Exploring membrane protein dynamics by multicolor single quantum dot imaging using wide field, TIRF, and hyperspectral microscopy

Abstract

The development of colloidal quantum dots (QDs) for biological imaging has brought a new level of sensitivity to live cell imaging. Single particle tracking (SPT) techniques in particular benefit from the superior photostability, high extinction coefficient and distinct emission spectra of QDs. Here we describe the use of QDs for SPT to study the dynamics of membrane proteins in living cells. We work with the RBL-2H3 mast cell model that signals through the high affinity IgE receptor, FcVRI. Using wide field or Total Internal Reflection Fluorescence (TIRF) microscopy we have achieved simultaneous imaging of two spectrally distinct QDs with frame rates of up to 750 frames/s and localization accuracy of ~10 nm. We also describe the imaging and analysis of QDs using a novel hyperspectral microscope and multivariate curve resolution analysis for multi-color QD tracking. The same QD-tag used for SPT is used to localize proteins at <10 nm resolution by electron microscopy (EM) on fixed membrane sheets.