A novel frequency domain FLIM microscope for the dynamic study of protein function in live cells

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.

With the current proposal we seek funds to develop a novel and versatile Frequency Domain Fluorescence Lifetime Imaging Microscope (FD FLIM) which is flexible to operate at 1) high measurement speed by a method we term phi2FLIM, or, 2) at a precision hitherto difficult to achieve with available FD FLIM instrumentation in a variant of FD FLIM we term mh-FLIM (multi harmonic FLIM). The technology we propose is motivated by our wish to promote research on the role of key mitotic regulators during cell division. It represents a substantial improvement over existing technologies and, although specifically applied to mitosis research, will find widespread applications for biophysical studies of molecular function in living cells. Beneficiaries will include life scientists who may already have FD FLIM instrumentation available in their laboratories: The proposed technologies are relatively straight forward to implement on current, commercially available FD FLIM systems. The ideas behind the proposed research arose from ongoing collaborations between the groups of Dr. Clemens Kaminski at the department of Chemical Engineering and Biotechnology and Dr. Catherine Lindon at the department of Genetics at the University of Cambridge. We have conducted extensive feasibility studies to assess the feasibility of the proposed tools and methods and the current grant would permit us to: 1) set up a fully functioning microscopy unit which permits, at user's choice and essentially 'at the flick of a switch', either mh FLIM or phi2FLIM to be performed, 2) Validate and calibrate the developed techniques, 3) demonstrate the use of the instrumentation to generate insights into the ubiquitination of substrates in living cells. If successful, this will generate preliminary results to pump prime a larger scale research programme for which separate funding will be sought.

This research will develop a capability to perform frequency domain fluorescence lifetime microscopy 1) at rapid speed without the problem of frequency aliasing and 2) at high accuracy, with the capability to resolve multiple lifetime components from a sample. Who will benefit from this research (see also academic beneficiaries section above)? The tools to be developed will help the UK maintain its leading role in applied photonics research. There is great commercialisation potential (see attached letter of support from Fianium). European manufacturers of diagnostic equipment are likely to incorporate the proposed technology into future platforms for example for drug screening. This idea is currently being explored with Astra Zeneca and the technology transfer office in Cambridge to develop a screening platform for anti aggregation drugs against neurodegenerative disease. The general potential to generate impact for the economic sector is maximised through the PI's role as director of CamBridgeSens (http://sensors.cam.ac.uk) which facilitates liaison with, and technology diffusion to, leading manufacturers of biomedical equipment. The technology proposed is enabled through the use of supercontinuum radiation generated in Photonic Crystal Fibres, an invention that originates from the UK. The leading manufacturer of supercontinuum products (Fianium Ltd, southampton) is also situated in the UK and collaborates with us, ensuring a rapid translation of concepts to products which are of general value to the community. The research will enable the study of dynamic molecular interactions in living systems and initially be used for fundamental research related to cancer and protein aggregation disease. Outcomes from such research will increase fundamental knowledge that in the longer term may lead to novel treatments of disease.