Olympus’ FluoView FV1000 confocal laser scanning microscope (cLSM) and FV1000MPE Multiphoton ranges can now be fitted with the PicoQuant module for fluorescence lifetime imaging (FLIM) and fluorescence correlation spectroscopy (FCS). FLIM and FCS are advanced methods for measuring minute changes in fluorescence that occur in cells over very short periods of time. FLIM is often used to accurately determine factors such as: O2, H2O or Ca2+ concentration; intracellular signal transduction; as well as molecular structure and dynamics. FCS applications include: molecular association/dissociation; concentration (fL range); kinetic rate constants; as well as in vitro and in vivo intramolecular dynamics.

The new PicoQuant module is based on time-correlated single photon counting (TCSPC) using time-tagged time-resolved (TTTR) data handling. This is considered to be the most precise technique with the highest temporal resolution. The unique pulsed diode lasers provide picosecond or femtosecond pulses at high repetition rates from 375 nm-900 nm with controllable power and pulse frequency. The multichannel version can also perform pulsed interleaved excitation (PIE), and up to four pulsed laser units can be combined into one optical fiber via a coupling unit, making wavelength changes or simultaneous excitation schemes very easy.

The TCSPC data acquisition unit has a very high temporal resolution of 4 ps. Data acquisition is done in the unique TTTR mode in which the temporal information of each detected photon is preserved, which allows very sophisticated offline analyses. Two different detector technologies are available in either single- or dual-channels, providing greater system flexibility. Photon-multiplier tubes (PMTs) offer excellent properties for FLIM techniques, whereas the higher detection efficiency of single-photon avalanche diodes (SPADs) makes them ideal for FCS. Furthermore, for measurements that require high detection efficiencies, two types of SPADs are available. These detectors ensure the system has the versatility to resolve fluorescence lifetimes well below 100 picoseconds and perform ‘online FLIM’ depending on the setup in use.

All the components of the PicoQuant module are controlled via the SymPho Time software. This easy to use software also provides the powerful data collection, handling and analysis processes required to maximize any FCS or FLIM technique. The software also supports a number other related procedures including fluorescence lifetime correlation spectroscopy (FLCS), fluorescence resonance energy transfer (FRET) and PIE-FRET.

Olympus Life Science Europa GmbH, www.microscopy.olympus.eu