Optical and acoustic detection of laser-generated microbubbles in single cells

Acoustically monitored laser-induced optical breakdown (LIOB) has potential as an important tool to diagnose and treat living cells. Laser-induced intracellular microbubbles are readily detectable using high-frequency ultrasound, and LIOB can be controlled to operate within two distinct regimes. In the nondestructive regime, a single, short-lived bubble can be generated within a cell, without affecting its immediate viability. In the destructive regime, the induced photodisruption quickly can kill a targeted cell. To generate and monitor this range of bioeffects in real time, we have developed a system integrating an ultrafast laser source with optical and acoustic microscopy. Experiments were performed on monolayers of Chinese hamster ovary (CHO) cells. A 793 nm, 100 fs laser pulsed at 3.8 kHz was tightly focused within each cell to produce the photodisruption, and a 50 MHz ultrasonic transducer monitored the resultant bubble via continuous pulse-echo recordings. Photodisruption was also observed using bright field microscopy, and cell viability was assessed following laser exposure with a trypan blue assay. By controlling laser pulse fluence and exposure duration, either nondestructive or destructive LIOB could be produced. The intracellular position of the laser focus was also varied to demonstrate that cell viability was affected by the specific location of material breakdown.     

Bubble-based acoustic radiation force elasticity imaging

Acoustic radiation force is applied to bubbles generated by laser-induced optical breakdown (LIOB) to study viscoelastic properties of the surrounding medium. In this investigation, femtosecond laser pulses are focused in the volume of gelatin phantoms of different concentrations to form bubbles. A two-element confocal ultrasonic transducer generates acoustic radiation force on individual bubbles while monitoring their displacement within a viscoelastic medium. Tone burst pushes of varying duration have been applied by the outer element at 1.5 MHz. The inner element receives pulse-echo recordings at 7.44 MHz before, during, and after the excitation bursts, and crosscorrelation processing is performed offline to monitor bubble position. Maximum bubble displacements are inversely related to the Young's moduli for different gel phantoms, with a maximum bubble displacement of over 200 microm in a gel phantom with a Young's modulus of 1.7 kPa. Bubble displacements scale with the applied acoustic radiation force and displacements can be normalized to correct for differences in bubble size. Exponential time constants for bubble displacement curves are independent of bubble radius and follow a decreasing trend with the Young's modulus of the surrounding medium. These results demonstrate the potential for bubble-based acoustic radiation force methods to measure tissue viscoelastic properties.     

High frequency ultrasound imaging with optical arrays

Dynamically focused and steered high frequency ultrasound imaging systems require arrays with fine element spacing, wide bandwidths, and large apertures. However, these characteristics are difficult to achieve at frequencies greater than 30 MHz using conventional array construction methods. Optical schemes offer a solution. Focused laser beams incident on a suitable surface can generate and detect acoustic radiation. Precisely controlling the position and size of the beams defines points of transmission and detection, making it possible for pulse-echo image formation by synthetic aperture methods. An optical detection array was built, relying on a conventional piezoelectric transducer as an ultrasound source. The detection system, with near optimal resolution over a wide depth of field, demonstrates the potential for high frequency array implementation using optical techniques. A possible application is in pathology, where 2-D or 3-D fine resolution pulse-echo imaging can be performed in situ without the need for biopsies.     

The measurement of backscatter coefficient from a broadband pulse-echo system: a new formulation

A new formulation for obtaining the absolute backscatter coefficient from pulse-echo measurements is presented. Using this formulation, performing the diffraction correction and system calibration is straightforward. The diffraction correction function for the measurement of backscatter coefficient and the acoustic coupling function for a pulse-echo system are defined. Details of these functions for two very useful cases are presented: a flat disk transducer and a spherically focused transducer. Approximations of these functions are also provided. For a flat disk transducer, the final formulation appears as a modification to the established Sigelmann-Reid formulation. For a focused transducer, the final correction is a weak function of frequency when the scattering volume is near the focal area, rather than the frequency squared dependence proposed by earlier investigators.     

