The features that make Tolansky interference method so suitable for cell cultures are:
1) Contrast enhancement. Filopodia, are very fine but also very flat, i.e., thin in the Z direction. The technique enhances their visibility in two ways: a) because of light interference, a difference in thickness of <3 nm is visible, b) the refractive index difference between air and the cell content is greater than between cells and water (this makes the optical path length greater).
2) The repeat of interference fringes at about 1 micrometer intervals in the Z-direction provides information about the change in cell shape in height, i.e., it allows for 3-dimensional sampling of the cell. The colors are in reproducible order as illustrated in the Michel- Lévy color chart (below).
The technique relies upon having an oxide layer formed on the surface of the metal, ordinarily by oxidation in an electrochemical bath. Due to reflection, one ray of light from a metallic surface and another from the surface of its oxide interfere above the oxide surface. Tantalum has nice properties for this, and is commonly used. The result is a standing wave from metal and oxide surfaces. If, on the oxide surface, one attaches a dielectric material, a shift in interference color will be observed. The actual color of the shifted interference varies with the background color from the standing wave as well as the thickness of the adsorbed layer. The thickness of the oxide can be adjusted but is most sensitive when the background color is bronze or gold. A limit of ~3 nm height difference was shown by a researcher named Leo Vroman [1]. The lateral resolution is unaffected, so that it is still limited by the Abbe diffraction limit.
Conversion of the colored images to gray-level images provides nice contrast at the boundary of the contours (see below). These digital images are easy to segment automatically.
[1] Vroman, L.; Adams, A. L. Surf. Sci. 1969, 16, 438-446