General Defocusing Particle Tracking: fundamentals and uncertainty assessment

General Defocusing Particle Tracking (GDPT) is a single-camera, three-dimensional particle tracking method that determines the particle depth positions from the defocusing patterns of the corresponding particle images. GDPT relies on a reference set of experimental particle images which is used to predict the depth position of measured particle images of similar shape. While several implementations of the method are possible, its accuracy is ultimately limited by some intrinsic properties of the acquired data, such as the signal-to-noise ratio, the particle concentration, as well as the characteristics of the defocusing patterns. GDPT has been applied in different fields by different research groups, however, a deeper description and analysis of the method fundamentals has hitherto not been available. In this work, we first identity the fundamental elements that characterize a GDPT measurement. Afterwards, we present a standardized framework based on synthetic images to assess the performance of GDPT implementations in terms of measurement uncertainty and relative number of measured particles. Finally, we provide guidelines to assess the uncertainty of experimental GDPT measurements, where true values are not accessible and additional image aberrations can lead to bias errors. The data were processed using DefocusTracker, an open-source GDPT software. The datasets were created using the synthetic image generator MicroSIG and have been shared in a freely-accessible repository

Diving with microparticles in acoustic fields

Sound can move particles. A good example of this phenomenon is the Chladni plate, in which an acoustic wave is induced in a metallic plate and particles migrate to the nodes of the acoustic wave. For several years, acoustophoresis has been used to manipulate microparticles in microscopic scales. In this fluid dynamics video, submitted to the 30th Annual Gallery of Fluid Motion, we show the basic mechanism of the technique and a simple way of visualize it. Since acoustophoretic phenomena is essentially a three-dimensional effect, we employ a simple technique to visualize the particles in 3D. The technique is called Astigmatism Particle Tracking Velocimetry and it consists in the use of cylindrical lenses to induce a deformation in the particle shape, which will be then correlated with its distance from the observer. With this method we are able to dive with the particles and observe in detail particle motion that would otherwise be missed. The technique not only permits visualization but also precise quantitative measurements that can be compared with theory and simulations.Comment: Fluid dynamics video for the 30th Annual Gallery of Fluid Motion, Entry #84160 65th Annual Meeting of the American Physical Society, Division of Fluid Dynamics San Diego, CA, Nov 201

Ultrasound-induced acoustophoretic motion of microparticles in three dimensions

We derive analytical expressions for the three-dimensional (3D) acoustophoretic motion of spherical microparticles in rectangular microchannels. The motion is generated by the acoustic radiation force and the acoustic streaming-induced drag force. In contrast to the classical theory of Rayleigh streaming in shallow, infinite, parallel-plate channels, our theory does include the effect of the microchannel side walls. The resulting predictions agree well with numerics and experimental measurements of the acoustophoretic motion of polystyrene spheres with nominal diameters of 0.537 um and 5.33 um. The 3D particle motion was recorded using astigmatism particle tracking velocimetry under controlled thermal and acoustic conditions in a long, straight, rectangular microchannel actuated in one of its transverse standing ultrasound-wave resonance modes with one or two half-wavelengths. The acoustic energy density is calibrated in situ based on measurements of the radiation dominated motion of large 5-um-diam particles, allowing for quantitative comparison between theoretical predictions and measurements of the streaming induced motion of small 0.5-um-diam particles.Comment: 13 pages, 8 figures, Revtex 4.

Acoustic radiation- and streaming-induced microparticle velocities determined by micro-PIV in an ultrasound symmetry plane

We present micro-PIV measurements of suspended microparticles of diameters from 0.6 um to 10 um undergoing acoustophoresis in an ultrasound symmetry plane in a microchannel. The motion of the smallest particles are dominated by the Stokes drag from the induced acoustic streaming flow, while the motion of the largest particles are dominated by the acoustic radiation force. For all particle sizes we predict theoretically how much of the particle velocity is due to radiation and streaming, respectively. These predictions include corrections for particle-wall interactions and ultrasonic thermoviscous effects, and they match our measurements within the experimental uncertainty. Finally, we predict theoretically and confirm experimentally that the ratio between the acoustic radiation- and streaming-induced particle velocities is proportional to the square of the particle size, the actuation frequency and the acoustic contrast factor, while it is inversely proportional to the kinematic viscosity.Comment: 11 pages, 9 figures, RevTex 4-

