Waveguide Evanescent Field Fluorescence Microscopy Images of Osteoblast Cells: The Effect of Trypsin and Image Processing Using TrackMate.

Waveguide evanescent field fluorescence microscopy (WEFF) is an evanescent-based microscopy that utilizes a confined thin film of light, around 100 nm, to image the plasma membrane of cells attached to a waveguide. Low photobleaching and low background besides its high axial resolution allows time-lapse imaging to investigate changes in cell morphology in the presence or absence of chemical agents. Both large field of view (FOV) and uniform illumination are very important while imaging cell-substrate contacts with an evanescent field. In the current study, we demonstrate that the WEFF microscope is capable of large FOVs with a uniform illumination source and imaging over a very long time period with a simple and inexpensive experimental setup. The interaction of the trypsin with plasma membranes of live osteoblast cells is investigated. To analyze cell images (250 images), instead of relying on manual tracking, which is time-consuming and can introduce numerous errors, we performed image processing using TrackMate to investigate the dynamic response of cells upon exposure to trypsin. This helps to save time and increase the accuracy of the analysis. The powerful tracking and analysis capabilities of the TrackMate plugin in ImageJ are used to automatically detect the cells border and trace each cluster of cells. The reduction in cell area is accompanied by a notable increase in mean intensity, reflecting changes in the intracellular environment. However, the background did not change during the experiment, which proves that the fluorescence material remains attached to the cell membrane and does not leak into the cell medium.