Imaging Nanoscale Axial Dynamics at the Basal Plasma Membrane

Understanding of how energetically unfavorable plasma membrane shapes form, especially in the context of dynamic processes in living cells or tissues like clathrin-mediated endocytosis is in its infancy. Even though cutting-edge microscopy techniques that bridge this gap exist, they remain underused in biomedical sciences. Here, we demystify the perceived complexity of these advanced microscopy approaches and demonstrate their power in resolving nanometer axial dynamics in living cells. Total internal reflection fluorescence microscopy based approaches are the main focus of this review. We present clathrin-mediated endocytosis as a model system when describing the principles, data acquisition requirements, data interpretation strategies, and limitations of the described techniques. We hope this standardized description will bring the approaches for measuring nanoscale axial dynamics closer to the potential users and help in choosing the right approach to the right question.