Fluorescence Lifetime Imaging for Chemical Sensing

Abstract

Fluorescence Lifetime Imaging Microscopy (FLIM) has become an important tool in biological research because it enables accurate and sensitive measurements of molecular level processes. FLIM is non-invasive, independent of a fluorophore’s concentration, and sensitive to changes in molecular environments. There is a wide variety of FLIM techniques, including time-domain and frequency-domain methods which can be combined with widefield and scanning illumination options. Several methods are capable of optical sectioning but more advanced three-dimensional FLIM setups are still under development. In this thesis, theory of fluorescence lifetime and principles of FLIM are reviewed based on literature search. The goal is to provide information about different FLIM methods and implementations, about how FLIM can be used in three-dimensional imaging, and about FLIM applications in cell biology and chemical sensing. First, fluorescence and fluorescence lifetime are introduced so that the reader can understand on what phenomena FLIM is based on. Time- and frequency-domain methods are described, current FLIM instrumentations are presented, and FLIM implementation possibilities to three-dimensional microscopy methods are considered. This is followed by an introduction to FLIM data analysis methods. Chemical sensing possibilities are examined by discussing FLIM applications for oxygen sensing, ion concentration measurements, pH monitoring and Förster resonance energy transfer measurements. Literature reveals that there are many different approaches to determine fluorescence lifetimes. The decision about which method is the best depends on the application, desired lifetime accuracy, sample properties, and length of acquisition time. The review shows that also the used detector or camera affects the measurement outcome. By comparing different FLIM methods and detector types, it seems that megahertz frame rate cameras combined with time-domain approaches are most suitable for three-dimensional FLIM. Frequency-domain methods might be faster but less accurate. With the help of different analysis algorithms, fluorescence lifetime values can be determined from FLIM data. Moreover, noise and background signals can be reduced. This thesis reveals that FLIM has already been used successfully in many biological applications and that it can provide information of molecular environments. Future development might result in even more accurate and versatile FLIM methods which can be applied to image three-dimensional cell cultures and monitor their culturing conditions.