Olympus Adds Further Advanced Capabilities to the FluoView FV1000 Confocal Laser Scanning Microscope
04 Mar 2009

Olympus has 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 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.

In fluorescence microscopy, fluorescence intensity is displayed using a false colour scale, whereas for FLIM the fluorescence lifetime is used. It can be performed with just one detector and is not affected by fluctuations in the fluorescence intensity. There is also no influence from different fluorophore concentrations or sample thicknesses and therefore further structural insight can often be gained. FLIM also enables discrimination between fluorophores with similar emission spectra (e.g. GFP and YFP) and from autofluorescence and can be combined very successfully with fluorescence resonance energy transfer (FRET). FLIM can therefore be used to determine: Oxygen, water or Ca2+ concentration, pH, distances in the ‘nm’ range, intracellular signal transduction, as well as molecular structure and dynamics and other factors.

FCS can be used to measure molecular properties, diffusion and concentrations in solution at the single molecule level. It is a highly precise and versatile method which has demonstrated its great potential for many different applications and can also be used for luminescent signals. The method records the temporal changes in the fluorescence emission intensity caused by single fluorophores passing through the excitation volume. These intensity changes can be quantified in their strength and duration by the temporal autocorrelation of the recorded intensity signals. Typical FCS applications include: molecular association and dissociation, sample concentration in the ‘fL’ range, investigation of complex fluorophore diffusion, conformational dynamics, kinetic rate constants, enzyme dynamics and intramolecular dynamics in vitro and in vivo.

Image Legend:
Fluorescence intensity (left) and fluorescence lifetime image (right) of the autofluorescence of a pollen grain (from a daisy). Regions that look very uniform in the intensity image clearly show pronounced differences in the FLIM image.

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