announced that its 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 addition of FLIM and FCS capabilities further increases the versatility
of the Olympus FluoView FV1000 cLSM and ensures that users can bring a new dimension
to their research.
The new PicoQuant module available from Olympus enables advanced FLIM and FCS,
and 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 fibre 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 maximise 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.