臺大機械 王建凱 AML

Expansion microscopy is an innovative technique that employs swellable polymers to physically enlarge biological specimens, enabling high-resolution imaging beyond the traditional optical diffraction limits. However, the sample expansion process may result in non-uniform, anisotropic deformation across the sample dilation process. A method to quantify and correct the anisotropic expansion is required. Herein, we used a cultured cell, HeLa (a cancer cell line) with its microtubules stained and followed by 4X expansion. Lattice lightsheet microscopy captured 3D pre- and post- expansion images on the identical cell. The present imaging analysis method is based on these two set of 3D images.

Taking advantage of the good mechanical strength of expanded Drosophila brains and to tackle their relatively large size that can complicate imaging, we apply potassium (poly)acrylate-based hydrogels for expansion microscopy (ExM), resulting in a 40x plus increased resolution of transgenic fluorescent proteins preserved by glutaraldehyde fixation in the nervous system. Large-volume ExM is realized by using an axicon-based Bessel lightsheet microscope, featuring gentle multi-color fluorophore excitation and intrinsic optical sectioning capability, enabling visualization of Tm5a neurites and L3 lamina neurons with photoreceptors in the optic lobe. We also image nanometer-sized dopaminergic neurons across the same intact iteratively expanded Drosophila brain, enabling us to measure the 3D expansion ratio. Here we show that at a tile scanning speed of  1 min/mm^3 with 10^12 pixels over 14 hours, we image the centimeter-sized fly brain at an effective resolution comparable to electron microscopy, allowing us to visualize mitochondria within presynaptic compartments and Bruchpilot (Brp) scaffold proteins distributed in the central complex, enabling robust analyses of neurobiological topics.

This study investigates the mechanical properties of pressure-sensitive adhesive (PSA) and PSA blended with polyethylene terephthalate (PET) film (PBP) through a series of mechanical tests. The combination of a linear-elastic and viscoelastic material model was employed and calibrated to accurately characterize their mechanical behavior. The tensile test and dynamic mechanical analysis (DMA) yielded contrasting results regarding the elastic properties of the two polymer thin films. The experimental data revealed that Young’s modulus of PBP is significantly higher than that of PSA, whereas the shear modulus of PBP is notably lower than PSA. This behavior can be attributed to the sandwich structure of the PBP composite, where the PSA and PET components interact in different configurations either in parallel or in series during the tensile test and DMA, respectively. This paper presents a systematic approach for the precise and efficient application of material models for PSA and PBP, providing valuable insights for future use in various composite polymer film adhesives. Comprehensive calibration and finite element analysis were conducted for both PSA and PBP materials. Based on these experimental findings, we propose a systematic method for measuring and calibrating the mechanical properties of thin film composite materials. This method offers a reliable reference for future engineering applications, as it can be applied to other material systems to expedite the development process.

Endothelium lining interior surface of blood vessels experiences various physical stimulations in vivo. Its physical properties, especially elasticity, play important roles in regulating the physiological functions of vascular systems. In this paper, an integrated approach is developed to characterize the anisotropic elasticity of the endothelium under physiological-level fluid shear stress. A pressure sensor-embedded microfluidic device is developed to provide fluid shear stress on the perfusion-cultured endothelium and to measure transverse in-plane elasticities in the directions parallel and perpendicular to the flow direction. Biological atomic force microscopy (Bio-AFM) is further exploited to measure the vertical elasticity of the endothelium in its out-of-plane direction. The results show that the transverse elasticity of the endothelium in the direction parallel to the perfusion culture flow direction is about 70% higher than that in the direction perpendicular to the flow direction. Moreover, the transverse elasticities of the endothelium are estimated to be approximately 120 times larger than the vertical one. The results indicate the effects of fluid shear stress on the transverse elasticity anisotropy of the endothelium, and the difference between the elasticities in transverse and vertical directions. The quantitative measurement of the endothelium anisotropic elasticity in different directions at the tissue level under the fluid shear stress provides biologists insightful information for the advanced vascular system studies from biophysical and biomaterial viewpoints.

Oxygen is necessary for cellular respiration in aerobic organisms. In animals, such as human, inhaled oxygen moves from the alveoli to the blood through alveolar epithelium into pulmonary capillaries. Up to now, different studies have been reported to examine experimental oxygen diffusivity for simple membrane or single-celled organisms; however, devices capable of precisely characterizing oxygen transportation through cell layers with dimensions similar to their physiological ones have not been developed. In this study, we establish an integrated approach exploiting a multi-layer microfluidic device and relative fluorescence lifetime detection apparatus to reliably measure oxygen diffusivity through a cell layer. In the experiments, different types of cells, including A549 and 3T3 cell lines, lung stem/progenitor cells, and the differentiated type I pneumocyte-like cells, are used to form cell layers within the devices for their oxygen diffusivity evaluation. A distinct facilitated oxygen transportation behavior of the differentiated type I pneumocyte-like cells that has never been discussed before is identified using the approach. The study offered a new in vitro approach to evaluate the oxygen diffusivity across cell layers in a microfluidic device and open a door to construct more physiologically meaningful in vitro model system to study respiratory systems.

Formation of 3D networks is a crucial process for endothelial cells during development of primary blood vessels under both normal and pathological conditions. In order to investigate effects of oxygen microenvironment and matrix composition on the 3D network formation, an upside-down microfluidic cell culture device capable of generating oxygen gradients is developed in this paper. In cell experiments, network formation of human umbilical vein endothelial cells (HUVECs) within fibrinogen-based hydrogels with different concentrations of hyaluronic acid (HA) is systematically studied. In addition, five different oxygen microenvironments (uniform normoxia, 5%, and 1% O2; oxygen gradients under normoxia and 5% O2) are also applied for the cell culture. The generated oxygen gradients are characterized based on fluorescence lifetime measurements. The experimental results show increased 3D cell network length when the cells are cultured under the oxygen gradients within the hydrogels with the HA addition suggesting their roles in promoting network formation. Furthermore, the formed networks tend to align along the direction of the oxygen gradients indicating the presence of gradient-driven cellular response. The results demonstrate that the developed upside-down microfluidic device can provide an advanced platform to investigate 3D cell culture under the controlled oxygen microenvironments for various biomedical studies in vitro.