lifa specifications

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Why Fluorescence
Lifetime Imaging Microscopy?
Upon excitation, fluorescent molecules emit light that decays
exponentially with time according to the decay rates of their excited
state. This fluorescence lifetime is a telltale signature of the molecules
and their immediate environment. FLIM - Fluorescence Lifetime
Imaging Microscopy - is the technique to map – next to the regular
fluorescence intensity – the spatial distribution of lifetimes in living
cells and in inorganic material, such as natural rock samples or semiconductor materials. A key advantage of the fluorescence lifetime
above the intensity is that fluorescence lifetime is independent of
concentration, bleaching and intensity variations.
In cell-biology and in pharmacy FLIM is a functional imaging technique
used to probe a range of biophysical phenomena. FLIM is applied in
oxygen imaging, ion imaging, and protein-protein interactions through
FRET - Förster Resonance Energy Transfer. FRET is a non-radiative
process of energy transfer between a donor fluorescent molecule and
an acceptor molecule that is used to quantify molecular interactions
and molecular conformation changes at the nanoscale. FRET causes
the lifetime of the donor molecule to decrease and it is this change
that is quantitatively measured with FLIM.
TECHNOLOGY
Frequency Domain Lifetime Imaging
2
3
4
5
Time (ns)
retains the phase shift and the demodulation of the fluorescence emission.
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. By using a stateof-the-art ICCD camera, the fluorescence lifetime is obtained simultaneously
for all pixels in the field-of-view.
Phase Shift
1
5
Demodulation
2
4
Intensity
1
In frequency domain FLIM the fluorescence lifetime of your sample can be
acquired very rapidly by controlling the frequency of a pulsed laser-diode or
LED (left picture, blue curve) and the frequency and phase of an ICCD camera
(left picture, green curve). The ICCD camera sensitivity is modulated by operating it as an ultra-fast electro-optical shutter. Due to the fluorescence lifetime,
the fluorescence emission (left picture,
red curve) is phase-shifted and reduced in
amplitude (i.e. de-modulated).
For both the excitation and the detection
the same frequency is used (i.e. homodyne detection), and at different camera
phase settings (1-5 in the figures), a series of images of the fluorescence emission is taken. 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 fluorescence emission.
This results in a cross-correlation function
(right picture, red curve) for each of the
0
20
40
pixels in the image. This function exactly
3
3
Phase
The LIFA:
Frequency Domain FLIM
The LIFA is a camera-based FLIM system for fast fluorescence and/
or phosphorescence lifetime imaging in the frequency domain, and is
compatible with Leica®, Nikon®, Olympus® and Zeiss® fluorescence
microscopes. Its well-established homodyne detection technology together
with the parallelism offered by the FLIM camera allows near instantaneous
acquisition of full field lifetime images at high accuracy.
The LIFA is especially well-suited for live cell imaging. Using a Laser Box
it can be combined with Total Internal Reflection Fluorescence (TIRF) for
TIRF-FLIM, and with multi-beam confocal spinning disk, for confocal FLIM.
The standard, widefield system includes a Multi-LED modulated light source
with high-power LEDs that can be modulated across a broad frequency
range for good lifetime sensitivity. The other key components of the LIFA
are the LI²CAM modulated ICCD camera, a control unit and the LI-FLIM
software package. The LI²CAM can be equipped with a range of state-ofthe-art Gen II or Gen III image intensifiers to match your specific application.
The Lambert Instruments LIFA is easy to install - within the hour - and very
easy to operate. Its open architecture is particularly well suited for highcontent screening applications. The LIFA system has been judged “easy
and highly quantitative” for a.o. FLIM-FRET studies.
KEY FEATURES
Frequency domain FLIM – used on wide field fluorescence microscopes.
Fast acquisition – down to several lifetime images per
second: convenient for moving objects in living cell samples.
Low excitation intensity – reduces photo toxicity.
Broad lifetime range – up to phosphorescence.
Near real-time lifetime image acquisition –
dedicated system that allows generation and acquisition of
lifetime images in a matter of seconds.
Higly cusomizable – compatible with TIRF and confocal spinning disk.
Polar plot – for distinguishing lifetime components and
retrieving FRET efficiencies
Advanced scientific software package LI-FLIM –
includes: single frequency, multi-frequency, time lapses, polar plot, multi-lifetime-component-fit, and more...
Applications
Analysis
For further analysis frequency-domain lifetime
data can be decomposed classically into exponential components. A popular alternative is to
plot the measured phase shift and de-modulation 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-linear fitting procedures of the
fluorescence decay using exponentials. The
polar plot analysis is instantaneous and makes
FLIM accessible to the non-expert in spectroscopy and data analysis.
Molecular Interactions
Protein Conformation
Biosensors
Oxygen Imaging
Cells showing localized expression of CFP1 and CFP4, and
the corresponding polar plot. Courtesy of Prof. T.W.J. Gadella
(VU Amsterdam, The Netherlands).
NADH / FAD Fluorescence
Dynamics
Viscosity Imaging
Membrane Dynamics
Membrane Trafficking
LED Inspection
Crude Oil Characterization
COMPONENTS
LI²CAM modulated ICCD camera
Multi-LED light source (optionally: laser diode)
FLIM control unit (signal generator / high voltage power supply)
USB 2.0 interface
LI-FLIM software package
Personal computer and LCD screen
Installation, training and support
LIFA SPECIFICATIONS*
Lifetime range
0-300 ns (LIFA) / 0-1 ms (LIFA-X)
Lifetime resolution
80 ps single pixel
Lifetime drift
less than 30ps
Modulation frequency
1-120 MHz
Speed
Two lifetime images per second
Spatial resolution
21 lp / mm or 450 nm FWHM (100x objective)
Sensitivity
single photon
Detection range GenII, S20
200 - 600 nm
Detection range GenII, Super S25
450 - 850 nm
Detection range GenIII, GaAs
450 - 900 nm
Intrascence dynamic range
12 bit
Camera field of view
14.4 x 10.8 mm
Camera CCD pixel lay-out (H x W)
1392 x 1040 pixels
Effective pixel size
10.3 micron square
Camera mount
C-mount 1x
Wavelength availability Multi-LED
350-800 nm, except for 540-580 nm
Wavelength availability Laser Box
375-515 nm, 635-785 nm
Dimensions FLIM Camera (L x W x H)
133 x 116 x 80 mm
Dimensions Control Unit (L x W x H)
470 x 420 x 200 mm
Dimensions Multi-LED (L x W x H)
346 x 109 x 183 mm
*LIFA Multi-LED for widefield FLIM. Please contact our Sales & Support team for detailed specifications of the LIFA confocal,
LIFA TIRF and LIFA-X products.
Oosteinde 16, 9301 ZP Roden
The Netherlands
Tel. +31 (0)50 501 84 61
Fax +31 (0)50 501 00 34
Email [email protected]
Website www.lambertinstruments.com