Focused high frequency needle transducer for ultrasonic imaging and trapping

A miniature focused needle transducer (<1?mm) was fabricated using the press-focusing technique. The measured pulse-echo waveform showed the transducer had center frequency of 57.5 MHz with 54% bandwidth and 14?dB insertion loss. To evaluate the performance of this type of transducer, invitro ultrasonic biomicroscopy imaging on the rabbit eye was obtained. Moreover, a single beam acoustic trapping experiment was performed using this transducer. Trapping of targeted particle size smaller than the ultrasonic wavelength was observed. Potential applications of these devices include minimally invasive measurements of retinal blood flow and single beam acoustic trapping of microparticles.     

脉冲激光击穿水介质特性的实验研究

针对激光击穿水介质过程中的微观及宏观特性研究,利用调QNd：YAG激光聚焦击穿水介质形成激光声源,采用高速摄像机、高频测量水听器对激光击穿水介质过程中的等离子腔体闪光、空泡脉动、近／远场声波特性等综合效应进行了实验测量。实验表明：激光空泡的特征与水动力空化空泡相似;激光声信号强度在光击穿条件下与入射激光能量具有一定的线性关系;声脉冲高频段占声能的主要部分。研究结果可为水下激光加工、激光医学、激光声的研究提供一定的理论和实验支持。     

铁电驻极体空气耦合声换能器的研制

基于多孔聚丙烯铁电驻极体薄膜系统研制了平面型和球型聚焦空气耦合超声波换能器。平面型换能器孔径为20mm,两个球型聚焦换能器的孔径和焦距分别为20mm和35mm、30mm和40mm。使用激光干涉仪测得了三个换能器作为发射器工作时的带宽和谐振频率,并且将在脉冲回波模式下测得的换能器作为接收机工作时的响应与激光干涉仪测试结果进行比较。最后选择孔径为20mm的球型聚焦换能器,在脉冲回波模式下对不同直径孔的聚乙烯阶梯楔进行扫描成像。     

Characterization of a broadband all-optical ultrasound transducer-from optical and acoustical properties to imaging

A broadband all-optical ultrasound transducer has been designed, fabricated, and evaluated for high- frequency ultrasound imaging. The device consists of a 2-D gold nanostructure imprinted on top of a glass substrate, followed by a 3 microm PDMS layer and a 30 nm gold layer. A laser pulse at the resonance wavelength of the gold nanostructure is focused onto the surface for ultrasound generation, while the gold nanostructure, together with the 30 nm thick gold layer and the PDMS layer in between, forms an etalon for ultrasound detection, which uses a CW laser at a wavelength far from resonance as the probing beam. The center frequency of a pulse-echo signal recorded in the far field of the transducer is 40 MHz with -6 dB bandwidth of 57 MHz. The signal to noise ratio (SNR) from a 70 microm diameter transmit element combined with a 20 microm diameter receive element probing a near perfect reflector positioned 1.5 mm from the transducer surface is more than 10 dB and has the potential to be improved by at least another 40 dB. A high-frequency ultrasound array has been emulated using multiple measurements from the transducer while mechanically scanning an imaging target. Characterization of the device's optical and acoustical properties, as well as preliminary imaging results, strongly suggest that all-optical ultrasound transducers can be used to build high-frequency arrays for real-time high-resolution ultrasound imaging.     

Characterization of high-frequency, single-element focused transducers with wire target and hydrophone

In this paper, a wire-target technique was used for lateral beam profile measurements for a single-element, focused transducers in the very high-frequency range (35-60 MHz). Two wire targets made from 9-cm long tungsten wires with diameters of 8 microm and 20 microm were used as the pulse-echo targets to measure the lateral beam profiles at the focal plane of two single-element, focused transducers, a spherically focused 40 MHz transducer and a lens-focused in-house lithium niobate (LiNbO3) 60 MHz transducer. For comparison, measurements on the same transducers were performed by three small-aperture hydrophones with geometrical diameters varying from 37 microm to 150 microm. Tomographic reconstruction of the acoustic field from the spherically focused transducer also was conducted. Results obtained with the wire-target technique are comparable to those obtained with small-aperture hydrophones in characterizing lateral radiation patterns of a single-element, focused transducer in the high-frequency range (35-60 MHz). However, the wire-target method may overestimate pulse length because of the additional attenuation caused by the return path. Compared to small-aperture hydrophones, the wire-target technique is simpler and more cost effective. Its major advantage, however, is in the frequency range above 100 MHz in which commercial hydrophones are not yet available.     

