Targeted cell immobilization by ultrasound microbeam

Various techniques exerting mechanical stress on cells have been developed to investigate cellular responses to externally controlled stimuli. Fundamental mechanotransduction processes about how applied physical forces are converted into biochemical signals have often been examined by transmitting such forces through cells and probing its pathway at cellular levels. In fact, many cellular biomechanics studies have been performed by trapping (or immobilizing) individual cells, either attached to solid substrates or suspended in liquid media. In that context, we demonstrated two‐dimensional acoustic trapping, where a lipid droplet of 125 µm in diameter was directed transversely toward the focus (or the trap center) similar to that of optical tweezers. Under the influence of restoring forces created by a 30 MHz focused ultrasound beam, the trapped droplet behaved as if tethered to the focus by a linear spring. In order to apply this method to cellular manipulation in the Mie regime (cell diameter > wavelength), the availability of sound beams with its beamwidth approaching cell size is crucial. This can only be achieved at a frequency higher than 100 MHz. We define ultrasound beams in the frequency range from 100 MHz to a few GHz as ultrasound microbeams because the lateral beamwidth at the focus would be in the micron range. Hence a zinc oxide (ZnO) transducer that was designed and fabricated to transmit a 200 MHz focused sound beam was employed to immobilize a 10 µm human leukemia cell (K‐562) within the trap. The cell was laterally displaced with respect to the trap center by mechanically translating the transducer over the focal plane. Both lateral displacement and position trajectory of the trapped cell were probed in a two‐dimensional space, indicating that the retracting motion of these cells was similar to that of the lipid droplets at 30 MHz. The potential of this tool for studying cellular adhesion between white blood cells and endothelial cells was discussed, suggesting its capability as a single cell manipulator. Biotechnol. Bioeng. 2011; 108:1643–1650. © 2011 Wiley Periodicals, Inc.     

Three-Dimensional Mid-Air Acoustic Manipulation by Ultrasonic Phased Arrays

The essence of levitation technology is the countervailing of gravity. It is known that an ultrasound standing wave is capable of suspending small particles at its sound pressure nodes. The acoustic axis of the ultrasound beam in conventional studies was parallel to the gravitational force, and the levitated objects were manipulated along the fixed axis (i.e. one-dimensionally) by controlling the phases or frequencies of bolted Langevin-type transducers. In the present study, we considered extended acoustic manipulation whereby millimetre-sized particles were levitated and moved three-dimensionally by localised ultrasonic standing waves, which were generated by ultrasonic phased arrays. Our manipulation system has two original features. One is the direction of the ultrasound beam, which is arbitrary because the force acting toward its centre is also utilised. The other is the manipulation principle by which a localised standing wave is generated at an arbitrary position and moved three-dimensionally by opposed and ultrasonic phased arrays. We experimentally confirmed that expanded-polystyrene particles of 0.6 mm, 1 mm, and 2 mm in diameter could be manipulated by our proposed method.     

Laser-induced tobacco protoplast fusion

Laser tweezers can manipulate small particles, such as cells and organdies. When coupling them with laser microbeam selective fusion of two tobacco protoplasts containing some chloroplast was achieved. Physical and biological variables that affect laser trapping and laser-induced fusion were also discussed. The results show that the effect of chloroplast content and distribution on the yield of cell fusion is remarkable.     

A simple uniformity test for ultrasound phased arrays

PurposeIt is difficult to test phased array ultrasound transducers for non functioning elements. We aimed to modify a widely performed test to improve its ease and effectiveness for these arrays.MethodsA paperclip was slowly moved along the transducer array, with the scanner operating in M-mode, imaging at a fundamental frequency with automatic gain and grey scale adjustment disabled. Non-functioning elements are identified by a dark vertical line in the image. The test was repeated several times for each transducer, looking for consistency of results. 2 transducers, with faults already shown by electronic transducer testing, were used to validate the method. 23 transducers in clinical use were tested.ResultsThe results of the modified test on the 2 faulty transducers agreed closely with electronic transducer testing results. The test indicated faults in 5 of the 23 transducers in clinical use: 3 with a single failed element and 2 with non-uniform sensitivity. 1 transducer with non-uniform sensitivity had undergone lens repair; the new lens was visibly non-uniform in thickness and further testing showed a reduction in depth of penetration and a loss of elevational focus in comparison with a new transducer.ConclusionsThe modified test is capable of detecting non-functioning elements. Further work is required to provide a better understanding of more subtle faults.     

