Direct frequency domain fluorescence lifetime imaging using field programmable gate arrays for real time processing

Frequency domain (FD) fluorescence lifetime imaging (FLIM) involves the excitation of the sample of interest with a modulated light source and digitization of the fluorescence emission for further analysis. Traditional FD-FLIM systems use heterodyne or homodyne detection, where the excitation light source and detector are modulated at specific frequency(s). More recently, FD-FLIM systems that use reflection of the light source as a trigger or phase reference for lifetime calculations have been developed. These detection schemes, however, require extra components that increase the cost and complexity of the FD-FLIM system. Here, we report a novel FD-FLIM detection scheme whereby the light source modulation and emission digitization are implemented using Field Programmable Gate Arrays (FPGAs), and fixed gain avalanche photodiodes are used for fluorescence detection. The reported FD-FLIM system was designed for probing nanosecond lifetime fluorophores (2-10 ns) at three emission bands simultaneously. The system utilizes a 375 nm diode laser for excitation at multiple simultaneous modulation frequencies (between 1 MHz and 83 MHz, bandwidth limited intentionally by using a lowpass filter) and three fixed gain avalanche photodiodes for simultaneous detection of three emission bands: 405/20 nm, 440/40 nm, and 525/50 nm (center/FWHM). Real-time computation of the modulation and phase lifetimes is simply performed by direct application of the discrete Fourier transform (max. of 10 frequencies) to the digitized fluorescence emission signals. The accuracy and sensitivity of this novel FD-FLIM detection scheme was demonstrated by imaging standard fluorophores and ex vivo unfixed human coronary artery tissue samples.