Total internal reflection fluorescence microscopy is an evanescent based fluorescence microscope providing a selective visualization of cell-substrate contacts without interference from other, deeper cellular regions. Total internal reflection fluorescence microscope is used extensively to visualize cell-substrate contacts. However, quantifying these contacts - in particular the measurement of cell-substrate distances - has not been performed often. In order to quantify the cell-substrate distances we have developed a new theoretical method which is based on a change in the penetration depth of the evanescent field by tuning the angle of incidence slightly above the angle of total internal reflection for s-polarized light. This is simpler and much more accurate in comparison to the few existing approaches.
