We use advanced microscopy and spectroscopy techniques to study cell signaling events and
cytoskeletal dynamics with high spatial and temporal resolution. Our expertise is predominantly in
advanced functional imaging and Super Resolution microscopy. Functional imaging techniques like
FRET, FLIM and FCCS aim to provide information about the function of molecules, rather than just static images of their position within the cell. We also develop methods, hard- and software for
various advanced microscopy applications. These techniques are used in research projects in our
group as well as in collaborations within and outside our institute.
FLIM imaging on wide-field and confocal microscopes
Having completed construction and testing of instruments for ultrafast Fluorescence Lifetime Imaging (FLIM) in our lab on both confocal and widefield microscopes, we have now moved forward to apply these developments in automated FLIM screens. FLIM records the fluorescence lifetime of a fluorophore, i.e. the average time that a fluorophore remains in the excited state following excitation
and is an intrinsically quantitative method to detect molecular interactions in living cells. We use
FLIM, in conjunction with a variety of Fluorescence Resonance Energy Transfer (FRET) sensors to dynamically read out various signal transduction pathways in individual single cells. In single cells we have characterized the sensitivity, response kinetics and cell-to-cell variability in well-known signaling pathways, including the cAMP-PKA axis, ERK signaling, PLC/Ca2+ signals, receptor tyrosine kinase activity and caspase signaling. Of particular interest is that with the current palette of FRET sensors, we can now follow activity of different nodes within the same signaling cascade, for example following GPCR activation, activation of the G-protein Gq, PIP2 hydrolysis, IP3 formation and Ca2+ release with individual FRET sensors. Being able to study these with sub-second temporal and micro-meter spatial resolutions will allow us to uncover cross-talk between signaling cascades, as well as feedback- and feed-forward mechanism that affect intensity and longevity of these signals. We also study the distribution of signaling activity in multi-cell preparations such as spheroids and
organoids.
How does hypoxia affect activity of signal transduction cascades?
Until now, microscopy studies on living cells have been conducted exclusively at atmospheric oxygen levels (i.e., 20% of oxygen). This is remarkable, because the cells in our body are never exposed to 20% O2; rather, they experience between 2% and 7 % of O2 (normoxia). Solid tumours in our bodies almost invariably lack well-developed blood vessels, and large parts of the tumor are therefore devoid of O2 (<1 % of O2; hypoxia). Importantly, a large body of literature shows that hypoxic tumors are much less sensitive to clinical therapies, particularly to irradiation and to systemic cytostatic agents. We therefore adapted our screening microscopes to allow imaging at arbitrarily set O2 and CO2 levels, thus mimicking the natural conditions of cells in our body much better. With these adaptations, we are studying cell growth and differentiation, signal transduction and migration in several model systems, partly with collaborators at the NKI-AVL.