ArticlePloS one2019
Can we use rapid lifetime determination for fast, fluorescence lifetime based, metabolic imaging? Precision and accuracy of double-exponential decay measurements with low total counts.
Article in PloS one, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
What it found
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Who cites it
3 citing papers in PubMed.
- Fluorescence Lifetime Microscopy Methods for Studying Dynamics of Fluorescent Proteins In Vivo and In Vitro.Advances in experimental medicine and biology · 2026Review
- Real-time open-source FLIM analysis.Frontiers in bioinformatics · 2023Article
- A Review of New High-Throughput Methods Designed for Fluorescence Lifetime Sensing From Cells and Tissues.Frontiers in physics · 2021Article
Corrections and comments
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Authors and funding
3 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Fluorescence lifetime imaging microscopy (FLIM) can assess cell's metabolism through the fluorescence of the co-enzymes NADH and FAD, which exhibit a double-exponential decay, with components related to free and protein-bound conditions. In vivo real time clinical imaging applications demand fast acquisition. As photodamage limits excitation power, this is best achieved using wide-field techniques, like time-gated FLIM, and algorithms that require few images to calculate the decay parameters. The rapid lifetime determination (RLD) algorithm requires only four images to analyze a double-exponential decay. Using computational simulations, we evaluated the accuracy and precision of RLD when measuring endogenous fluorescence lifetimes and metabolic free to protein-bound ratios, for total counts per pixel (TC) lower than 104. The simulations were based on a time-gated FLIM instrument, accounting for its instrument response function, gain and noise. While the optimal acquisition setting depends on the values being measured, the accuracy of the free to protein-bound ratio α2/α1 is stable for low gains and gate separations larger than 1000 ps, while its precision is almost constant for gate separations between 1500 and 2500 ps. For the gate separations and free to protein-bound ratios considered, the accuracy error can be as high as 30% and the precision error can reach 60%. Precision errors lower than 10% cannot be obtained. The best performance occurs for low camera gains and gate separations near 1800 ps. When considering the narrow physiological ranges for the free to protein-bound ratio, the precision errors can be confined to an interval between 10% and 20%. RLD is a valid option when for real time FLIM. The simulations and methodology presented here can be applied to any time-gated FLIM instrument and are useful to obtain the accuracy and precision limits for RLD in the demanding conditions of TC lower than 104.
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