The Application of Multi-dimensional Fluorescence Imaging to Microfluidic Systems

Abstract

This thesis describes the application of multidimensional fluorescence imaging to microfluidic systems. The work focuses on time- and polarisation-resolved fluorescence microscopy to extract information from microchannel environments. The methods are applied to polymerase chain reaction (PCR) and a DNA repair enzyme, uracil DNA glycosylase (UDG). The fluorescence lifetimes Rhodamine B are calibrated over a thermal gradient using time correlated single photon counting. The dye is then introduced in solution into a novel microfluidic PCR device. Fluorescence lifetime imaging microscopy (FLIM) is then performed, and using the calibration curve, the temperature distributions are accurately determined. The device is subsequently optimised for efficient DNA amplification. A line-scanning FLIM microscope is used to characterise a rapid microfluidic mixer via a fluorescence quenching experiment. Fluorescein and sodium iodide are mixed in a continuous flow format and imaged in 3-D. The spatial distributions of the fluorescence lifetimes are converted to the concentrations of sodium iodide to quantify mixing. Computational fluid dynamic (CFD) simulations are validated by comparison to the quantitative concentrations obtained experimentally. The binding reaction between UDG and a hexachlorofluorescein (HEX) labelled DNA strand is characterised spectrally. As well as an increase in fluorescence polarisation anisotropy, a 700 ps increase in the fluorescence lifetime is measured. Confocal microscopy shows the same spectral properties when the reaction is performed in both simple and rapid microfluidic mixers. In the latter experiment, a concentration series allows the determination of kinetics, which agree with conventional stopped-flow data. A two-colour two-photon (2c2p) FLIM microscope is developed and applied to the UDG-DNA system. An oligonucleotide containing 2-aminopurine, a reporter of DNA base flipping, and HEX is mixed with UDG in a microfluidic Y-mixer. The 2c2p excitation allows FLIM of both fluorophores and hence detection of binding and base flipping. Comparison to CFD with known kinetic rate constants confirms the experimental observations.