The combination of the LIFA (Lambert Instruments Lifetime Attachment) with laser-TIRF enables lifetime imaging microscopy (FLIM) while detecting just the first 100nm of the sample. TIRF FLIM images provide better resolution and are more specific regarding lifetime values per region of interest which is important e.g. in case of FLIM FRET studies of the cellular plasma membrane.
Reference: Paper describing the LIFA in TIRF mode; Beatson Institute for Cancer Research, Glasgow, UK.
Components LIFA combined to laser-TIRF
A directly modulated laser diode is directed by a similar control unit and same software package as the standard LIFA system. 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 is connected to the laser-TIRF attachment directly.
Different models have been evaluated in combination to the LIFA system: Leica, Nikon, and Olympus. The laser-TIRF is mounted to the epi-attachment of the microscope. The modulated ICCD camera (LI2CAM MD) is mounted to the camera port of the wide field fluorescence microscope.
The system is offered as one package with the LIFA system; however, users can also use their own laser-TIRF set-up and wide field fluorescence microscope to which the LIFA system will be attached.
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 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: