Systematic Enzyme Mapping of Cellular Metabolism by Phasor-Analyzed Label-Free NAD(P)H Fluorescence Lifetime Imaging
Abstract
:1. Introduction
2. Results
2.1. Benchmarking of NAD(P)H-FLIM Data Evaluated Using the Phasor Approach
2.1.1. Low Signal-to-Noise Ratio of the Image Affects the NAD(P)H-FLIM Data
2.1.2. Validation of Phasor-Evaluated FLIM Data on Mixtures of NADH and MDH or LDH
2.2. Enzyme-Based Reference System to Interpret Label-Free NAD(P)H-FLIM Data in Cells
2.3. Analysis Framework of NAD(P)H-FLIM Data: Validation on Homogeneous NAD(P)H-Enzyme Mixtures
2.4. Validation of the NAD(P)H-FLIM Analysis Framework on Stromal-Like 3T3-L1 Cells
3. Discussion
4. Material and Methods
4.1. Two-Photon Microscope Setup Adequate for FLIM
4.2. Phasor Analysis of Time-Domain NAD(P)H-FLIM Data
4.3. Enzyme and NAD(P)H Solutions
4.4. T3-L1 Cell Culture and Preparation for Imaging
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Leben, R.; Köhler, M.; Radbruch, H.; Hauser, A.E.; Niesner, R.A. Systematic Enzyme Mapping of Cellular Metabolism by Phasor-Analyzed Label-Free NAD(P)H Fluorescence Lifetime Imaging. Int. J. Mol. Sci. 2019, 20, 5565. https://doi.org/10.3390/ijms20225565
Leben R, Köhler M, Radbruch H, Hauser AE, Niesner RA. Systematic Enzyme Mapping of Cellular Metabolism by Phasor-Analyzed Label-Free NAD(P)H Fluorescence Lifetime Imaging. International Journal of Molecular Sciences. 2019; 20(22):5565. https://doi.org/10.3390/ijms20225565
Chicago/Turabian StyleLeben, Ruth, Markus Köhler, Helena Radbruch, Anja E. Hauser, and Raluca A. Niesner. 2019. "Systematic Enzyme Mapping of Cellular Metabolism by Phasor-Analyzed Label-Free NAD(P)H Fluorescence Lifetime Imaging" International Journal of Molecular Sciences 20, no. 22: 5565. https://doi.org/10.3390/ijms20225565