Physics & Astronomy ETDs

Publication Date

7-11-2013

Abstract

One of the challenges in studying protein interactions in live cells lies in the capacity to obtain both spatial and temporal information that is sufficient to extend existing knowledge of the dynamics and interactions, especially when tracking proteins at high density. Here we introduce a high-speed laser line-scanning hyperspectral microscope that is designed to track quantum dot labeled proteins at 27 frames/sec over an area of 28 um2 using 128 spectral channels spanning the range from 500 to 750 nm. This instrument simultaneously excites 8 species of quantum dots and employs a custom prism spectrometer and high speed EMCCD to obtain spectral information that is then used to distinguish and track individual probes at high density. These emitters are localized to within 10s of nm in each frame and reconstructed trajectories yield information of the protein dynamics and interactions. This manuscript describes the design, implementation, characterization, and application of a high-speed laser line-scanning hyperspectral microscope (HSM). The intended primary application is that of investigating the dynamics of transmembrane antibody receptors using quantum dot labeled immunoglobulin E (QD-IgE). Several additional examples demonstrate other advantages and applications of this method, including 3D hyperspectral imaging of live cells and hyperspectral superresolution imaging.

Degree Name

Physics

Level of Degree

Doctoral

Department Name

Physics & Astronomy

First Advisor

Lidke, Keith A.

First Committee Member (Chair)

Lidke, Diane S.

Second Committee Member

Thomas, James L.

Third Committee Member

Prasad, Sudhakar

Language

English

Keywords

confocal, hyperspectral, line-scanning, QD-IgE, quantum dot, SPT

Document Type

Dissertation

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