Direct frequency domain fluorescence lifetime imaging using simultaneous ultraviolet and visible excitation.

Due to the complexity, limited practicality, and cost of conventional fluorescence lifetime imaging/microscopy (FLIM) instrumentation, FLIM adoption has been mostly limited to academic settings. We present a novel point scanning frequency-domain (FD) FLIM instrumentation design capable of simultaneous multi-wavelength excitation, simultaneous multispectral detection, and sub-nanosecond to nanosecond fluorescence lifetime estimation. Fluorescence excitation is implemented using intensity-modulated CW diode lasers that are available in a selection of wavelengths spanning the UV-VI-NIR range (375-1064 nm). Digital laser intensity modulation was adopted to enable simultaneous frequency interrogation at the fundamental frequency and corresponding harmonics. Time-resolved fluorescence detection is implemented using low-cost, fixed-gain, narrow bandwidth (100 MHz) avalanche photodiodes, thus, enabling cost-effective fluorescence lifetime measurements at multiple emission spectral bands simultaneously. Synchronized laser modulation and fluorescence signal digitization (250 MHz) is implemented using a common field-programmable gate array (FPGA). This synchronization reduces temporal jitter, which simplifies instrumentation, system calibration, and data processing. The FPGA also allows for the implementation of the real-time processing of the fluorescence emission phase and modulation at up to 13 modulation frequencies (processing rate matching the sampling rate of 250 MHz). Rigorous validation experiments have demonstrated the capabilities of this novel FD-FLIM implementation to accurately measure fluorescence lifetimes in the range of 0.5-12 ns.