FPGA-based multi-channel fluorescence lifetime analysis of Fourier multiplexed frequency-sweeping lifetime imaging

Abstract

We report a fast non-iterative lifetime data analysis method for the Fourier multiplexed frequency-sweeping confocal FLIM (Fm-FLIM) system [\nOpt. Express22, \n10221 (\n2014)24921725]. The new method, named R-method, allows fast multi-channel lifetime image analysis in the system’s FPGA data processing board. Experimental tests proved that the performance of the R-method is equivalent to that of single-exponential iterative fitting, and its sensitivity is well suited for time-lapse FLIM-FRET imaging of live cells, for example cyclic adenosine monophosphate (cAMP) level imaging with GFP-Epac-mCherry sensors. With the R-method and its FPGA implementation, multi-channel lifetime images can now be generated in real time on the multi-channel frequency-sweeping FLIM system, and live readout of FRET sensors can be performed during time-lapse imaging.