Counting single molecules in living cells at high resolution by spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) and coincidence analysis

2005 ◽  
Author(s):  
Thomas Heinlein ◽  
Pia Schlueter ◽  
Dirk P. Herten ◽  
Markus Sauer ◽  
Juergen Wolfrum
Keyword(s):  
High Resolution ◽  
Fluorescence Lifetime ◽  
Single Molecules ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Spectrally Resolved

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.

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