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Advanced Fluorescence Lifetime Imaging and Spectroscopy Techniques for Biological Samples

Student thesis: Doctoral ThesisDoctor of Philosophy

Intracellular viscosity is correlated with cause of diseases, and it also affects the metabolism and chemical signalling. In this thesis, fluorescence-based studies were conducted on a fluorescent molecular rotor, BODIPY-Ci2, which was used to measure the intracellular viscosity. BODIPY-C12 is insensitive to the surrounding polarity using solvatochromic methods. It was demonstrated that the fluorescence lifetime of BODIPY-Ci2 in methanol/glycerol mixtures is related to viscosity via the Forster-Hoffmann equation, and the rotational correlation time depends on viscosity based on the Stokes-Einstein-Debye equation. The intracellular viscosity is measured via FLIM, time-resolved and steady-state fluorescence anisotropy. The relationship between the fluorescence lifetime and rotational correlation time of BODIPY-Ci2 in methanol/glycerol mixtures agrees with the combination of the Forster-Hoffmann equation and the Stokes-Einstein-Debye equation and is different to that of a rigid fluorophore. From the fluorescence imaging and counterstaining experiments, it appears that BODIPY-Ci2 is located in lipid droplets and the endoplasmic reticulum of cells. Particle tracking provided further evidence of lipid droplet staining. It seems that there are two different lifetimes in lipid droplets and the endoplasmic reticulum via time-resolved fluorescence anisotropy measurements, which indicate of two different environments for both locations. The lifetime is not a function of rotational correlation time for BODIPY-Ci2 in cells. An appropriate medium has to use to create a calibration of lifetime versus viscosity for measuring the microviscosity of cells. It is also found that BODIPY-Ci2 fails to measure viscosity in non-polar silicone oils.
In addition, spectroscopic studies of Nile red, Rhodamine 123, Pyrromethene 546 and BODIPY-C12 in methanol/glycerol mixtures were carried out to examine the relationship between the radiative rate constant and refractive index according to the Strickler-Berg equation and an advanced model by Toptygin et al. The transition dipole moment and the shape of the fluorophore can be analyzed using a model also by Toptygin et al. Time-resolved fluorescence imaging and spectroscopy offer a very powerful and versatile method for applications in the life sciences.
Original languageEnglish
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Award date1 Aug 2012

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