Wide-field time-gated FLIM
We have worked with Kentech Instruments Ltd since 1996 to develop wide-field time-gated imaging technology and this has resulted in what we believe to be a robust platform for FLIM-based studies.
The figure below shows a generic configuration for wide-field time-gated FLIM comprising the core elements : (i) ultrafast excitation source, (ii) GOI and computer-controlled delay generator, (iii) computer for data acquisition and analysis, and (iv) the optical instrument. Here the optical instrument is a wide-field epifluorescence microscope and the excitation radiation is homogenized using a holographic flat-top diffuser that is rotated to time-average speckle.
For the ultrafast laser source, we typically use a fibre-laser pumped supercontunuum source (e.g. Fianium UK Ltd, model SC 400-6) or a femtosecond mode-locked Ti:Sapphire laser (e.g. Spectra-Physics, model Mai-Tai). We have also used gain -switched picosecond diode lasers (e.g. PicoQuant GmbH, model LDHP-C-440M with driver PDL-800-B) or a frequency-tripled Nd:YVO4 laser (Spectra-Physics, model Vanguard 350-HMD355) to provide exciation at 355 nm. The "top-hat" holographic diffuser (e.g. Physical Optics Corp, 60° FWHM) is rotated at 10-50 Hz using a simple d.c. motor and is imaged to the back focal plane of the microscope objective lens.
To realise time-gated detection, we use a GOI (Kentech Instruments Ltd, model HRI) and a computer-controlled delay generator (Kentech Instruments Ltd, agile delay unit). A cooled camera is required to read out the time-gated iamges from the phosphor of the GOI, for which we use a scientific CCD camera (e.g. Teledyne QImaging, model Retiga R1).
For FLIM data acquisition, the computer can be a standard personal computer requiring ~8 GB RAM with USB and serial ports to interface with the equipment components. We have written our FLIM acquisition software in MicroManager. For FLIM data analysis, we use FLIMfit, our open source MATLAB-based software that provides a range of fitting techniques inclusing global analysis. This will run on a standard personal computer or laptop but we recommend investing in at least 64 GB RAM and a reasonable multicore processor if you intend to analyse large FLIM data sets. The FLIMfit software package is available as a client for the OMERO platform and can also be used as a stand-alone MATLAB application. All our open soruce software for FLIM can be found here.