Waveguide Evanescent Field Fluorescence Microscopy Images of Osteoblast
Cells: The effect of trypsin and image processing using TrackMate
Abstract
Waveguide evanescent field fluorescence microscopy (WEFF) is an
evanescent based microscopy 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 beside 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
work, 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 an 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 is used to automatically detect the cells boarder and traces 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.