Imaging in diffuse media with pulsed-ultrasound-modulated light and the photorefractive effect

Acousto-optic imaging in diffuse media is a dual wave-sensing technique in which an acoustic field interacts with multiply scattered laser light. The acoustic field causes a phase modulation in the optical field emanating from the interaction region, and this phase-modulated optical field carries with it information about the local optomechanical properties of the media. We report on the use of a pulsed ultrasound transducer to modulate the optical field and the use of a photorefractive-crystal-based interferometry system to detect ultrasound-modulated light. The use of short pulses of focused ultrasound allows for a one-dimensional acousto-optic image to be obtained along the transducer axis from a single, time-averaged acousto-optic signal. The axial and lateral resolutions of the system are controlled by the spatial pulse length and width of the ultrasound beam, respectively. In addition, scanning the ultrasound transducer in one dimension yields two-dimensional images of optical inhomogeneities buried in turbid media.     

Liquid lens using acoustic radiation force

A liquid lens is proposed that uses acoustic radiation force with no mechanical moving parts. It consists of a cylindrical acrylic cell filled with two immiscible liquids (degassed water and silicone oil) and a concave ultrasound transducer. The focal point of the transducer is located on the oil-water interface, which functions as a lens. The acoustic radiation force is generated when there is a difference in the acoustic energy densities of different media. An acoustic standing wave was generated in the axial direction of the lens and the variation of the shape of the oil-water interface was observed by optical coherence tomography (OCT). The lens profile can be rapidly changed by varying the acoustic radiation force from the transducer. The kinematic viscosity of silicone oil was optimized to minimize the response times of the lens. Response times of 40 and 80 ms when switching ultrasonic radiation on and off were obtained with a kinematic viscosity of 200 cSt. The path of a laser beam transmitted through the lens was calculated by ray-tracing simulations based on the experimental results obtained by OCT. The transmitted laser beam could be focused by applying an input voltage. The liquid lens could be operated as a variable-focus lens by varying the input voltage.     

Three-dimensional photoacoustic imaging using a two-dimensional CMUT array

In this paper, we describe using a 2-D array of capacitive micromachined ultrasonic transducers (CMUTs) to perform 3-D photoacoustic and acoustic imaging. A tunable optical parametric oscillator laser system that generates nanosecond laser pulses was used to induce the photoacoustic signals. To demonstrate the feasibility of the system, 2 different phantoms were imaged. The first phantom consisted of alternating black and transparent fishing lines of 180 μm and 150 μm diameter, respectively. The second phantom comprised polyethylene tubes, embedded in chicken breast tissue, filled with liquids such as the dye indocyanine green, pig blood, and a mixture of the 2. The tubes were embedded at a depth of 0.8 cm inside the tissue and were at an overall distance of 1.8 cm from the CMUT array. Two-dimensional cross-sectional slices and 3-D volume rendered images of pulse-echo data as well as photoacoustic data are presented. The profile and beamwidths of the fishing line are analyzed and compared with a numerical simulation carried out using the Field II ultrasound simulation software. We investigated using a large aperture (64 x 64 element array) to perform photoacoustic and acoustic imaging by mechanically scanning a smaller CMUT array (16 x 16 elements). Two-dimensional transducer arrays overcome many of the limitations of a mechanically scanned system and enable volumetric imaging. Advantages of CMUT technology for photoacoustic imaging include the ease of integration with electronics, ability to fabricate large, fully populated 2-D arrays with arbitrary geometries, wide-bandwidth arrays and high-frequency arrays. A CMUT based photoacoustic system is proposed as a viable alternative to a piezoelectric transducer based photoacoustic systems.     

