Fluorescence Lifetime Imaging Microscopy (FLIM)
4Pages

Why lifetime imaging? 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. Because of the fact that some phenomena do affect fluorescence lifetimes, the lifetime is used to detect these phenomena leading to various applications such as: ion imaging (pH measurements), oxygen imaging, probing microenvironment, and medical diagnosis. Moreover, the most powerful FLIM-application in biology is Fluorescence Resonance Energy Transfer (FRET). When two fluorescent molecules (or two fluorescent labeled epitopes within a protein) are in very close proximity, i.e. less than 9 nm, the energy of the one fluorescent (donor) molecule (e.g. GFP) is transferred in a nonradiative process to the other fluorescent (acceptor) molecule (e.g. mCherry). In this way, the lifetime of the donor molecule decreases and this change can be measured quantitatively by FLIM. Frequency domain method The homodyne frequency domain FLIM method requires a modulated light source and a modulated detector. In the LIFA system these are the LED and the intensified CCD camera. Both are modulated at exactly the same frequency, but with an adjustable difference in phase. The emission intensity shows a phase-shift (or delay) with respect to the excitation as well as a decrease of modulation-depth. These two parameters depend on the fluorescence lifetime of the sample and the modulation frequency and are measured to calculate the fluorescence lifetime in each pixel of the image. Fluorescence Lifetime Imaging Microscopy (FLIM) Pseudo colored lifetime image of mammalian cells stably expressing GFP in the nuclei. Additionally, two cells are transiently transfected with GFP-RFP at the plasmamembrane. The lifetime of the GFP in the membrane is shorter (blue) compared to the lifetime of GFP in the nuclei (green). This is due to FRET between GFP and RFP. (Courtesy of BCF/ IRI Lille, France)

The Lambert Instruments FLIM Attachment (LIFA)… what is it? The Lambert Instruments Fluorescence lifetime imaging microscopy Attachment (LIFA) is a dedicated system that allows image acquisition and generation of lifetime images in a matter of seconds. The system can be simply attached to any wide field fluorescence microscope and is easy to operate. Standard 3 LED’s are mounted in the Multi-LED lamp house and wavelength selection is done by the LI-FLIM software. The software allows acquisition at single-frequency or multifrequency, time lapses and includes analysis features such as...

Features 1. Frequency domain FLIM, used on wide field fluorescence microscopes. 2. Fast acquisition down to several lifetime images per second: convenient for moving objects in living cell samples. 3. The use of modulated LEDs or diode lasers allows excitation wavelengths from 340nm until 640nm. 4. Moderate excitation intensity reduces the chance to photo toxicity. 5. Multi frequency measurement allows the analysis of multi exponential lifetime components. 6. Lifetime range up to phosphorescence. 7. Combination with other microscopy techniques like multibeam confocal, TIRF, etc. Build your...

Reference sites Europe: Amsterdam, The Netherlands; Heidelberg, Germany; Cambridge, United Kingdom; Paris, France; Albacete, Spain; Goteborg, Sweden Other continents: New York, USA; Toronto, Canada; Melbourne, Australia; Singapore, Singapore; Okinawa, Japan. Full details of test sites (address, system components) are available at our website. Quick reference LIFA system specification Lifetime range : 0-300 ns (LIFA) and 0-1 ms (LIFA-X) (in selectable ranges) Lifetime resolution : 80 ps Modulation frequency : 120 MHz max. Measurement time : 1 second typical Speed : 12 lifetime images/sec...