光镊和拉曼光镊在生物医学研究中的应用

光镊是由美国科学家Arthur Ashkin于1986年发明的,是一种利用高度汇聚的激光束产生的三维梯度势阱来俘获、操纵微小粒子的技术。因其可俘获、操纵单个细胞,并在细胞和亚细胞层次上为生物医学研究提供方便,近年来,已越来越多地被应用于生物医学研究中。本文在介绍光镊的原理和特点的基础上,阐述了光镊(尤其是拉曼光镊)技术在生物医学领域中的研究进展、现状和展望。     

Efficient Driving of Piezoelectric Transducers Using a Biaxial Driving Technique

Efficient driving of piezoelectric materials is desirable when operating transducers for biomedical applications such as high intensity focused ultrasound (HIFU) or ultrasound imaging. More efficient operation reduces the electric power required to produce the desired bioeffect or contrast. Our preliminary work [Cole et al. Journal of Physics: Condensed Matter. 2014;26(13):135901.] suggested that driving transducers by applying orthogonal electric fields can significantly reduce the coercivity that opposes ferroelectric switching. We present here the experimental validation of this biaxial driving technique using piezoelectric ceramics typically used in HIFU. A set of narrow-band transducers was fabricated with two sets of electrodes placed in an orthogonal configuration (following the propagation and the lateral mode). The geometry of the ceramic was chosen to have a resonance frequency similar for the propagation and the lateral mode. The average (± s.d.) resonance frequency of the samples was 465.1 (± 1.5) kHz. Experiments were conducted in which each pair of electrodes was driven independently and measurements of effective acoustic power were obtained using the radiation force method. The efficiency (acoustic/electric power) of the biaxial driving method was compared to the results obtained when driving the ceramic using electrodes placed only in the pole direction. Our results indicate that the biaxial method increases efficiency from 50% to 125% relative to the using a single electric field.     

Multiple three‐dimensional mammalian cell aggregates formed away from solid substrata in ultrasound standing waves

Single and multiple three‐dimensional cell aggregates of human red blood cells (RBCs) and HepG2 cells were formed rapidly in low mega‐Hertz ultrasound standing wave fields of different geometries. A single discoid aggregate was formed in a half‐wavelength pathlength resonator at a cell concentration sufficient to produce a 3D structure. Multiple cell aggregates were formed on the axis of a cylindrical resonator with a plane transducer (discoid aggregates); in a resonator with a tubular transducer and in the cross‐fields of plane and tubular transducers and two plane orthogonal transducers (all cylindrical aggregates). Mechanically strong RBC aggregates were obtained by crosslinking with wheat germ agglutinin (WGA, a lectin). Scanning electron microscopy showed aggregate surface porous structures when RBCs were mixed with WGA before sonication and tighter packing when ultrasonically preformed aggregates were subsequently exposed to a flow containing WGA. HepG2 cell aggregates showed strong accumulation of F‐actin at sites of cell–cell contact consistent with increased mechanical stability. The aggregates had a porous surface, and yet confocal microscopy revealed a tight packing of cells in the aggregate's inner core. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Vibrot,a Simple Device for the Conversion of Vibration into Rotation Mediated by Friction: Preliminary Evaluation

While “vibrational noise” induced by rotating components of machinery is a common problem constantly faced by engineers, the controlled conversion of translational into rotational motion or vice-versa is a desirable goal in many scenarios ranging from internal combustion engines to ultrasonic motors. In this work, we describe the underlying physics after isolating a single degree of freedom, focusing on devices that convert a vibration along the vertical axis into a rotation around this axis. A typical Vibrot (as we label these devices) consists of a rigid body with three or more cantilevered elastic legs attached to its bottom at an angle. We show that these legs are capable of transforming vibration into rotation by a “ratchet effect”, which is caused by the anisotropic stick-slip-flight motion of the leg tips against the ground. Drawing an analogy with the Froude number used to classify the locomotion dynamics of legged animals, we discuss the walking regime of these robots. We are able to control the rotation frequency of the Vibrot by manipulating the shaking amplitude, frequency or waveform. Furthermore, we have been able to excite Vibrots with acoustic waves, which allows speculating about the possibility of reducing the size of the devices so they can perform tasks into the human body, excited by ultrasound waves from the outside.     

