Optimisation of a novel technique for measuring microviscous environments in living cells

Thesis

Microviscosity is a critical physical parameter to be taken into account when modelling cellular environments, and yet it has largely been ignored in cell biology because of the technical impediments to measuring it in cells. Traditional methods of measuring viscosity operate on far too gross a scale to be useful for making measurements on the cellular level, while more granular techniques all suffer from their own limitations. Molecular rotors, in combination with Fluorescence Lifetime Imaging Microscopy (FLIM), present a means of overcoming many of these limitations. However, to date, most molecular rotors for use in cell biology rely upon the addition of specific chemical groups for delivery to discrete organelles. In this thesis, I endeavour to optimise a novel BODIPY-based molecular rotor that has a benzyl guanine group, allowing it to be targeted to any protein tagged with the SNAP-tag enzyme. We show that BG-BODIPY is well tolerated by cells, and can be targeted to a range of different organelles, both membrane bound and membraneless. We then go on to show that it can be targeted to much smaller targets than whole organelles, namely the nuclear pore complex. Finally, we show that BG-BODIPY can stain whole semi-terrestrial invertebrates and monitor their internal states during desiccation, making it one of the most versatile molecular rotors developed to date.

Authors: Foley, A

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: tardigrade; microviscosity; BODIPY; nuclear pore complex; FLIM
• Subjects: Molecular biology