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.