Tunable-angle wedge transducer for improved acoustophoretic control in a microfluidic chip

We present a tunable-angle wedge ultrasound transducer for improved control of microparticle acoustophoresis in a microfluidic chip. The transducer is investigated by analyzing the pattern of aligned particles and induced acoustic energy density while varying the transducer geometry, transducer coupling angle, and transducer actuation method (single-frequency actuation or frequency-modulation actuation). The energy-density analysis is based on measuring the transmitted light intensity through a microfluidic channel filled with a suspension of 5 mu m diameter beads and the results with the tunable-angle transducer are compared with the results from actuation by a standard planar transducer in order to decouple the influence from change in coupling angle and change in transducer geometry. We find in this work that the transducer coupling angle is the more important parameter compared to the concomitant change in geometry and that the coupling angle may be used as an additional tuning parameter for improved acoustophoretic control with single-frequency actuation. Further, we find that frequency-modulation actuation is suitable for diminishing such tuning effects and that it is a robust method to produce uniform particle patterns with average acoustic energy densities comparable to those obtained using single-frequency actuation.</p

Differential Requirement for CD70 and CD80/CD86 in Dendritic Cellmediated Activation of Tumor Tolerized CD8 T Cells

A major obstacle to efficacious T cell-based cancer immunotherapy is the tolerizing-tumor microenvironment that rapidly inactivates tumor-infiltrating lymphocytes. In an autochthonous model of prostate cancer, we have previously shown that intratumoral injection of Ag-loaded dendritic cells (DCs) delays T cell tolerance induction as well as refunctionalizes already tolerized T cells in the tumor tissue. In this study, we have defined molecular interactions that mediate the effects of DCs. We show that pretreating Ag-loaded DCs with anti-CD70 Ab abolishes the ability of DCs to delay tumor-mediated T cell tolerance induction, whereas interfering with 4-1BBL, CD80, CD86, or both CD80 and CD86 had no significant effect. In contrast, CD80[superscript −/−] or CD80[superscript −/−]CD86[superscript −/−] DCs failed to reactivate already tolerized T cells in the tumor tissue, whereas interfering with CD70 and 4-1BBL had no effect. Furthermore, despite a high level of programmed death 1 expression by tumor-infiltrating T cells and programmed death ligand 1 expression in the prostate, disrupting programmed death 1/programmed death ligand 1 interaction did not enhance T cell function in this model. These findings reveal dynamic requirements for costimulatory signals to overcome tumor-induced tolerance and have significant implications for developing more effective cancer immunotherapies.American Cancer Society (Postdoctoral Fellowship 12109-PF-11-025-01-LIB)John D. Proctor Foundation (Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship)United States. Army Medical Research and Materiel Command. Prostate Cancer Research Program (Grant

Acoustic force mapping in a hybrid acousticoptical micromanipulation device supporting high resolution optical imaging

Many applications in the life-sciences demand non-contact manipulation tools for forceful but nevertheless delicate handling of various types of sample. Moreover, the system should support high-resolution optical imaging. Here we present a hybrid acoustic/optical manipulation system which utilizes a transparent transducer, making it compatible with high-NA imaging in a microfluidic environment. The powerful acoustic trapping within a layered resonator, which is suitable for highly parallel particle handling, is complemented by the flexibility and selectivity of holographic optical tweezers, with the specimens being under high quality optical monitoring at all times. The dual acoustic/optical nature of the system lends itself to optically measure the exact acoustic force map, by means of direct force measurements on an optically trapped particle. For applications with (ultra-)high demand on the precision of the force measurements, the position of the objective used for the high-NA imaging may have significant influence on the acoustic force map in the probe chamber. We have characterized this influence experimentally and the findings were confirmed by model simulations. We show that it is possible to design the chamber and to choose the operating point in such a way as to avoid perturbations due to the objective lens. Moreover, we found that measuring the electrical impedance of the transducer provides an easy indicator for the acoustic resonances