The LIFA is a camera-based fast fluorescence lifetime imaging microscopy system that operates in the frequency domain and is compatible with Total Internal Reflection Fluorescence (TIRF). TIRF-FLIM enables selective lifetime imaging down to 100nm from the coverslip, allowing exciting applications including quantitative membrane trafficking studies using FLIM-FRET and Multi-Color fluorescence lifetime imaging microscopy.
The LIFA is easy to install - within the hour - and very easy to operate. The attachment is compatible with Leica, Nikon, Olympus, and Zeiss fluorescence microscopes. The LIFA is in addition well suited for high-content screening applications. The LIFA system has been judged "easy and highly quantitative" for a.o. FLIM-FRET studies.
Reference: In the paper titled Neuropilin-1/GIPC1 Signaling Regulates a5B1 Integrin Traffic and Function in Endothelial Cells by the Beatson Institute for Cancer Research (Glasgow, UK) the LIFA has been applied in TIRF mode.
Supported TIRF modules
Different TIRF modules have been succesfully evaluated in combination to the LIFA system including Leica, Nikon, and Olympus. The laser-TIRF unit is mounted to the epi-attachment of the microscope. The system's directly modulated laser diodes are connected to the laser-TIRF unit using fiber optics. The TRiCAM modulated intensified CCD camera is mounted to the camera port of the wide field fluorescence microscope.
TIRF users can upgrade their existing laser-TIRF setup with a LIFA system. For excitation a modulated laser source is required. Lambert Instruments offers the Multi-LASER, which is available with a range of excitation wavelengths among which 405 nm, 445 nm, 488 nm, 515 nm and 639 nm.
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Fast TIRF-FLIM, up to two lifetime images per second
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Lifetime sensitivity 0-300 ns
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Lifetime accuracy 50 ps r.m.s.
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High quantum efficiency image intensifier
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Easy integration into specialized image analysis pipelines
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Time-lapses
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FRET efficiency mapping
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Multi-frequency acquisition and analysis for separation of multiple lifetimes
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Polar (Phasor) plot inspection and separation of multiple lifetimes
Lambert Instruments provides the following LIFA models, which cover a wide range in spectral sensitivity and in lifetime sensitivity. Each model is based on a specific high-resolution Gen II or Gen III image intensifier, see also our fluorescence lifetime imaging microscopy properties of modulated intensifiers document.
Model
|
Photocathode
|
Modulation frequency
|
Lifetime sensitivity
|
|
LIFA Gen II S20 |
S20 |
1-120 MHz |
0-300 ns |
|
LIFA Gen II S25 |
S25 |
1-120 MHz |
0-300 ns |
|
LIFA Gen III GaAs |
GaAs |
1-120 MHz |
0-300 ns |
|
LIFA X Gen II S20 |
S20 |
0-100 kHz, 1-120 MHz |
0 ns - 1 ms |
|
LIFA X Gen II S25 |
S25 |
0-100 kHz, 1-120 MHz |
0 ns - 1 ms |
|
LIFA X Gen III GaAs |
GaAs |
0-100 kHz, 1-120 MHz |
0 ns - 1 ms |
|
LIFA P Gen II S20 |
S20 |
0-100 kHz |
100 ns - 1 ms |
|
LIFA P Gen II S25 |
S25 |
0-100 kHz |
100 ns - 1 ms |
|
LIFA P Gen III GaAs |
GaAs |
0-100 kHz |
100 ns - 1 ms |
|
LIFA P Gen III GaAsP |
GaAsP |
0-100 kHz |
100 ns - 1 ms |
Photocathode spectral sensitivity
Spectral sensivity as a function of wavelength for each of the photocathodes is shown below.
For more information, please refer to our technologies page on the operating principle of image intensifiers for lifetime imaging.
The LIFA - TIRF combination has the following applications:
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Molecular interactions
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Protein conformation
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Biosensors
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Oxygen concentration imaging in cells and tissue
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NADH / FAD fluorescence dynamics
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Viscosity imaging
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Membrane dynamics
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Membrane trafficking