Techniques and physical properties of 10MHz short pulse focused ultrasonic transducer

A focused ultrasonic transducer used for biomedical purposes with a fundamental frequency of 10MHz and a pulse width of one and a half periods is described in this paper. Its physical properties are given including (1) focused acoustic field recorded by an optical means, (2) electric waveform for triggering the transducer and the corresponding waveform of the wave received by another transducer, and (3) result of tests on a sample object.     

Pulse-echo field distribution measurement technique forhigh-frequency ultrasound sources

A simple technique for the determination of the spatial and temporal transmit-receive field distributions of spherically focused high-frequency transducers is described. Instead of a point-like target, tungsten wires (line-like targets) with diameters less than the acoustic wavelength are used as pulse-echo targets. Spatial and temporal field quantities were determined for spherically focused transducers in the frequency range from 3 to 17 MHz, and a comparison with hydrophone measurements showed that both techniques yielded comparable results for the low-frequency transducer. However, for the higher frequency transducers, hydrophone measurements did not yield satisfactory results compared to the wire-target technique due to the hydrophone's aperture size, while the results from the wire-target technique were in general agreement with theory     

Modeling of transient ultrasonic wave propagation using the distributed point

Transient ultrasonic waves in a fluid medium generated by a flat circular and a point-focused transducer of finite size are modeled by the distributed point source method (DPSM). DPSM is a Green's-function-based semi-analytical mesh-free technique which is modified here to incorporate the transient loading from a finite-sized acoustic transducer. Conventional DPSM solves acoustic problems in steady-state frequency domain. Here, DPSM is extended to the time domain without the fast Fourier transform (FFT) but using the Green's function in the time domain. This modified method is denoted t-DPSM. Harmonic point sources of DPSM are replaced by time-dependent point sources in t-DPSM. Generated t-DPSM results are compared with the finite element (FE) results for both focused and flat circular transducers. The developed method is used to solve the transient problem of wave scattering by an air bubble in a fluid as the bubble is moved horizontally or vertically from the focal point of the focused transducer. The received energy signal is compared for different eccentricities.     

Focusing with an optoacoustic transducer

A prototype of an ultrasonic transducer has been developed that uses an optical input to control the ultrasonic output. This transducer is called an optoacoustic transducer (OAT) and provides an ultrasonic pattern that is spatially similar to the optical pattern used to illuminate it. When a focus-inducing optical pattern, such as a zone plate, is used, an acoustic focus is achieved. The success of this procedure depends on the use of amplitude-modulated light at the input and on filtering the received signal to eliminate the primary frequency. This provides an increase, from 30 dB to 70 dB, in the ratio of acoustic pressure in the illuminated regions to that in the dark regions. The prototype operates at 2.8 MHz and has been used to provide a good acoustic focus in water. A 3-dB beamwidth of 3.5 mm was measured at a range of 92 mm. The construction techniques and materials used are discussed.     

Optical breakdown in fused silica and argon gas: application to Nd:YAG laser limiter

A gas cell filed with argon gas under pressure is placed in a tightly focused laser beam to provide a limiter for laser pulses above a certain peak power, corresponding to the optical breakdown threshold for the creation of a laser-induced plasma. Measurements of the threshold intensity as a function of argon gas pressure are given for a laser wavelength of 1.064 microm (Nd:YAG) and a pulse length of 6.4 ns. Threshold intensities for optical breakdown in fused silica were measured with the same optical system, enabling a relative comparison of breakdown thresholds, of interest for protecting fused-silica optical components in fiber-optic delivery systems for laser material processing applications. The threshold intensity was measured to 220 GW/cm2 in Ar at 1.0 x 10(5) N/m2 (1 atm), 80 GW/cm2 in Ar at 8.0 x 10(5) N/m2 (7.9 atm), and 55 GW/cm2 in fused silica. Even though the threshold in argon is higher than that in fused silica, the limiter will protect the optical components if the laser beam is focused to a tighter spot in the gas cell than at the input end of the fiber.     

