ŷü  
   
 
 

 

LIFA, Lambert Instruments Fluorescence Attachment

 
 

The LIFA (Lambert Instruments Fluorescence Attachment) for lifetime imaging microscopy (FLIM) can be attached to any widefield fluorescence microscope and works in the homodyne frequency domain. This means that it consists of a modulated light source and a modulated image intensifier as detector (i.e. ICCD camera). The system is easy to install (within 1 hour) and to operate and does not require an optical bench with laser. The LIFA system has been judged "easy and highly quantitative" for a.o. FLIM FRET studies.

 
LIFA: Lambert Instruments
Fluorescence lifetime imaging
Attachment
 


Components

Light source Multi-LED
A multiple LED system specifically designed for use in Fluorescence Lifetime Imaging Microscopy (FLIM). With up to four high-power LEDs for modulated fluorescence excitation, the Multi-LED can be connected to the epi-fluorescence port of the wide field fluorescence microscope. The use of a LED has several advantages over a laser as light source: LEDs are inexpensive, robust, can be modulated over a broad frequency range (1 - 80 MHz), have no interference effects or speckles, and are available in many wavelengths in the UV and VIS spectrum. Existing LIFA systems can be upgraded with this excitation source.

Light source laser diode
Additionally, a directly modulated laser diode can be used as light source, especially when higher intensities are required or high modulation frequencies (up to 120MHz). The laser diode is directed by a similar control unit and same software package as the standard LIFA system with LED. The laser diode can be offered as single-wavelength, as well as multi-wavelength and several lines are available like 405nm, 445nm, 473nm, 488nm, 639nm.
The laser light can be used as widefield light source by attaching the fiber directly to a so-called scrambler at the epi-attachment of the wide field fluorescence microscope. Eventually a CW laser modulated by an external modulator (Pockel cell or AOM) can be used. The use of a modulated laser diode is essential when combining the LIFA to a spinning disk to obtain optical sectioning and 3D imaging or to laser-TIRF for e.g. membrane studies.

Image Intensifier
The high-resolution modulated image intensifier is the key component of the LIFA. 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 (Gen 2) we normally offer either the S20 or the SuperS25 photocathode. For higher quantum efficiency (25% instead of 10%) the GenIII (Gen 3) intensifier is offered, with the GaAs photocathode. See here for the photocathode characteristics:

 
       
 


Digital CCD camera

The image intensifier is fiber-optic coupled to a CCD camera and integrated in the LI2CAM MD camera that is made by Lambert Instruments. Optionally we offer a lens-coupled intensified CCD camera. The intensified CCD camera LI2CAM MD 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 however 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 (up to 120MHz), amplitude and phase difference to the LED or laser 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 single- and multifrequency lifetime images, process them and do analyses by statistics or polar plot. Time series can be done as well, for which the frequency domain FLIM is highly appropriate because of its fast acquisition possibilities.
It automatically recognizes connected devices. Among others different light sources are integrated in LI FLIM like the LEDs, the directly modulated laser diodes, and the Cobolt DPSS laser (not modulated). Additionally various detectors are integrated like the standard LI2CAM MD, but also the Andor Ixon EMCCD and a Roper Scientific / Photometrics camera for the lens-coupled option of the LIFA.

Basic support for

Customers receive the password for the newest software release which can be downloaded from the LI FLIM software page directly.

LIFA set-up

 
       
 
Patents
Patents owned by Photonic Research Systems Ltd.