Applications of fluorescence lifetime imaging in biology

Supervisors:
Dr Angus Bain, Department of Physics & Astronomy, CoMPLEX, UCL
Prof Michael Duchen, Department of Cell and Developmental Biology, UCL

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful tool in the study of biological processes. One significant opportunity and challenge for FLIM though, is its application on the minimally invasive or non-invasive in situ biopsy, based on natural fluorophores, such as the reduced nicotinamide adenine dinucleotide (NADH) and tryptophan. This has to be combined with data analysis models, whether in time or in frequency domain, that are efficient, reliable and increase the signal-to-noise ratio (especially when the acquisition times are short), such as Global Analysis. Furthermore, mathematical models, such as the Stretched Exponential Function and the Maximum Entropy Method that require less processing load and represent the biophysical mechanisms better than the multiexponential model, particularly when a range of microenvironments is imaged, are necessary in order to provide better tissue differentiation.

Screenshot from the software package SPCImage analyzing FLIM microscopy data. The microscope used was a two-photon Zeiss LSM 510 with Coherent Chameleon laser at 700nm, Becker and Hickl FLIM system. Measurement of the lifetime of free and bound NADH in cultured HEK (Human Embryonic Kidney) cells was conducted in the Lab of the UCL Research Department of Cell and Developmental Biology, with the guidance and help of PhD student Thomas Blacker. The cells were NADK gene knock-out mutants, so the fluorescence measured originated only from NADH and not NADPH.