© 2015 Macmillan Publishers Limited. All rights reserved. Measuring the nanoscale organization of protein structures near the plasma membrane of live cells is challenging, especially when the structure is dynamic. Here we present the development of a two-wavelength total internal reflection fluorescence method capable of real-time imaging of cellular structure height with nanometre resolution. The method employs a protein of interest tagged with two different fluorophores and imaged to obtain the ratio of emission in the two channels. We use this approach to visualize the nanoscale organization of microtubules and endocytosis of the epidermal growth factor receptor.