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.

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

A fast and robust algorithm for general defocusing particle tracking

The increasing use of microfluidics in industrial, biomedical, and clinical applications requires a more and more precise control of the microfluidic flows and suspended particles or cells. This leads to higher demands in three-dimensional and automated particle tracking methods, e.g. for use in feedback-control systems. General defocusing particle tracking (GDPT) is a 3D particle tracking method based on defocused particle images which is easy to use and requires standard laboratory equipment. In this work, we describe in detail a fast and robust algorithm for performing GDPT, which is suitable for automatized and real-time applications. Its key feature is a fast, segmentation-free approach to identify particles and estimate their 3D position. This detection step is followed by a refinement and iteration step to improve accuracy and identification of overlapping particles. We show that the algorithm is versatile and can be applied to different types of images (darkfield and brightfield). We use synthetic image sets of varying particle concentration to evaluate the performance of the algorithm in terms of detected depth coordinate uncertainty, particle detection rate, and processing time. The algorithm is applied and validated on experimental images showing that it is robust towards background or illumination fluctuations. Finally, to test the algorithm on real-time applications, we use synthetic images to set up a simulation framework with experimentally-relevant parameters and where the true particle positions are known

DefocusTracker: A Modular Toolbox for Defocusing-based, Single-Camera, 3D Particle Tracking

The need for single-camera 3D particle tracking methods is growing, among others, due to the increasing focus in biomedical research often relying on single-plane microscopy imaging. Defocusing-based methods are ideal for a wide-spread use as they rely on basic microscopy imaging rather than requiring additional non-standard optics. However, a wide-spread use has been limited by the lack of accessible and easy-to-use software. DefocusTracker is an open-source toolbox based on the universal principles of General Defocusing Particle Tracking (GDPT) relying solely on a reference look-up table and image recognition to connect a particle’s image and its respective out-of-plane depth coordinate. The toolbox is built in a modular fashion, allowing for easy addition of new image recognition methods, while maintaining the same workflow and external user interface. DefocusTracker is implemented in MATLAB, while a parallel implementation in Python is in the preparation

Characterization of human melanoma skin cancer models:A step towards model-based melanoma research

Advancing 3D in vitro human tissue models is crucial for biomedical research and drug development to address the ethical and biological limitations of animal testing. Recently, 3D skin models have proven to be effective for studying serious skin conditions, such as melanoma. For these advanced models to be applicable in preclinical studies, thorough characterization is essential to understand their applicability and limitations. In this study, we used bioimaging and RNA sequencing to assess the architecture and transcriptomic profiles of skin models, including models with melanoma. Our results indicated that these models closely mimicked skin morphology and gene expression patterns. The full-thickness (FT) model shows a superior resemblance to the human skin, particularly in basement membrane formation and cellular interactions. The integrity of the skin-like properties and gene expression signatures of both skin and melanoma cells were preserved upon the integration of melanoma cells, establishing these models as robust platforms for cancer research. The responsiveness of the FT melanoma models to vemurafenib treatment was successfully monitored, demonstrating their validity as a reliable, reproducible, and humane tool for pharmacological testing and drug development. Furthermore, the transcriptomic data showed that skin models with cancer spheroids had upregulated genes linked to aggressive and resilient cancer behavior compared to spheroids alone. This emphasizes the importance of the microenvironment in cancer progression and suggests that 3D skin models can serve to uncover mechanisms and therapeutic targets that are not detectable in simpler systems. Statement of significance: This study introduces advanced, ethically sound skin and melanoma models as alternatives to animal testing in drug discovery. By thoroughly characterizing these models using bioimaging and RNA sequencing, we demonstrate their close resemblance to human skin, particularly in full-thickness models. These models not only replicate the complex cellular interactions and gene expression patterns of human tissue but also maintain robustness after melanoma integration. Our findings highlight the potential of these models in revealing cancer mechanisms and therapeutic targets, offering a significant impact on melanoma research and preclinical testing.</p

Post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson's disease GBA-N370S dopamine neurons

Variants at the GBA locus, encoding glucocerebrosidase, are the strongest common genetic risk factor for Parkinson's disease (PD). To understand GBA-related disease mechanisms, we use a multi-part-enrichment proteomics and post-translational modification (PTM) workflow, identifying large numbers of dysregulated proteins and PTMs in heterozygous GBA-N370S PD patient induced pluripotent stem cell (iPSC) dopamine neurons. Alterations in glycosylation status show disturbances in the autophagy-lysosomal pathway, which concur with upstream perturbations in mammalian target of rapamycin (mTOR) activation in GBA-PD neurons. Several native and modified proteins encoded by PD-associated genes are dysregulated in GBA-PD neurons. Integrated pathway analysis reveals impaired neuritogenesis in GBA-PD neurons and identify tau as a key pathway mediator. Functional assays confirm neurite outgrowth deficits and identify impaired mitochondrial movement in GBA-PD neurons. Furthermore, pharmacological rescue of glucocerebrosidase activity in GBA-PD neurons improves the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate neurodegeneration-associated pathways and potential drug targets in complex disease models.</p

MicroBubble activated acoustic cell sorting

Acoustophoresis, the ability to acoustically manipulate particles and cells inside a microfluidic channel, is a critical enabling technology for cell-sorting applications. However, one of the major impediments for routine use of acoustophoresis at clinical laboratory has been the reliance on the inherent physical properties of cells for separation. Here, we present a microfluidic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method that rely on the specific binding of target cells to microbubbles conjugated with specific antibodies on their surface for continuous cell separation using ultrasonic standing wave. In acoustophoresis, cells being positive acoustic contrast particles migrate to pressure nodes. On the contrary, air-filled polymer-shelled microbubbles being strong negative acoustic contrast particles migrate to pressure antinodes and can be used to selectively migrate target cells. As a proof of principle, we demonstrate the separation of cancer cell line in a suspension with better than 75% efficiency. Moreover, 100% of the microbubble-cell conjugates migrated to the anti-node. Hence a better upstream affinity-capture has the potential to provide higher sorting efficiency. The BAACS technique expands the acoustic cell manipulation possibilities and offers cell-sorting solutions suited for applications at point of care. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10544-017-0157-4) contains supplementary material, which is available to authorized users