Frequency Domain Wide-field-FLIM

Leutingewolde, The Netherlands - Sept. 12, 2005…Lambert Instruments has developed a dedicated system that allows image acquisition and generation of lifetime images in a matter of seconds - the Lambert Instruments Fluorescence Lifetime Imaging Attachment (LIFA). The LIFA can be attached to any fluorescence wide-field microscope and the system is fast and highly efficient because excitation and detection are running simultaneously. The system is easy to install and to operate and does not require an optical bench with a laser.

The system works in the frequency domain, which means that the intensity of the excitation light is modulated at a certain frequency. Therefore, the induced fluorescence emission will be intensity-modulated as well, but due to the decay of the emission, the emitted light will show a phase-shift and a decrease in modulation-depth with respect to the excitation light. This phase-shift and decrease in modulation-depth depend on the decay constants of the fluorescent material and the modulation frequency. The amplification (or gain) of the image intensifier is modulated at the same frequency as the excitation light and the intensified (emission) light can be monitored continuously by the CCD camera. By taking images at different phase settings of the intensifier gain modulation relative to the excitation light modulation, phase images are acquired from which the nanosecond lifetime information can be extracted by a pixel-by-pixel image analysis procedure.

The fluorescence lifetime is the signature of a fluorescent material; it is the exponential decay in emission after the excitation of a fluorescent material has been stopped. FLIM (Fluorescence Lifetime Imaging Microscopy) is a technique to map the spatial distribution of lifetimes within microscopic images and it allows measurements in living cells as well as in fixed materials.

Fluorescence lifetime is independent of bleaching and intensity variations in a sample. Because of the fact that some phenomena do affect fluorescence lifetimes, the applications of FLIM are various: ion imaging, oxygen imaging, probing microenvironment, and medical diagnosis. Moreover, the most powerful FLIM-application in biology is fluorescence resonance energy transfer (FRET)-microscopy. When two fluorescent molecules (or e.g. two fluorescent labelled epitopes within a protein) are in very close proximity, i.e. less than 10 nm, the energy of the one fluorescent (donor) molecule is transferred in a nonradiative process to the other fluorescent (acceptor) molecule. In this way, the lifetime of the donor molecule decreases and this change can be measured by FLIM.

For more information, please contact Advanced Labs.

Tags: fluorescence, FLIM, FRET, microscopy