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.
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