Optimisation of the transmit beam parameters for generation of subharmonic signals in native and altered populations of a commercial microbubble contrast agent SonoVue®

The aim of this work was to establish the optimum acoustic characterisation approach and insonation transmit beam parameters for subharmonic signal generation with ‘native’ and ‘altered’ populations of a commonly-used microbubble contrast agent. Dynamic contrast-enhanced (DCE) ultrasound is a non-invasive method of imaging the microvasculature, typically implemented using harmonic imaging. Subharmonic imaging, in which echoes at half the fundamental frequency are detected, detects signals which are generated by the ultrasound contrast agents (UCAs) but not by tissue. However, optimal transmission parameters and furthermore, the optimum acoustic characterisation method have not been established. The subharmonic response of ‘native’ and ‘altered’ UCA, altered through decantation, was investigated at transmit centre frequencies 1.8–5 MHz and pulse lengths 1–8 cycles. The ‘altered’ UCA had reduced polydispersity (1–4 µm: 82% bubble volume), compared to ‘native’ (4–10 µm: 57% bubble volume). A custom-built narrow-band acoustic characterisation system was found to be more appropriate for acoustic characterisation compared to the commonly used broadband pulse-echo approach. Both UCA generated the highest subharmonic signal at pulse length of 3-cycles. The maximum ‘native’ subharmonic signal was generated at a transmit centre frequency of 1.9 MHz, corresponding to a subharmonic at 0.95 MHz. This optimal frequency increased in the ‘altered’ population to 2.3–2.5 MHz, bringing the subharmonic above 1 MHz and hence into a range amenable to clinical abdominal imaging transducers. The use of subharmonic signal detection coupled with a modified UCA size distribution has potential to significantly improve the quantification sensitivity and accuracy of DCE ultrasound imaging.     

Optical tweezers in biology and medicine

Optical trapping techniques provide unique means to manipulate biological particles such as virus, living cells and subcellular organelles. Another area of interest is the measurement of mechanical (elastic) properties of cell membranes, long strands of single DNA molecule, and filamentous proteins. One of the most attractive applications is the study of single motor molecules. With optical tweezers traps, one can measure the forces generated by single motor molecules such as kinesin and myosin, in the piconewton range and, for the first time, resolve their detailed stepping motion.     

A toolbox for generating single‐stranded DNA in optical tweezers experiments

Essential genomic transactions such as DNA‐damage repair and DNA replication take place on single‐stranded DNA (ssDNA) or require specific single‐stranded/double‐stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single‐molecule studies of DNA–protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to dsDNA, only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single‐molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force‐induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force‐induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA. © 2013 Wiley Periodicals, Inc. Biopolymers 99:611–620, 2013.     

Mechanical manipulation of bone and cartilage cells with 'optical tweezers'.

The single beam optical gradient trap (optical tweezers) uses a single beam of laser light to non-invasively manipulate microscopic particles. Optical tweezers exerting a force of approximately 7 pN were applied to single bone and cartilage derived cells in culture and changes in intracellular calcium levels were observed using Fluo-3 labelling. Human derived osteoblasts responded to optical tweezers with an immediate increase in [Ca2+]i that was inhibited by the addition of a calcium channel blocker nifedipine. Force applied to different regions of cells resulted in a variable response. [Ca2+]i elevation in response to load was lower in rat femur derived osteoblasts, and not apparent in primary chondrocytes and the osteocytic cell line (MLO Y4).     

