Hyperspectral Line Scanning Microscopy for High-Speed Multicolor Quantum Dot Tracking

Abstract

One of the fundamental goals in observing protein-protein interactions on the cell membrane is achieving nanometer scale spatial resolution along with temporal resolution sufficient to study live cell behavior. Traditional fluorescence microscopy methods have been unsuccessful in studying these interactions due to the diffraction limit with visible light. Single particle tracking techniques using quantum dots have provided single particle localizations to well below the diffraction limit, however, clustering of multiple particles limits the unique identification and thus tracking of individual particles throughout the (possibly dynamic) clustering process. This problem can be solved by tracking multiple quantum dot colors using a high-speed hyperspectral microscope.We have developed a high-speed hyperspectral microscope based on a line scanning design that is capable of up to 30 frames/sec with 128 spectral channels per pixel (ranging from 500-800 nm) with a sample area of ∼30 υm2. We describe the details of the microscope optical design details and layout, and show the results of excitation and emission path characterization. We image RBL-2H3 cells with IgE that has been conjugated with various colors of QDs (to give a near 1:1 QD:IgE ratio) and image at 30 frames per second. By imaging in both the presence and absence of crosslinking DNP-BSA, we demonstrate the ability to identify dynamic interactions and/or oligomers at spatial scales below the diffraction limit.