Real-time Fluorescence Lifetime Imaging Microscopy Implementation by Analog Mean-Delay Method through Parallel Data Processing

Fluorescence lifetime imaging microscopy (FLIM) has been considered an effective technique to investigate chemical properties of the specimens, especially of biological samples. Despite of this advantageous trait, researchers in this field have had difficulties applying FLIM to their systems because acquiring an image using FLIM consumes too much time. Although analog mean-delay (AMD) method was introduced to enhance the imaging speed of commonly used FLIM based on time-correlated single photon counting (TCSPC), a real-time image reconstruction using AMD method has not been implemented due to its data processing obstacles. In this paper, we introduce a real-time image restoration of AMD-FLIM through fast parallel data processing by using Threading Building Blocks (TBB; Intel) and octa-core processor (i7-5960x; Intel). Frame rate of 3.8 frames per second was achieved in $1,024{\times}1,024$ resolution with over 4 million lifetime determinations per second and measurement error within 10%. This image acquisition speed is 184 times faster than that of single-channel TCSPC and 9.2 times faster than that of 8-channel TCSPC (state-of-art photon counting rate of 80 million counts per second) with the same lifetime accuracy of 10% and the same pixel resolution.