Core B: MATERIAL CORE

核心B：核心材料

• 批准号: 8234233
• 负责人: Alex Groisman
• 金额: $ 11.88万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8234233
• 关键词: Adhesions; Animals; Area; Atomic Force Microscopy; Automobile Driving; Cell Adhesion; Cell membrane; Cells; Collaborations; Detection; Development; Devices; Elasticity; Event; Fluorescence; Fluorescence Microscopy; Gel; Glass; Human Resources; Knowledge; Location; Magnetism; Maintenance; Mammalian Cell; Maps; Measurement; Measures; Mediating; Membrane; Methods; Microscope; Microscopy; Molecular; Pattern; Physiological; Production; Recruitment Activity; Refractive Indices; Resolution; Scanning Probe Microscopes; Silicone Elastomers; Silicones; Stretching; Structure; System; Techniques; Testing; Thick; Total Internal Reflection Fluorescent; Traction; Training; Variant; base; cell motility; cellular imaging; design; improved; mathematical model; molecular marker; particle; polyacrylamide gels; research study; soft tissue; trafficking; tumorigenesis

When grown on a substrate, animal cells sense its rigidity, especially in a range corresponding to soft tissues, with elastic moduli, E, of 0.1 - 100 kPa.[1] Variations of substrate rigidity are important in development (2-4), tumorigenesis(5,6), and cell migration (7,8). To better understand the rigidity sensing, it is essential to selectively visualize cellular adhesion structures that exert traction forces on the substrate and mediate the rigidity sensing. For substrates with E of 0.1 - 100 kPa, substrate deformations caused by the cell traction forces can be measured under a microscope and the traction forces can be reconstructed. Because substrate deformation in a given area often results from traction forces applied at multiple adhesion points, the conversion ofa map of substrate deformation into a cell traction force map is complicated, especially when the locations of the adhesion points are not known(9). Adhesion points can be detected with molecular markers recruited to cellular adhesion structures using wide-field or confocal fluorescence microscopy, but identification of adhesion points exerting traction forces can be challenging. In addition, the accurate assessment of the adhesion area and the detection of small adhesion points can be limited by the fluorescence background. The level of fluorescence background is substantially lower in TIRF microscopy, which selectively visualizes fluorescent molecules in an -100 nm thick layer above the substrate and is the method of choice to image the cell-to-substrate adhesion structures^" and to study molecular trafficking events at the plasma membrane Application of TIRF microscopy to cell imaging requires the refractive index ofthe substrate to be substantially higher than that of cells, n = 1.36 - 1.38. In particular, for optimal TIRF microscopy with a specialized TIRF objective, the substrate refractive index must be higher than the numerical aperture (NA) ofthe objective, which is typically 1.45-1,49, The most commonly used cell substrates that have the rigidity of soft tissue, polyacrylamide gels, have a refractive index of ~1.33, making them unsuitable for TIRF microscopy. Gels made of silicone elastomer polydimethylsyloxane (PDMS)(12), usually have a refractive index of ~1.41 and are not very practical as substrates for cell TIRF microscopy either. The elastic modulus of bulk gels can be evaluated with a variety of techniques and systems, from measurements of indentations produced by heavy beads to the use of specialized stretching machines or indenters(13,14). For thin gel layers on cover glasses for experiments on animal cells, the method of choice is usually the application of an atomic force microscope (AFM), However AFM systems are expensive and the interpretation of results of the measurements depends on mathematical models of gel elasticity and on the exact knowledge of the curvature of the AFM tip.

当在基板上生长时，动物细胞会感觉到其刚性，尤其是在某个范围内 对应于软组织，弹性模量为0.1-100 kpa。[1]底物刚性的变化在发育（2-4），肿瘤发生（5,6）和细胞迁移（7,8）中很重要。为了更好地理解刚性感应，至关重要的是，可以选择性地可视化在基板上施加牵引力并介导刚性感应的细胞粘附结构。对于E 0.1-100 kPa的底物，可以在显微镜下测量由细胞牵引力引起的底物变形，并且可以重建牵引力。由于给定区域中的底物变形通常是由于在多个粘附点上施加的牵引力而导致的，因此底物变形向细胞牵引力图的转换很复杂，尤其是当粘附点的位置未知时（9）。可以用分子标记检测粘附点 使用宽视野或共聚焦荧光显微镜募集到细胞粘附结构，但鉴定发挥牵引力的粘附点可能是具有挑战性的。另外，可以通过荧光背景限制对粘附区域的准确评估和小粘附点的检测。 TIRF显微镜的荧光背景水平显着较低，该水平在基板上方的-100 nm厚层中有选择地可视化荧光分子，并且是对细胞到覆盖结构进行成像的选择方法 TIRF显微镜在细胞成像中的应用要求底物的折射率大大高于细胞n = 1.36-1.38。特别是，对于具有专门TIRF物镜的最佳TIRF显微镜，底物折射率必须高于物体的数值孔径（Na），该指数通常是1.45-1,49，最常用的细胞底物是最常用的细胞底物，这些底物具有软组织，多酰胺胶质的固定性，可抗逆转型，使其具有固定性，使其具有1.33，使其具有〜1.33，使其成为〜1.33，是〜1.33。凝胶制成 硅酮弹性体聚二甲氧基（PDMS）（12）通常具有〜1.41的折射率，并且不像细胞TIRF显微镜的底物一样实用。可以通过多种技术和系统评估散装凝胶的弹性模量，从重珠产生的压痕的测量到使用专业的拉伸机或凹痕（13,14）。对于盖玻璃上的薄凝胶层以进行动物细胞实验，选择的方法通常是原子力显微镜（AFM）的应用，但是AFM系统很昂贵，测量结果的解释取决于凝胶弹性的数学模型以及AFM尖端的曲率知识。