| In 2018,the Nobel Prize in physics was awarded to "Optical tweezers and their application to biological systems",and non-contact manipulation technologies have great prospects in scientific research and application fields.Similar to optical tweezers,acoustic tweezers also allow non-contact,label-free manipulation of particles by applying acoustic radiation forces to the particles.In addition,acoustic tweezers also have the advantages of large radiation force,small thermal damage,strong penetration and can be used in non-transparent media,which makes it possible to become a powerful tool in biomedical applications,especially in the field of in-vivo manipulation applications.Cells,drugs,micro robots,targeted-drug carriers（nanoparticles,droplets）,and other objects,can provide micro medical tools for biomedical applications,such as disease surveillance,targeted drug delivery,non-invasive surgery,single-cell analysis in circulation and so on,which has the significant values of research and application.Acoustic tweezers based on acoustic radiation force can provide a non-contact means for these targets to enter the living bodys.However,the realization of acoustic tweezers in vivo faces many challenges:（1）Opaque tissue hinders visualization of particle manipulation based on optical images;（2）The complex spatial structure requires flexible programmable manipulation ability of acoustic tweezers;（3）Acoustic distortion caused by heterogeneous tissue poses a challenge to accurate manipulation.At present,among all the methods,acoustic tweezers based on phased array can accurately regulate the acoustic fields in time and space,and can complete complex spatial manipulation behavior,which is the most likely acoustic manipulation method to realize in-vivo applications.Three-dimensional holographic acoustic tweezers based on arrays have been achieved in air,but studies in water and in vivo are rarely reported.Aiming at the challenges of the in-vivo applications of acoustic tweezers,this project mainly carried out the following work around the goal of realizing threedimensional acoustic tweezers and their applications in vivo:（1）Hardware condition design and characterization of the acoustic fields: the development and acoustic field characterization of 2D planar array ultrasonic array.By studying the design and manufacturing process of the arrays,1.04 MHz,256-element arrays（16*16）suitable for the manipulation of macroscopic particles（diameter≥1mm）and 3MHz,64-element arrays（8*8）suitable for the manipulation of micro-cells and micro-organisms（diameter<800μm）were developed respectively.The free-field,transcranial and coherent acoustic fields of a 256-element array are characterized.The results show that the peak acoustic pressure decreases to 18.1% of the free-field situation when the focused beam emitted by a single array passes through the skull.The transcranial transverse steering distance was up to 10 mm and the axial was up to 40 mm.In addition,by simultaneously emitting a focused acoustic beam from two vertically and symmetrically placed arrays,a tiny focus can be obtained in the coherent region with an axial size reduced from 29.7mm in the case of a single array to 3.3mm,and the "stripe" pattern caused by standing waves in the coherent region can be eliminated by using a different-frequency-transmitting-strategy.The transcranial focusing and beam steering capabilities of the array also pave the way for transcranial manipulation.（2）Acoustic manipulation method and theoretical basis: spatiotemporal precise modulation of complex acoustic field and force analysis of particle acoustophoretic motion.This project makes full use of the advantages of cooperative and independent control of multiple elements,and generates multi-foci,spherical vortex,twin trap and other acoustic fields in space by using the iterative backpropagation method and the holographic acoustic element framework method（HAEFM）,and realizes the accurate regulation of 3D movement and time-multiplexing of the acoustic field by flexibly controlling the sequence of transmitted pulses in time;At the same time,the acoustic radiation force and Stokes force caused by acoustic streaming are analyzed and calculated,and the acoustophoretic motion of particles under the combined action of the two forces is analyzed by using simulation software,and the trajectories of particles under different parameter settings are predicted successfully.（3）Breakthrough in key technology of 3D acoustic tweezers: Image-guided 3D acoustic tweezers in complex media using time-reversal.Firstly,based on the manipulation method and theory,the dynamic 3D manipulation of multiple particles with different acoustic properties（PDMS and PS respectively represent negative and positive acoustic contrast particles）was realized with different acoustic traps（multifoci and vortex）using 256-element array.Secondly,by utilizing the ultrasonic volume imaging capability in water,acoustic manipulation of particle can be guided and monitored in real time.3D images can track PDMS particle falling freely from any initial position,and navigated the system to trap the falling particle and then manipulate it along the preset path.Finally,a time-reversal method is put forward for phase and amplitude correction in which the distortions are caused by inhomogeneous media,and the PDMS particle is successfully manipulated along a 3D trajectory through SIAT baffles,ex-vivo monkey and human skulls.These breakthroughs in key technologies,such as manipulation visualization and acoustic distortion correction,have cleared the way for in-vivo applications of 3D acoustic tweezers.（4）Basic experiments of biological manipulation: research on 3D acoustic manipulation of microscopic organisms.Multi-dimensional translation,rotation,orientation and levitation of living cells and micro-organisms were realized in a common Petri dish using the 64-element array.Spherical vortex and twin trap fields are generated by HAEFM.By controlling the center position of the vortex,the eggs and larvae of brine shrimp could be manipulated to move along the preset trajectory.By time multiplexing of the counterclockwise vortices,clockwise vortices and the twin trap fields,the rotation direction of the eggs can be changed in real time,while the orientation of the non-spherical larvae can be reoriented.The eggs and larvae could be pulled up from the bottom of the dish and manipulated further vertically and horizontally after being levitated by the combined action of standing wave and Eckart streaming formed by the reflection of the beam.The relationship between the rotation frequency of shrimp eggs and the focal depth,topological charge and excitation voltage was also analyzed quantitatively.（5）In-vivo applications of 3D acoustic tweezers: In-vivo acoustic manipulation of bacterial microswimmers.Using gene editing technology to generate gas vesicles in bacteria,ultrasonic sensitive bacterial microswimmers were prepared and manipulated acoustically.Firstly,based on the 3MHz,64-element array,microswimmers can be aggregated by focal beam in a silicone tube while ordinary bacteria cannot.Under the scenarios of PDMS cavity and Petri dish,the microswimmer clusters were successfully manipulated to move according to the specified trajectory.Secondly,combined with microscopic imaging,the window chamber mice model was used to trap microswimmers in the superficial blood vessels of different diameters of mice,and further to manipulate the microswimmers to travel in the bifurcation vessels.Through the combination of high-frequency ultrasound imaging,the aggregation and manipulation of the microswimmers in the deep tissue of mice,such as the intestinal tract,was successfully achieved,thus realizing the in-vivo applications of 3D acoustic tweezers.In conclusion,based on the 2D planar arrays,this project studied the spatiotemporal precise regulation mechanism of complex acoustic fields and the characteristics of particle acoustophoretic motion,and proposed a self-navigated 3D acoustic tweezers based on time-reversal in complex environment,and further advanced towards the direction of biological manipulation,and finally realized the invivo applications of 3D acoustic tweezers.It has important guidance and reference significance for the future development and applications of acoustic tweezers technology. |
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