Förster (Fluorescence) Resonance Energy Transfer (FRET) has become a powerful tool to study protein-protein interactions and signal transduction in living cells. FRET is commonly read out either by detecting the ratio of the donor and acceptor intensities (sensitized emission) or by detecting the excited state lifetime of the donor, which decreases with increasing FRET (Fluorescence Lifetime IMaging or FLIM). FLIM is robust, immune to bleaching and inherently quantitative. On confocal microscopes, FLIM is typically read out by Time-Correlated Single Photon Counting (TCSPC). This requires expensive add-on hardware and is inherently very slow, necessitating accumulation of many consecutive scans to arrive at low-noise lifetime images. For this reason fast lifetime changes, such as those encountered when reading out live-cell signaling events with FRET sensors, cannot be detected. Accumulation of images and/or very slow scanning can also cause morphological artifacts in the acquired images.
We present the Leica SP8 FALCON, a new high-end confocal instrument with built-in very fast FLIM capabilities. The instrument applies a novel technology to records photon arrival times at up to 80 MHz (160 MHz if two detectors are used) using the built-in spectral HyD detectors. To arrive at such high counts rates, the instrument uses real-time 10 GHz sampling and analysis algorithms that render it largely immune to pulse pile-up. Falcon FLIM has been fully integrated in the LAS-X software, allowing convenient acquisition of FLIM time-lapses, stitched FLIM overview images, FLIM XYZ-stacks and spectral FLIM stacks, among others, as well as combinations thereof.
Kees JalinkProfessor, The Netherlands Cancer Institute
Kees Jalink studied biology, biophysics and archeology at Leiden University. He did his PhD at the NKI where he discovered that the lipid metabolite lysophosphatidate doubles as a GPCR agonist, opening up a new field. He went on to do a postdoc in the labs of Drs Roger Tsien and Charles Zucker, both at San Diego, CA, working on phototransduction and participating in the FRET assays that were pioneered at that time. In 1998 he took a position as group leader at the NKI where he has focused on all aspects of FRET ever since. The lab is most famous for contributing various FRET sensors, for developing FRET and FLIM instrumentation and paradigms, and for applying that technology to learn about cells. Kees has a long history of collaborating and advising industry, in particular in microscopy and pharmacological screening. Kees loves new technology, inside and outside the lab, and spends every free hour building new setups and tinkering with electronics.