Abstract
Single molecule tracking and localization has become a powerful strategy for noninvasive imaging of nanoscale structures in biology. The aim of this study is to develop a scanning astigmatism module for 3D Stochastic-Optical-Reconstruction-Microscopy (STORM). For 3D STORM imaging, a weak cylindrical lens is placed in the imaging path to induce astigmatism. The specific location of the lens in the optical path can affect the extent of correction and in our approach, the ability to scan the lens position allows for finer control and thus optimization for the reconstruction. Our design uses an Arduino-based circuit along with a stepper motor controlled by a Python interface to position the cylindrical lens. The ellipticity and orientation of fluorophores’ localization are obtained using the ImageJ data analysis plugin ThunderSTORM. We demonstrate the feasibility of this approach through 3D reconstruction of the mitochondrial morphology of HeLa cells. Improvement in the visualization of cellular structures and their relationships with nanoscale resolution in all three dimensions will contribute to a better understanding of molecular processes occurring in cells.
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