| Abstract |
Optical techniques are powerful tools to probe the function of proteins in living cells. Proteins can be fluorescently labelled with high specificity and this permits one to study where and when they are produced or degraded within living cells with high spatial and temporal resolution. The fluorescence signatures may also report on subtle changes in the molecular environment of the tagged proteins. For example interactions between proteins may lead to subtle intensity, colour and lifetime changes in the emission of the reporting fluorophores. There is therefore a constant demand in the life sciences for novel, better, and more flexible instrumentation to measure fluorescence from within living cells. The fluorescence lifetime in particular is a key parameter in such efforts. Molecular proximity can quench fluorescence and this in turn results in a reduction of the lifetime. Measuring lifetime from molecules in cells is however a difficult task: There is always a trade-off between the precision and the speed at which a lifetime measurement can be performed. In the current proposal we seek to improve on a technique called frequency domain lifetime imaging microscopy (FD FLIM): This technique is popular in conjunction with widefield fluorescence microscopy because it is simple and flexible in application but it has drawbacks, because in its conventional form, the speed of measurement and the precision are limited. In the present work we aim to overcome these limitations and vastly improve on the capability of FD FLIM for measurements of protein-protein interactions in living systems. The instrument to be developed here will be used in the study of the molecular processes, which regulate cell division and we will be able to do this in much more detail than before: The features we study are highly dynamic and occur over small spatial scales and the improved temporal resolution and precision will quantify events that we have not been able to observe so far. The capability we are developing here will be available for researchers from various departments and be useful in situations where either molecular scale events need to be monitored at rapid speed, such as signalling events in cells, or in situations where high measurement precision is required, for example to record subtle lifetime changes that may report on different oligomeric states of aggregating monomers. |
