Abstract

Macromolecular complexes are nanomachines that mediate cellular function. A mechanistic understanding of their architecture, spatial organization and dynamic changes at the nanoscale level is key to deciphering their functions and to designing biomedical interventions. The recent emergence of various superresolution microscopy techniques has allowed scientists to assess the complexity of the nanoscale world inside living cells. However, such techniques remain largely inaccessible due to the need for sophisticated instrumentation and specialized technical skillsets. Furthermore, laborious acquisition and analysis pipelines result in low data throughput and narrow the spectrum of potential applications. In this chapter, we will illustrate the working principle and the detailed workflow for a particular surface-generated structured illumination technique, known as Scanning Angle Interference Microscopy (SAIM). As compared to more popular superresolution techniques, which offer optimal performance in x-y dimensions, SAIM is designed to localize molecules along the optical axis with 5–10 nm precision while maintaining diffraction-limited lateral resolution (~250 nm with a high numerical aperture). Importantly, SAIM can be easily implemented on commercially available Total Internal Reflection Fluorescence (TIRF) microscopes. Finally, we discuss current applications of SAIM in mechano-molecular cell biology.