On Synthetic Instrument Response Functions of Time-Correlated Single-Photon Counting Based Fluorescence Lifetime Imaging Analysis

Abstract

Time-correlated single-photon counting (TCSPC) has been the gold standard for fluorescence lifetime imaging (FLIM) techniques due to its high signal-to-noize ratio and high temporal resolution. The sensor system's temporal instrument response function (IRF) should be considered in the deconvolution procedure to extract the real fluorescence decay to compensate for the distortion on measured decays contributed by the system imperfections. However, to measure the instrument response function is not trivial, and the measurement setup is different from measuring the real fluorescence. On the other hand, automatic synthetic IRFs can be directly derived from the recorded decay profiles and provide appropriate accuracy. This paper proposed and examined a synthetic IRF strategy. Compared with traditional automatic synthetic IRFs, the new proposed automatic synthetic IRF shows a broader dynamic range and better accuracy. To evaluate its performance, we examined simulated data using nonlinear least square deconvolution based on both the Levenberg-Marquardt algorithm and the Laguerre expansion method for bi-exponential fluorescence decays. Furthermore, experimental FLIM data of cells were also analyzed using the proposed synthetic IRF. The results from both the simulated data and experimental FLIM data show that the proposed synthetic IRF has a better performance compared to traditional synthetic IRFs. Our work provides a faster and precise method to obtain IRF, which may find various FLIM-based applications. We also reported in which conditions a measured or a synthesized IRF can be applied.

Time-Resolved Silica Luminescence Probes Photoinduced Electron Transfer from Heterobinuclear Units to 2,2′-Bipyridine and O2

“…Because the strongest PL occurs from samples that feature the complete electron transfer chain described in Figure , the rate of onset of PL serves as an upper bound to the rate of charge transfer through the sequence. The time scale of this rise time behavior was ascertained by fitting data from Figure a–d with a convolution of a synthetic instrument response function (a Gaussian function, simulating the laser pulse and instrument response) and a simulated kinetics function (the sum of a single exponential growth function and an appropriate decay function, either exponential or Albery-type as discussed in greater detail below); an example fit is shown in Figure . While the 390 and 405 nm excitation sources had temporal full-width-at-half-max values of 1.08 ns and 520 ps, respectively, the TCSPC detector bins could be as short as 27 ps/bin, allowing for fitting results with uncertainties substantially lower than the laser pulse limit when sufficient intensity was available (Table ).…”

Beyond conventional wisdom: unveiling quantitative insights in fluorescence lifetime imaging via realistic simulation of biological systems

“…1D), and the ability to detect a specific amount of fluorescence lifetime response increases. Although the number of photons required to achieve a certain amount of SNR was analyzed previously 15,[26][27][28][29][30][31][32][33][34] , such analysis had not been performed with consideration of biological samples in realistic experiments with autofluorescence, background, and afterpulse.…”

“…An effective tool to explore how experimental parameters contribute to outcome is simulation. Both analytical and simulation methods have provided insights into issues such as SNR 15,[26][27][28][29][30][31][32][33][34] . However, prior work usually assumes the presence of sensor fluorescence only without considering the important contributions to noise and bias due to other factors such as autofluorescence and afterpulse of the PMT.…”

Non‐fitting FLIM‐FRET facilitates analysis of protein interactions in live zebrafish embryos

“…The instrument's contribution to photon arrival times (Instrument Response Function, IRF) must also be measured and corrected for. 15 TSCPC FLIM-FRET requires high photon counts, dependent on the τ precision and dynamic range and signal-to-noise ratio. 16 For example, photon counts in the order of 10 6 -10 7 have been reported in cells.…”

“…For example, the donor's characteristic fluorescence decay may itself be multiexponential and can be affected in donor–acceptor fusions. The instrument's contribution to photon arrival times (Instrument Response Function, IRF) must also be measured and corrected for 15 . TSCPC FLIM‐FRET requires high photon counts, dependent on the τ precision and dynamic range and signal‐to‐noise ratio 16 .…”