| dc.description.abstract | Recently, single-molecule microscopy techniques have increasingly been used to study the subcellular trafficking of biomolecules due to their nanometer-scale resolution and multicolor imaging capabilities. These techniques rely on the accurate localization of single molecules. Therefore, calibrating the microscope and its optical components is important in the field of single-molecule microscopy because accurate and careful calibration helps guarantee a high level of accuracy that is often required in the analysis of single-molecule image data. In this study, we develop an approach for calibrating single-molecule microscopy systems at the nanometer scale. Using this approach, we assess the performance of single-molecule microscopes and their optical elements by detecting geometric aberrations in the emission light path.
Another contribution that we make in this dissertation is that we develop a method for evaluating and optimizing remote-focusing multifocal plane microscopy (rMUM). Generally, rMUM is an imaging technique that allows the acquisition of single-molecule trajectories in three dimensions while simultaneously acquiring z-stack images of the cellular context. However, in practical implementations of rMUM, it is challenging to evaluate and optimize rMUM data with a high accuracy because of the complex image data set generated by the rMUM experiments. Therefore, in this study, we develop an experimental protocol for evaluating and optimizing the performance of rMUM. This protocol relies on determining the localization accuracy of single molecules, measuring the spatial registration accuracy, and correcting for the focal shift due to the refractive index mismatch. | en |
