FLIM-Fluorescence Lifetime Imaging Microscopy
Fluorescence-lifetime imaging microscopy or FLIM is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography.
The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample.
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Fluorescence Lifetime Imaging -FLIM
With the Lambert Instruments FLIM Attachment (LIFA), you record quantitative lifetime data in a matter of seconds. It’s easy to connect our FLIM camera and light source to your microscope. And our specialized software records the images, analyzes the data instantly and presents the results visually for easy interpretation.
Record fluorescence lifetime imaging microscopy (FLIM) data in a matter of seconds. Compared to alternative methods like time-correlated single photon counting (TCSPC), the LIFA is over 100 times faster.
Their advanced software instantly analyzes your data and presents the calculated fluorescence lifetimes visually. Recorded images are compatible with ImageJ, FIJI, Matlab and MetaMorph. Detailed statistical data can be exported to Excel worksheets.
The LIFA is compatible with every fluorescence microscope with a camera output. This includes fluorescence microscopes by Leica, Nikon, Olympus, TILL and Zeiss, as well as confocal microscopes and TIRF microscopes.
- Non-phototoxic illumination
- High quantum efficiency with the optional Gen III GaAs intensifier
- Time-lapse recording mode
- Forster Resonance Energy Transfer (FRET) efficiency mapping
- Multi-frequency acquisition for separation of multiple lifetimes
- Polar (Phasor) plot inspection and separation of multiple lifetimes
- Easy integration into specialized image analysis pipelines
The LIFA is compatible with several types of microscopes:
- Widefield fluorescence microscopes
- Confocal spinning disk fluorescence microscopes
- Total Internal Reflection Fluorescence (TIRF) microscopes
- Hyperspectral imaging system by Gooch & Housego
- Prior filter wheels and XY stage for multi-channel acquisition
All LIFA components and the microscope hardware are shown in the illustration above.
An image intensifier is a device that intensifies low light-level images to light levels that can be seen with the human eye or can be detected by a video camera. An image intensifier is a vacuum tube, having an input window on which inside surface a light sensitive layer called the photocathode has been deposited. Photons are absorbed in the photocathode and give rise to emission of electrons into the vacuum. These electrons are accelerated by an electric field to increase their energy and focus them. After multiplication by an MCP (multi channel plate) these electrons will finally be accelerated towards the anode screen. The anode screen contains a layer of phosphorescent material that is covered by a thin aluminium film. When striking the anode the energy of the electrons is converted into photons again. Because of the multiplication and increased energy of the electrons the output brightness is higher as compared to the original input light intensity.
The different “generations” of image intensifiers are described here:
First Generation Image Intensifier
A first generation works in principle as described above but does not use an MCP. The electrons however are “guided” from the
input to the output by means of electrostatic focussing. Two types can be distinguished: proximity focussed diodes and
electrostatic inverters. In the latter a structure of electrodes form an electrostatic lens that focus the electrons coming from
cathode onto the anode. The advantage of electrostatic focussing is that it allows de-magnification of the image. This is
especially interesting when these devices are coupled to small CCDs.
The advantages of first generation tubes in cameras are:
- available in de-magnifying formats
- therefore no fiber optic taper required
- no MCP noise
- high intra-scene dynamic range
- low cost (standard models)
- electrostatic inverters show a few percent of image distortion
- relatively low gain
- gating not possible
- no UV sensitivity
- limited external gain control
- poor over-illumination protection
Second Generation Image Intensifier
Because first generation image intensifiers have a relatively low gain something had to be done to improve this. In a second-generation image intensifier a so-called “Micro Channel Plate” or MCP is added. This MCP is placed between the cathode and the anode and acts as an electron multiplier. It is a 0,5mm thick plate with a few million 6 micron wide holes. When an electron is leaving the photocathode it will be accelerated towards the MCP. When the electron hits the wall of one of the MCP channels it will generate a few secondary electrons. Due to the voltage over the MCP these electrons will also be accelerated and hit the surface deeper in the channel and again create secondary electrons. This process is repeated many times and results in an electron gain of several thousands. When leaving the MCP the electrons are accelerated to the phosphor screen where they will generate multiple photons. The overall gain will be a few thousand times. With two or three MCPs amplifications up to 10 million times is possible. The gain of the image intensifier can be controlled over a wide range by changing the voltage across the MCP.
Another important feature of the second-generation image intensifier is gating.
Gating offers the possibility to use the image intensifier as an ultra fast electro-optical shutter with minimum effective exposure times down to a few nanoseconds. Gating is done by controlling the photocathode voltage of the image intensifier. By applying a negative voltage to the photocathode, typically -200V, referenced to the MCP input, photoelectrons generated in the photocathode are emitted by the photocathode and accelerated to the MCP for multiplication. The intensifier is therefore “”gated on””. By applying a small positive voltage to the photocathode, typically 50V, the photoelectrons can not be emitted and the intensifier is therefore “”gated off””. With this gating option the input light range is extended significantly and it offers unique options for time resolved experiments. Furthermore gating can be used to reduce or prevent the effect of motion blur when capturing fast moving objects. In our Intensified cameras gating is standard synchronised with the exposure period of the CCD or CMOS sensor.
The advantages of a second generation image intensifiers are:
- fibre-optic/glass/quartz/MgF2 input windows
- many photocathode types from UV to NIR
- high gain
- fast shuttering is possible (gating)
- good over-illumination protection
- maximum output brightness control
- wide gain control range
- many types of output phosphors
- distortion free
- limited intra-scene dynamic ranges
- low maximum output brightness for fast phosphors
- no de-magnifying models
- MCP introduces extra noise
For FLIM-Fluorescence Lifetime Imaging Microscopy, a second-generation image intensifier is used as the detector of the system for lifetime imaging. The image intensifier, combined with a CCD camera, is attached to the widefield fluorescence microscope. The photocathode of the intensifier is located in the image (focal) plane of the microscope. In the frequency domain FLIM system the photocathode is switched from positive to negative at the same frequency as the light source is modulated.
Third Generation Image Intensifier
The next step in technology is the third generation (GenIII) image intensifier in which the multi-alkali photocathode is replaced by a Gallium Arsenide (GaAs) or GaAsP photocathode. The quantum efficiency of this type photocathode is much higher as compared to the multialkali photocathode of second-generation image intensifiers. Recently new filmless GenIII intensifiers have been developed that are using this high q.e. to its full extend. The higher quantum efficiency results in a better signal to noise ratio (S/N) or in a shorter exposure time at the same S/N. In the graph below spectral sensitivity curves of multialkali photocathodes, such as S25, S20 and broadband, are shown in comparison with GaAs and GaAsP photocathodes.
pco.flim – Luminescence lifetime imaging camera
The pco.flim camera system is the first luminescence lifetime imaging camera using a two tap CMOS image sensor.
The camera can internally generate a wide range of frequencies and will also accept external modulation signals within a limited
range of frequencies.
It features a variety of trigger input and output signals for easy system integration. Special software provides direct measurements and lifetime analysis.
Detailed information about fluorescence lifetime imaging using the pco.flim camera can be found in our new white paper explanation.
- 100ps – 100µs lifetimes measurable
- 5kHz – 40MHz modulation frequencies
- 500kHz – 40MHz external modulation signals
- modulation signal shape sinusoidal / rectangular
- 1008 x 1008 pixel resolution
- frequency domain FLIM
- USB 3.0 interface
- 39% quantum efficiency
- 1000:1 dynamic range
- 45 e- rms readout noise
- 90 fps frame rate (2 tap readout)
- 10 ns to 10 s selectable exposure times
- vibration-free water cooling
- special software for measurement and analysis