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FLIM-Fluorescence Lifetime Imaging Microscopy

Lambert Instruments

FLIM

Fluorescence Lifetime Imaging -FLIM


For doing fluorescence lifetime imaging microscopy -FLIM we offer the complete system LIFA -Lambert Instruments Fluorescence lifetime imaging Attachment allowing fluorescence image acquisition and the generation of lifetime images. Additionally the key components of this system, the modulated image intensifier II18MD and the modulated LED light source, are offered as separate products for users who decide to build their own FLIM -fluorescence lifetime imaging microscopy system including their own software.

The picture shows 4 HeLa cells with FRET (Alexa488 and Alexa568) only at the centrosomes (the microtubule organising centres of the cell): the lifetime of Alexa488 at the centrosomes has decreased to 2,3ns, and the lifetime of Alexa488 outside the centrosomes is 2,7ns.

Both the lifetime and the intensity image is shown, with the merge at the bottom. Courtesy of Prof. Diaspro, Genua, Italy.


The LIFA -Lambert Instruments Fluorescence Attachment for lifetime imaging microscopy can be attached to any widefield fluorescence microscope and works in the frequency domain, which means that it uses a modulated light source and a modulated image intensifier as detector. The system is easy to install (within 1 hour) and to operate and does not require an optical bench with laser. The LIFA -Lambert Instruments Fluorescence Attachment system has been judged "easy and highly quantitative". And last but not least, it's reasonable priced.


Components
Light source
Typically, the LIFA -Lambert Instruments Fluorescence Attachment uses high brightness LED light sources of different wavelengths. The use of an LED has several advantages over a laser as light source: LEDs are inexpensive, can be modulated over a broad frequency range (10 - 100 MHz), have no interference effects or speckles, and are available in many wavelengths in the UV and VIS spectrum.
Additionally, a modulated laser diode or a CW laser modulated by an external modulator (Pockel cell or AOM) can also be used as light source, especially when higher intensities are needed. This is advantageous when combining the LIFA -Lambert Instruments Fluorescence Attachment with a spinning disk to obtain optical sectioning and 3D imaging.

Image intensifier
The high-resolution image intensifier is the key component of the LIFA -Lambert Instruments Fluorescence Attachment . The gain of the image intensifier can be controlled by the voltage across the MCP and is typically in the range of 100 to 10,000 times. Also the voltage between cathode and MCP can be changed in order to control the gain. During modulation, the gain is modulated via the cathode voltage.
Depending on the application, the photocathode has to be chosen that has the appropriate sensitivity per emission wavelength. For GenII we normally offer either the S20 or the SuperS25 photocathode. For higher quantum efficiency (25% instead of 10%) the GenIII intensifier is offered, with the GaAs photocathode. See here for the photocathode characteristics.

Digital CCD camera
The image intensifier is either lens-coupled or fiber-coupled to a intensified CCD camera. The fiber-optic coupled image intensifier and the CCD unit is integrated in the LI2CAM MD camera that is made by Lambert Instruments. This intensified CCD camera is very compact and has a higher gain than the lens-coupled combination as a result of the more efficient and compact fiber coupling. The lens-coupled combination allows easy detachment and exchange of camera from image intensifier by the user.

Integrated Control unit / Signal generator / Power supply
This unit supplies the RF modulation signals at controlled frequency, amplitude and phase difference to the LED light source and to the image intensifier. The unit also supplies the DC power and high voltage to LED and image intensifier. An extra output to drive an external modulator is available as well as monitor outputs to check the modulation signals.

LI FLIM software
The user-friendly software is developed by Lambert Instruments. With this package you can acquire lifetime images, process them and do statistic analyses. Users that have the password for the new release, can download the software from the LI FLIM software page directly.

LIFA set-up

LIFA - FLIM



FLIM

Image intensifier

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)
>Disadvantages:
- 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
>Disadvantages:
- 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.

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