TECHNOLOGY - PDF document

1 LIFA TECHNOLOGY In frequency domain FLIM
LIFA TECHNOLOGY In frequency domain FLIM the �uorescence lifetime of your sample is acquired very rapidly by using a pulsed light source (left picture, blue curve) and a modulated ICCD camera (left picture, green curve). Due to the �uorescence decay, the �uorescence emission from the sample (left picture, red curve) is For both the excitation and the detection the same frequency is used, and at different camera phase settings (1-5 in the �gures) a series of images of the �uorescence emission is taken. This results in a frequency domain cross- correlation function (right picture, red curve) for each of the pixels in the image. The intensity of the emission image will depend on whether the detector sensitivity is partly (2 and 4 in the example) or fully (1 and 5 in the example) in phase with the �uorescence emission. The key is that this function exactly mirrors the phase shift and the demodulation of the �uorescence emission in the time domain. These two parameters can be translated to a lifetime value per pixel. This frequency- mixing approach is the basis of radio technology and is well known for its convenience, simplicity and strong noise suppression. This �uorescence lifetime is obtained simultaneously for all pixels in the �

2 0660069;eld-of-view by using a Frequenc
0660069;eld-of-view by using a Frequency Domain Lifetime Imaging state. The corresponding �uorescence lifetime is a telltale signature of A key protein interactions through FRET - Förster Resonance Energy Transfer. FRET is a non-radiative process of energy transfer between Why Fluorescence Lifetime Imaging Microscopy? 1 2 3 4 5 me 420n e 1 2 3 4 5 3 TECHNOLOGY Frequency domain FLIM – used on wide �eld �uorescence microscopes, from manual to highly automated. Fast acquisition – down to several lifetime images per Higly customizable – compatible with TIRF, confocal spinning disk, anisotropy, and hyperspectral. Low excitation intensity – reduces photo toxicity. Broad lifetime range – from picoseconds to 1 Polar plot – for distinguishing lifetime components and retrieving FRET ef�ciencies Extensive software package LI-FLIM – includes: single frequency, multi-frequency, time lapses, polar plot, Applications Analysis The LIFA is a camera-based FLIM system for fast �uorescence and/ d is compatible with Leica®, Nikon®, Olympus® and Zeiss® �uorescence together with the parallel detection offered by the TRiCAM modulated camera allows high accuracy. As it is a camera-based system the LIFA is especially well-suited for live cell imaging. The

3 standard, wide�eld system in
standard, wide�eld system includes a Multi-LED modulated a broad frequency range for good lifetime sensitivity. Using a Multi-LASER engine it can be easily combined with Total Internal Re�ection Fluorescence (TIRF) for TIRF-FLIM, and with multi-beam confocal spinning disk, for confocal FLIM. The other key components of the LIFA are the TRiCAM modulated package. The TRiCAM can be equipped with a state-of-the-art Gen II or Gen III image intensi�er to match your application. The Lambert Instruments LIFA is easy to install - within the hour - and very content screening applications. The LIFA system has been judged “easy and highly quantitative” for a.o. FLIM-FRET studies. The LIFA: Frequency Domain FLIM Why Fluorescence Lifetime Imaging Microscopy? KEY FEATURES For further analysis frequency-domain lifetime data can be decomposed classically into expo nential components. A popular alternative is to plot the measured phase shift and de-modula tion in a single diagram. This polar or phasor plot offers a direct, global view of the fluorescence decay at each pixel of an image. In the polar plot the presence of different molecular species or the occurrence of fluorescence resonance energy transfer is naturally recognized as data clustering in specific regions, as compared to performing non-

4 linear fitting procedures of the fluore
linear fitting procedures of the fluorescence decay using exponentials. The polar plot analysis is instantaneous and makes FLIM accessible to the non-expert in spectros Molecular Interactions Protein Conformation Biosensors Oxygen Imaging NADH / FAD Fluorescence Dynamics Viscosity Imaging Membrane Dynamics LED Inspection Crude Oil Characterization Solar Cell MCL Monitoring the corresponding polar plot. Courtesy of Prof. T.W.J. Gadella (VU Amsterdam, The Netherlands). LIFA SPECIFICATIONS 0-300 ns (LIFA) 0-1 ms (LIFA-X) 300 ns - 1ms (LIFA-P) Lifetime drift 1-120 MHz (LIFA) / 0,1 KHz - 120 MHz (LIFA-X) / 0,1 KHz - 100 KHz (LIFA-P) Two full-frame lifetime images per second Effective pixel size Wavelength availability Multi-LED Wavelength availability Multi-LASER Dimensions TRiCAM (L x W x H) 133 x 116 x 80 mm Dimensions Control Unit (L x W x H) Dimensions Multi-LED (L x W x H) Dimensions Multi-LASER (L x W x H) LIFA COMPONENTS Oosteinde 16, 9301 ZP Roden Tel. Fax Email Website www.lambertinstruments.com *LIFA Multi-LED for wide�eld FLIM. Please contact our Sales & Support team for detailed speci�cations of the LIFA confocal, LIFA TIRF and LIFA-X products. For contact information of our local distributors please visit the distributor section of our website. support team or our local distributor