Photoactivatable Quantum Dots in Super-Resolution 3D Microscopy of Myofibrils

Abstract

To image the relationships between immune-labeled myofibrilar proteins at sub-diffraction-limited resolutions, highly photostable quantum dots were chemically modified to make them photoactivatable. Although previous reports have used photoactivation of cyanine dyes and GFP variants for 2D super-resolution microscopy, photoactivatable quantum dots (PAQ dots) have sufficient brightness and photostability to enable 3D acquisitions of signals from individual quantum dots. The chemical synthesis of PAQ dots caused only minor changes in the spectroscopic properties and brightness of the activated PAQ dots relative to unmodified quantum dots as assessed by fluorescence lifetime imaging of single quantum dots. The PAQ dots were conjugated to Fab fragments for immunostaining of myofibrils. After optimizing conditions so that a balance between photoactivation and photobleaching of the PAQ dots occurred during 3D acquisition in a spinning disk confocal microscope, 3D images of individual quantum dots were reduced to the 3D center of mass and accumulated until sufficient data for a full image was generated. Initial results demonstrate sub-diffraction resolutions in XY and even more striking resolution improvements in Z. The superresolution images reveal finer structural details in the myofibrils than conventional confocal imaging. Unlike electron microscopy, all measurements are made in aqueous solutions. Furthermore, the ability to make PAQ dots with a variety of emission wavelengths enables multicolor 3D labeling that can be used for protein mapping at super-resolutions in myofibrils and other samples.