Spectrally resolved fluorescence microscopy and its applications for single molecule characterization

Abstract

Spectrally resolved fluorescence microscopy is an important tool for measuring biological systems at the single molecule level. Spectral resolution provides a means for unambiguous identification and a tool for characterizing heterogeneity. Fluorescence microscopy facilitates single molecule measurement by limiting contrast to only those molecules that are capable of producing fluorescence when illuminated with the excitation source of the system. In the following thesis I describe my work combining spectral resolution with a fluorescence microscope by developing a hyperspectral prism-type total internal reflection fluorescence microscope. The microscope has a tunable supercontinuum laser excitation source along with 3 CW solid state lasers and has the ability to separate a field of view into 4 spectral channels which are imaged simultaneously. We used the microscope to characterize the photoluminescence excitation spectrum of individual fluorescent molecules and measure F\"{o}rster resonant energy transfer from inter-domain motion of matrix metalloproteiase for the first time. This versatile platform facilitates high throughput single molecule measurements in which multiple properties can be studied simultaneously.