillation in a horizontal plane (x-y plane). An immobilized microbubble was studied using the developed system. Both temporally and spatially asymmetric oscillations of a bound microbubble were observed. Compared with a system based on an ultra-fast framing or streak camera, this system significantly reduces the cost of imaging microbubble oscillation, which makes it affordable for many research laboratories for characterizing and investigating targeted microbubble properties. Accordingly, the proposed system can be used as a valuable tool for investigating molecule-targeting ultrasound contrast agents, especially when a framing or streak camera system is unaffordable.