Assessment of shear modulus of tissue using ultrasound radiation force acting on a spherical acoustic inhomogeneity

An ultrasound-based method to locally assess the shear modulus of a medium is reported. The proposed approach is based on the application of an impulse acoustic radiation force to an inhomogeneity in the medium and subsequent monitoring of the spatio-temporal response. In our experimental studies, a short pulse produced by a 1.5-MHz highly focused ultrasound transducer was used to initiate the motion of a rigid sphere embedded into an elastic medium. Another 25 MHz focused ultrasound transducer operating in pulse-echo mode was used to track the displacement of the sphere. The experiments were performed in gel phantoms with varying shear modulus to demonstrate the relationship between the displacement of the sphere and shear modulus of the surrounding medium. Because the magnitude of acoustic force applied to sphere depends on the acoustic material properties and, therefore, cannot be used to assess the absolute value of shear modulus, the temporal behavior of the displacement of the sphere was analyzed. The results of this study indicate that there is a strong correlation between the shear modulus of a medium and spatio-temporal characteristics of the motion of the rigid sphere embedded in this medium.     

Synthetic aperture techniques with a virtual source element

A new imaging technique has been proposed that combines conventional B-mode and synthetic aperture imaging techniques to overcome the limited depth of field for a highly focused transducer. The new technique improves lateral resolution beyond the focus of the transducer by considering the focus a virtual element and applying synthetic aperture focusing techniques. In this paper, the use of the focus as a virtual element is examined, considering the issues that are of concern when imaging with an array of actual elements: the tradeoff between lateral resolution and sidelobe level, the tradeoff between system complexity (channel count/amount of computation) and the appearance of grating lobes, and the issue of signal to noise ratio (SNR) of the processed image. To examine these issues, pulse-echo RF signals were collected for a tungsten wire in degassed water, monofilament nylon wires in a tissue-mimicking phantom, and cyst targets in the phantom. Results show apodization lowers the sidelobes, but only at the expense of lateral resolution, as is the case for classical synthetic aperture imaging. Grating lobes are not significant until spatial sampling is more than one wavelength, when the beam is not steered. Resolution comparable to the resolution at the transducer focus can be achieved beyond the focal region while obtaining an acceptable SNR. Specifically, for a 15-MHz focused transducer, the 6-dB beamwidth at the focus is 157 mum, and with synthetic aperture processing the 6-dB beamwidths at 3, 5, and 7 mm beyond the focus are 189 mum, 184 mum, and 215 mum, respectively. The image SNR is 38.6 dB when the wire is at the focus, and it is 32.8 dB, 35.3 dB, and 38.1 dB after synthetic aperture processing when the wire is 3, 5, and 7 mm beyond the focus, respectively. With these experiments, the virtual source has been shown to exhibit the same behavior as an actual transducer element in response to synthetic aperture processing techniques.     

Acoustic detection of controlled laser-induced microbubble creation in gelatin

A high-frequency (85 MHz) acoustic technique is used to identify system parameters for controlled laser-induced microbubble creation inside tissue-mimicking, gelatin phantoms. Microbubbles are generated at the focus of an ultrafast 793-nm laser source and simultaneously monitored through ultrasonic pulse-echo recordings. Displayed in wavefield form, these recordings illustrate microbubble creation, and integrated backscatter plots provide specifics about microbubble characteristics and dissolution behavior. By varying laser parameters, including pulse fluence (or pulse energy flux, J/cm2), total number of pulses delivered, and the period between pulses, the size, lifetime, and dissolution dynamics of laser-induced microbubbles may be independently controlled. Pulse fluence is the main size-controlling parameter, whereas both increases in pulse fluence and pulse number can lengthen microbubble lifetime from tens to hundreds of milliseconds. In short, a microbubble of particular lifetime does not necessarily have to be of a particular size. Microbubble behavior, furthermore, is independent of pulse periods below a fluence-dependent threshold value, but it exhibits stochastic behavior if pulse repetition is too slow. These results demonstrate that laser pulse fluence, number, and period may be varied to deposit energy in a specific temporal manner, creating and stabilizing microbubbles with particular characteristics and, therefore, potential uses in sensitive acoustic detection and manipulation schemes.