Characterization and propagation of acoustic emission signals in woody plants: towards an improved acoustic emission counter

Abstract. The physics of ultrasonic acoustic emissions (AEs) was investigated for AE transmission through wood and transducers. The physical properties measured were velocity, attenuation and frequency composition of AEs produced by two sources: cavitation events in xylem and pencil lead breaks. The authors also measured the relative sensitivity of various combinations of ultrasound transducers and amplifiers to aid in the selection of a measuring system optimized for cavitation detection in woody plants. Some of the authors' conclusions are: (1) Softwoods ( Thuja, Pinus ) attenuate AEs more rapidly than hardwoods (maple, birch). (2) The velocity of AEs in wood exceeds that measured by others in water so the main medium of AE transmission must be the cellulose. (3) The strongest frequencies of AEs are in the range of 100–300 kHz. (4) Cavitation-induced AEs tend to shift to higher frequency as wood dehydration progresses. (5) One cannot determine the locus of origin of AEs from its frequency composition. (6) The frequency composition of the acoustic emissions probably cannot be determined at all with the sensors used because of their tendency to 'ring'. The data collected in this paper were used to aid in the design of an improved AE counter having a seven-fold increase in signal to noise ratio compared to counters previously used in our laboratory. The improved counter, model 4615 Drought Stress Monitor, is now commercially available from Physical Acoustics Corp., Princeton, NJ, U.S.A.     

Pure Ultrasonic Communication in an Endemic Bornean Frog

Huia cavitympanum, an endemic Bornean frog, is the first amphibian species known to emit exclusively ultrasonic (i.e., >20 kHz) vocal signals. To test the hypothesis that these frogs use purely ultrasonic vocalizations for intraspecific communication, we performed playback experiments with male frogs in their natural calling sites. We found that the frogs respond with increased calling to broadcasts of conspecific calls containing only ultrasound. The field study was complemented by electrophysiological recordings from the auditory midbrain and by laser Doppler vibrometer measurements of the tympanic membrane''s response to acoustic stimulation. These measurements revealed that the frog''s auditory system is broadly tuned over high frequencies, with peak sensitivity occurring within the ultrasonic frequency range. Our results demonstrate that H. cavitympanum is the first non-mammalian vertebrate described to communicate with purely ultrasonic acoustic signals. These data suggest that further examination of the similarities and differences in the high-frequency/ultrasonic communication systems of H. cavitympanum and Odorrana tormota, an unrelated frog species that produces and detects ultrasound but does not emit exclusively ultrasonic calls, will afford new insights into the mechanisms underlying vertebrate high-frequency communication.     

Theoretical evaluation of magnetoreception of power‐frequency fields

Several effects of power‐frequency (50/60 Hz) magnetic fields (PF‐MF) of weak intensity have been hypothesized in animals and humans. No valid mechanism, however, has been proposed for an interaction between PF‐MF and biological tissues and living beings at intensities relevant to animal and human exposure. Here we proposed to consider PF‐MF as disrupters of the natural magnetic signal. Under exposure to these fields, an oscillating field exists that results from the vectorial summation of both the PF‐MF and the geomagnetic field. At a PF‐MF intensity (rms) of 0.5 µT, the peak‐to‐peak amplitude of the axis and/or intensity variations of this resulting field exceeds the related discrimination threshold of magnetoreception (MR) in migrating animals. From our evaluation of the 50/60 Hz responsiveness of the putative mechanisms of MR, single domain particles (Kirschvink's model) appear unable to transduce that oscillating signal. On the contrary, radical pair reactions are able to, as well as interacting multidomain iron–mineral platelets and clusters of superparamagnetic particles (Fleissner/Solov'yov's model). It is, however, not yet known whether the reception of 50/60 Hz oscillations of the natural magnetic signal might be of consequence or not. Bioelectromagnetics 31:371–379, 2010. © 2010 Wiley‐Liss, Inc.     

Spatial and acoustic pressure dependence of microbubble-mediated gene delivery targeted using focused ultrasound

BACKGROUND: Ultrasound/microbubble-mediated gene delivery has the potential to be targeted to tissue deep in the body by directing the ultrasound beam following vector administration. Application of this technology would be minimally invasive and benefit from the widespread clinical experience of using ultrasound and microbubble contrast agents. In this study we evaluate the targeting ability and spatial distribution of gene delivery using focused ultrasound. METHODS: Using a custom-built exposure tank, Chinese hamster ovary cells in the presence of SonoVue microbubbles and plasmid encoding beta-galactosidase were exposed to ultrasound in the focal plane of a 1 MHz transducer. Gene delivery and cell viability were subsequently assessed. Characterisation of the acoustic field and high-resolution spatial analysis of transfection were used to examine the relationship between gene delivery efficiency and acoustic pressure. RESULTS: In contrast to that seen in the homogeneous field close to the transducer face, gene delivery in the focal plane was concentrated on the ultrasound beam axis. Above a minimum peak-to-peak value of 0.1 MPa, transfection efficiency increased as acoustic pressure increased towards the focus, reaching a maximum above 1 MPa. Delivery was microbubble-dependent and cell viability was maintained. CONCLUSIONS: Gene delivery can be targeted using focused ultrasound and microbubbles. Since delivery is dependent on acoustic pressure, the degree of targeting can be determined by appropriate transducer design to modify the ultrasound field. In contrast to other physical gene delivery approaches, the non-invasive targeting ability of ultrasound makes this technology an attractive option for clinical gene therapy.     

