Concentration gradients of ECM proteins play active roles in many areas of cell biology including wound healing and metastasis. They may also form the basis of tissue engineering scaffolds, as these can direct cell adhesion and migration and promote new matrix synthesis. To better understand cell–matrix interactions on attractive gradients, we have used multiphoton excited (MPE) photochemistry to fabricate covalently linked micro-structured gradients from fibronectin (FN). The gradient design is comprised of a parallel series of individual linear gradients with overall dimensions of approximately 800 × 800 μm, where a linear dynamic range of nearly 10-fold in concentration was achieved. The adhesion dynamics of 3T3 fibroblasts were investigated, where the cell morphology and actin cytoskeleton became increasingly elongated and aligned with the direction of the gradient at increasing protein concentration. Moreover, the cell morphologies are distinct when adhered to regions of differing FN concentration but with similar topography. These results show that the fabrication approach allows investigating the roles of contact guidance and ECM cues on the cell–matrix interactions. We suggest this design overcomes some of the limitations with other fabrication methods, especially in terms of 3D patterning capabilities, and will serve as a new tool to study cell–matrix interactions.
Surface plasmon resonance (SPR) and common-path phase-shift interferometry (PSI) techniques are integrated in a biosensing imaging system for measuring the two-dimensional spatial phase variation caused by biomolecular interactions on a sensing chip without the need for additional labeling. The common-path PSI technique has the advantage of long-term stability, even when it is subjected to external disturbances. Hence the system meets the requirements of the real-time kinetic studies involved in biomolecular interaction analysis. The proposed SPR-PSI imaging system demonstrates a detection limit of a 2 x 10(-7) refractive-index change, a long-term phase stability of 2.5 x 10(-4) pi rms for 4 h, and a spatial phase resolution of 10(-3) pi with a lateral resolution of 100 microm.
A surface plasmon-enhanced two-photon total-internal-reflection fluorescence (TIRF) microscope has been developed to provide fluorescent images of living cell membranes. The proposed microscope with the help of surface plasmons (SPs) not only provides brighter fluorescent images based on the mechanism of local electromagnetic field enhancement, but also reduces photobleaching due to having a shorter fluorophore lifetime. In comparison with a one-photon TIRF, the two-photon TIRF can achieve higher signal-to-noise ratio cell membrane imaging due its smaller excitation volume and lower scattering. By combining the SP enhancement and two-photon excitation TIRF, the microscope has demonstrated it's capability for brighter and more contrasted fluorescence membrane images of living monkey kidney COS-7 fibroblasts transfected with an EYFP-MEM or EGFP-WOX1 construct.
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