Fluorescence Lifetime Imaging Microscopy (FLIM) as a Tool to Investigate Hypoxia-Induced Protein-Protein Interaction in Living Cells

2018 ◽  
pp. 45-53 ◽  
Author(s):  
Vera Schützhold ◽  
Joachim Fandrey ◽  
Katrin Prost-Fingerle
Keyword(s):  
Fluorescence Lifetime ◽  
Protein Interaction ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Protein Protein Interaction ◽  
Lifetime Imaging

scholarly journals Coarse-grained modeling of mitochondrial metabolism enables subcellular flux inference from fluorescence lifetime imaging microscopy

2020 ◽  
Author(s):  
Xingbo Yang ◽  
Daniel J. Needleman
Keyword(s):  
Fluorescence Lifetime ◽  
Metabolic Flux ◽  
Living Cells ◽  
Coarse Grained ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Metabolic Fluxes ◽  
Subcellular Resolution

AbstractMitochondria are central to metabolism and their dysfunctions are associated with many diseases1–9. Metabolic flux, the rate of turnover of molecules through a metabolic pathway, is one of the most important quantities in metabolism, but it remains a challenge to measure spatiotemporal variations in mitochondrial metabolic fluxes in living cells. Fluorescence lifetime imaging microscopy (FLIM) of NADH is a label-free technique that is widely used to characterize the metabolic state of mitochondria in vivo10–18. However, the utility of this technique has been limited by the inability to relate FLIM measurement to the underlying metabolic activities in mitochondria. Here we show that, if properly interpreted, FLIM of NADH can be used to quantitatively measure the flux through a major mitochondrial metabolic pathway, the electron transport chain (ETC), in vivo with subcellular resolution. This result is based on the use of a coarse-grained NADH redox model, which we test in mouse oocytes subject to a wide variety of perturbations by comparing predicted fluxes to direct biochemical measurements and by self-consistency criterion. Using this method, we discovered a subcellular spatial gradient of mitochondrial metabolic flux in mouse oocytes. We showed that this subcellular variation in mitochondrial flux correlates with a corresponding subcellular variation in mitochondrial membrane potential. The developed model, and the resulting procedure for analyzing FLIM of NADH, are valid under nearly all circumstances of biological interest. Thus, this approach is a general procedure to measure metabolic fluxes dynamically in living cells, with subcellular resolution.

scholarly journals A simple yet effective AIE-based fluorescent nano-thermometer for temperature mapping in living cells using fluorescence lifetime imaging microscopy

2020 ◽  
Vol 5 (3) ◽  
pp. 488-494 ◽  
Author(s):  
Hui Gao ◽  
Chuen Kam ◽  
Tsu Yu Chou ◽  
Ming-Yu Wu ◽  
Xin Zhao ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Temperature Mapping ◽  
Lifetime Imaging

A novel nano-thermometer composed of butter and AIE molecules can be used for intracellular temperature mapping using fluorescence lifetime imaging.

scholarly journals Carbon Dots for Intracellular pH Sensing with Fluorescence Lifetime Imaging Microscopy

Nanomaterials ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 604 ◽  
Author(s):  
Maojia Huang ◽  
Xinyue Liang ◽  
Zixiao Zhang ◽  
Jing Wang ◽  
Yiyan Fei ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Fluorescence Lifetime ◽  
Fluorescence Intensity ◽  
Carbon Dots ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Gradual Change ◽  
Ph Sensing ◽  
Lifetime Imaging

The monitoring of intracellular pH is of great importance for understanding intracellular trafficking and functions. It has various limitations for biosensing based on the fluorescence intensity or spectra study. In this research, pH-sensitive carbon dots (CDs) were employed for intracellular pH sensing with fluorescence lifetime imaging microscopy (FLIM) for the first time. FLIM is a highly sensitive method that is used to detect a microenvironment and it can overcome the limitations of biosensing methods based on fluorescence intensity. The different groups on the CDs surfaces changing with pH environments led to different fluorescence lifetime values. The CDs aqueous solution had a gradual change from 1.6 ns to 3.7 ns in the fluorescence lifetime with a pH range of 2.6–8.6. Similar fluorescence lifetime changes were found in pH buffer-treated living cells. The detection of lysosomes, cytoplasm, and nuclei in living cells was achieved by measuring the fluorescence lifetime of CDs. In particular, a phasor FLIM analysis was used to improve the pH imaging. Moreover, the effects of the coenzymes, amino acids, and proteins on the fluorescence lifetime of CDs were examined in order to mimic the complex microenvironment inside the cells.

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