A simple image processing pipeline to sharpen topology maps in multi-wavelength interference microscopy

Multi-wavelength standing wave (SW) microscopy and interference reflection microscopy (IRM) are powerful techniques that use optical interference to study topographical structure. However, the use of more than two wavelengths to image the complex cell surface results in complicated topographical maps, and it can be difficult to resolve the three-dimensional contours. We present a simple image processing method to reduce the thickness and spacing of antinodal fringes in multi-wavelength interference microscopy by up to a factor of two to produce clearer and more precise topographical maps of cellular structures. We first demonstrate this improvement using model non-biological specimens, and we subsequently demonstrate the benefit of our method for reducing the ambiguity of surface topography and revealing obscured features in live and fixed-cell specimens.