THE APPLICATION OF SINGLE-POINT EDGE-EXCITATION SUB-DIFFRACTION MICROSCOPY FOR THE STUDY OF MACROMOLECULAR TRANSPORT

Abstract

The development of super-resolution microscopy made it possible to surpass the diffraction limit of optical microscopy, enabling researchers to gain a nanometer scale understanding of cellular structures. While many applications have benefited from standard super-resolution microscopy, gaps remained making high-speed dynamic imaging in live cells impossible. To address this problem, single-point edge-excitation sub-diffraction (SPEED) microscopy was developed. This methodology enables the nanometer imaging of dynamic cell processes within live cells, the evaluation of subcellular structural information, the capacity to derive three-dimensional information from two-dimensional images within rotationally symmetric structures, and the interrogation of novel questions regarding the transport dynamics of macromolecules in a variety of cellular structures. Here, I have described the theory and method behind the current iteration of SPEED microscopy that we have developed and validated via Monte Carlo simulation. Further, a detailed description of how we have further developed SPEED microscopy to derive structural information within the nuclear pore complex as well as how SPEED has been applied to evaluate the export kinetics of mRNA.