Waveguide evanescent field fluorescence microscopy: from cell-substratum distances to kinetic cell behaviour

摘要

We demonstrate an alternative to total internal reflection fluorescence (TIRF) microscopy. A method for imaging ultra thin films and living cells located on waveguides illuminated with their evanescent fields is introduced. Analysis of ion-exchanged waveguides focusing on their application as substrates for microscopic study of interfacial phenomena is presented. Various LB film stacks were imaged to verify the intensity interpretation due to the exponentially decaying evanescent fields of the waveguides. The paper gives an overview on the imaging applications of this technique. The fluorescence intensity has been used to determine quantitatively the cell attachment of osteoblasts (bone forming cells) to substrate surfaces. In live cell studies trypsin (a protease) was used to alter attachment of the cells to the substrate, as a means to demonstrate feasibility of the method in measuring attachment dynamics of cells in real time.