We present the SP8 FALCON from Leica Microsystems, 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
Precise and lightning-fast spectral Fluorescence Lifetime Imaging at video rate integrated in a high-end confocal microscope
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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.