Total internal reflection sets up an evanescent wave that – when used for microscopic imaging – renders the microscope short-sighted and enables the selective observation of the near-membrane space. But how deep is deep? In this Biophysical Perpective Article, Oheim, Brunstein and co-workers review existing techniques for calibrating the penetration depth of the evanescent field, allowing the quantitative comparison of fluorescence data across experiments, instruments and labs.
The article is now online as an AOP at the Biophysical Journal: Oheim M., et al. (2019) DOI: 10.1016/j.bpj.2017.07.048