Quantitative study of optical and mechanical bone status using multispectral photoacoustics

Osteoporosis is a major public health problem worldwide. Here, we present a quantitative multispectral photoacoustic method for the evaluation of bone pathologies which has significant advantages over pure ultrasonic or pure optical methods as it provides both molecular information and bone mechanical status. This is enabled via a simultaneous measurement of the bone's optical properties as well as the speed of sound and ultrasonic attenuation in the bone. To test the method's quantitative predictions, a combined ultrasonic and photoacoustic system was developed. Excitation was performed optically via a portable triple laser‐diode system and acoustically via a single element transducer. Additional dual transducers were used for detecting the acoustic waves that were generated by the two modalities. Both temporal and spectral parameters were compared between different excitation wavelengths and measurement modalities. Short photoacoustic excitation wavelengths allowed sensing of the cortical layer while longer wavelengths produced results which were compatible with the quantitative ultrasound measurements.

     

不同微生物的单光束激光陷阱操纵

本文报导了分别采用Ｈｅ－Ｎｅ和Ａｒ＾＋激光器与光不显微镜构成的单光束激光陷阱操纵酵母菌、青霉等不同微生物的实验观察结果,讨论了操纵条件。研究表明：用单光束高会聚激光产生的梯度力操纵微生物体,其有效作用力的大小不仅与激光功率、波长、束腰半径和光束会聚角有关,还与微生物的大小、吸收系数、菌龄及培养方法等因素有关。     

A dielectrophoretic microchip for controlled cell targeting with functionalized microspheres

Individual cell manipulation and targeting is of major interest in the field of diagnosis, phenotypic characterization and drug delivery. Lab-on-a-chip technologies open the possibility to work easily at the single cell level. We developed a dielectrophoretic microchip capable to trap and manipulate individual cells and microspheres. We targeted single cells with functionalized microspheres in a software-controlled way proving the efficiency and reliability of our chip. As an example, we demonstrate guided targeting and binding of cell lines expressing or not specific antigens with microspheres coated or not with the corresponding monoclonal antibodies.     

Acoustic Emissions from Plants: Ultrasonic and Audible Compared

Audible acoustic emissions (AAE) and ultrasonic acoustic emissions(UAE), produced by stem segments during dehydration in air,have been recorded and compared. We hypothesize that cavitationof xylem sap generally results in the production of a broadband acoustic emission (AE) with a lower cut-off frequency determinedby the dimensions of the resonating element. The larger a conduit'sdimensions, the lower is the frequency of its major resonance.Thus the vessels, the largest conduits, can be expected to produceboth AAE and UAE. Fibres and small cavitating elements suchas small tracheids are expected on the other hand to produceonly UAE. Most work utilized Acacia tissues but work was extended to otherplant tissues from a range of species with differing anatomicalcharacteristics. Evidence supporting our hypothesis shows thatAAE and UAE did not coincide in different tissue types or dependsimply upon the degree of dehydration. AAE were detected fromtissue with intact major conduits (vessels) but not in similartissue in which these major conduits had been severed, whereasUAE were detected from both types of tissue. In general, ourhypothesis that larger conduits produced lower frequency signalsand smaller units at the ultrasonic frequencies was supported.We are forced to conclude that some UAE are generated by eventsother than cavitating vessels or fibres. Possible interpretationof our data is discussed in terms of the size of the cavitatingconduits but including differential signal absorption withinthe tissue. Key words: Cavitation, acoustic emission, ultrasound, plants, water stress