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
| Autor: | José Paulo Domingues, António Miguel Morgado, Susana F. Silva |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Biochemistry 01 natural sciences Fluorescence Microscopy Microscopy Biochemical Simulations Physics 0303 health sciences Multidisciplinary Applied Mathematics Simulation and Modeling Optical Imaging Light Microscopy Power (physics) In Vivo Imaging Optical Equipment Physical Sciences Metabolome Engineering and Technology Medicine Algorithms Research Article Cell Physiology Accuracy and precision Imaging Techniques Science Equipment Research and Analysis Methods Noise (electronics) Fluorescence 010309 optics 03 medical and health sciences Fluorescence Imaging 0103 physical sciences Humans 030304 developmental biology Pixel Lasers Double exponential function Biology and Life Sciences Computational Biology Cell Biology Cell Metabolism Computational physics Microscopy Fluorescence Mathematics AND gate |
| Zdroj: | PLoS ONE, Vol 14, Iss 5, p e0216894 (2019) PLoS ONE |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0216894 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|