Book of abstract 2008

Towards high throughput, high content, fluorescence lifetime imaging
B. Vojnović1, P. R. Barber1, L. Huang1, G. Pierce1, R. G. Newman1, T. Ng2, S. Ameer Beg2
1Gray Cancer Institute, University of Oxford, Radiation Oncology and Biology Unit, Mount Vernon
Hospital, Northwood, Middlesex, HA6 2JR, United Kingdom; 2Randall Division of Cell & Molecular
Biophysics and Division of Cancer Studies, The Richard Dimbleby Dept. of Cancer Research, King’s
College London SE1 1UL, United Kingdom
Instrumentation to perform Fluorescence Lifetime Imaging (FLIM) at the single cell and
tissue levels is now well developed and the technique is finding ever-increasing applications
in the biomedical sciences. Nevertheless, some difficult issues remain to be solved. These
can be grouped in two broadly overlapping ‘problem’ areas: how to perform signal
acquisitions as fast as possible with minimal insult to the specimen and, having acquired
the information, what confidence can be placed on the result. This in turn suggests
observation of large fields (e.g. multi-well plates) or large numbers of tissue samples (e.g.
tissue micro-arrays).
Although there are numerous applications of FLIM, its ability to quantify protein
interactions, through the application of Főrster Resonance Energy Transfer (FRET)
is of particular interest. Robust analysis methods to determine fluorescence lifetime
components have been developed and their limitations when very low photon counts are
available will be presented. Global analysis methods are particularly advantageous in many
cases where the photophysics and kinetic models are well understood.
Recent and current developments in acquiring data from large areas will be discussed
and advantages and limitations of various approaches will be presented. While it is
always desirable to minimise instrumental limitations, biological variability very often
limits the eventual ‘noise’. This in turn dictates the imaging of large numbers of samples,
which demands a high degree of automation of the acquisition and analysis phases of the
experiment. Conventional ‘microscope’ platforms are not always optimal for such work
and systems to overcome limitations will be outlined. Technological solutions to provide
optimised imaging, and emerging parallel detection systems, specifically to overcome
the challenges
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imposed by optical proteomics will be discussed. Potential pitfalls in the
automated analyses of data acquired with automated platforms are numerous and must be
carefully avoided, partially by sound biological experiment design and partially by tight
quality control. The reward is worthwhile, since the reporting of fluorescence lifetime,
spectral content and other photophysical parameters allows us to explore the environment
of a molecule, and in the case of FRET, with a tool that provides near-field information with
a far-field technique. Various bottlenecks in data flow are, in general, inevitable and means
to minimise these will be explored. Directions of current and future work, specifications,
requirements and possibilities afforded by novel approaches will be discussed, with the
aim of setting the scene to apply FLIM in much wider areas of biological research.
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