Marko Tirri, Tampere University of Technology

Molecule counting with two-photon fluorescence excitation

The main goal of this thesis was to construct and test the equipment, by which it is possible to detect and count single molecules or molecule clusters in liquid. Technology and knowledge necessary for the construction of the equipment were principally known in advance. The design and realization of the assembly as well as the testing of the measurement system were performed in the experimental part of the thesis. The methods based on single molecule detection are important research tools, eg. in the discovery of new drugs.

The function of the constructed equipment is based on the measurement of fluorescence produced by two-photon excitation. The aim was to clarify the requirements for equipment and laser to realize dual-colour fluorescence correlation spectroscopy (FCS) by two-photon fluorescence excitation. Further aim was to clarify the possibility to realize a bioaffinity assay, in which fluorescent nanoparticles are used as label molecules and nanoparticle complexes produced by bioaffinity reaction are counted.

Two-photon fluorescence excitation was performed by a mode-locked Nd:glass femtosecond laser, which can produce laser pulses that have duration of 120 fs and wavelength of 1060 nm. Two-photon excited dual-colour FCS realized by that kind of laser has not been published earlier in scientific literature. In those experiences the lasers have been titanium-sapphire-lasers functioning in lower wavelength range.

The possibility to count biomolecules and nanoparticles in liquid with the constructed equipment was eluciated by measurements. It is possible to detect and count single photons by the measurement system. When calculating an auto-correlation function for the photon signal, it is possible to define size differences between molecules or molecule clusters. When measuring nanoparticles labeled with different fluorescent dyes cross-correlation can be calculated for these signals. Binding reactions between different particles can be observed by cross-correlation.