The Use of Quantum Dot Blinking to Optimize of 3D Nanoscopy

Abstract

Fluorescence microscopy is a widely used tool in many molecular and cellular biologically research, as it enables the observation of specific components or processes in living cells, tissues, and whole organisms. However, the limited resolution of fluorescence microscopy leaves many biological structures too small to be studied in detail. Although subcellular structures often ranges as small as nanometers, most optical microscopes have lateral and axial resolutions of ∼ 200 nm direction and ∼ 500 nm respectively. Currently, novel imaging methods, stochastic optical reconstruction microscopy (STORM), have broken the diffraction limit resulting in significant improvements in resolution by switching a fluorescent molecule ON (bright) or OFF (dark). Most recently, STORM has spread the observed area from two (XY) to three (XYZ) dimensions by applying cylindrical optics (3D-STORM). However, they are constrained by the specific optics or fluorescent probes.To simplify the 3D-STORM method, we optimized both of the optics and the probe. The Z position of the fluorophore was represented to be ellipticity of point spread function of it by setting cylindrical lens after imaging lens. This ellipticity dependence was responsible for the focusing length of the cylindrical lens. We optimized the ellipticity dependency by verifying a distance between two cylindrical lenses (concave and convex). The appropriate distance was 10 mm and the 3D resolutions of the position determination of fluorophore are 10 nm (XY) and 40 nm (Z) when the fluorophore emitted 1000 photons. Next, we applied quantum dot (Qdot) to be a 3D-STROM because Qdot has intense and stable fluorescence, and especially blinks stochastically. Since the fluorophore rarely emits fluorescence in STORM method, we improved the Qdot whose ON events were rare. In this meeting, we will discuss our method, which is performed with Qdot , in detail.