Abstract
Time-correlated single photon counting (TCSPC) enables acquisition of fluorescence lifetime decays with high temporal resolution within the fluorescence decay. However, many thousands of photons per pixel are required for accurate lifetime decay curve representation, instrument response deconvolution, and lifetime estimation, particularly for two-component lifetimes. TCSPC imaging speed is inherently limited due to the single photon per laser pulse nature and low fluorescence event efficiencies (<10%) required to reduce bias towards short lifetimes. Here, simulated fluorescence lifetime decays are analyzed by SPCImage and SLIM Curve software to determine the limiting lifetime parameters and photon requirements of fluorescence lifetime decays that can be accurately fit. Data analysis techniques to improve fitting accuracy for low photon count data were evaluated. Temporal binning of the decays from 256 time bins to 42 time bins significantly (p<0.0001) improved fit accuracy in SPCImage and enabled accurate fits with low photon counts (as low as 700 photons/decay), a 6-fold reduction in required photons and therefore improvement in imaging speed. Additionally, reducing the number of free parameters in the fitting algorithm by fixing the lifetimes to known values significantly reduced the lifetime component error from 27.3% to 3.2% in SPCImage (p<0.0001) and from 50.6% to 4.2% in SLIM Curve (p<0.0001). Analysis of nicotinamide adenine dinucleotide-lactate dehydrogenase (NADH-LDH) solutions confirmed temporal binning of TCSPC data and a reduced number of free parameters improves exponential decay fit accuracy in SPCImage. Altogether, temporal binning (in SPCImage) and reduced free parameters are data analysis techniques that enable accurate lifetime estimation from low photon count data and enable TCSPC imaging speeds up to 6x and 300x faster, respectively, than traditional TCSPC analysis.
Highlights
Fluorescence lifetime is the time a fluorophore remains in the excited state upon absorption of an incident photon, before emission of a subsequent photon and return to ground state
While gated cameras and fast oscilloscopes are advantageous for fast imaging of bright samples, both methods are limited in decay sampling to ~0.5-1 ns which does not provide sufficient sampling of the decay curve to accurately resolve multiexponential decays
The purpose of this study is to investigate the inherent limitations of photon count/SNR for accurate Time-correlated single photon counting (TCSPC) data analysis and identify strategies to reduce the number of required photons and image acquisition times
Summary
Fluorescence lifetime is the time a fluorophore remains in the excited state upon absorption of an incident photon, before emission of a subsequent photon and return to ground state. The fluorescence lifetime is dependent upon molecule conformation and binding, with short and long lifetimes depending on quenched or unquenched fluorophore configuration [1,2]. In this way, fluorescence lifetime can be used to evaluate protein interactions. Fluorescence lifetime decays are difficult to measure due to the required sub-nanosecond temporal resolution. Several methods have been developed for detecting fluorescence lifetime, including the use of gated cameras [10,11,12], fast oscilloscopes [13], and time-correlated single photon counting [14]. While gated cameras and fast oscilloscopes are advantageous for fast imaging of bright samples, both methods are limited in decay sampling (temporal resolution) to ~0.5-1 ns which does not provide sufficient sampling of the decay curve to accurately resolve multiexponential decays
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