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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
Published on: February 9, 2012
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Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells
Karolina Jahn1, Volker Buschmann2, Carsten Hille1
1Physical Chemistry/ALS ComBi, Institute of Chemistry, University of Potsdam, Potsdam, Germany.
Scientific Reports
|September 23, 2015
Summary
Simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM) effectively monitors cellular metabolic activity. Dopamine stimulation alters FAD fluorescence and oxygen-sensitive phosphorescence, revealing mitochondrial function in insect salivary glands.
Area of Science:
- Cellular biology
- Biophysics
- Microscopy
Background:
- Intracellular homeostasis relies on precise regulation of simultaneous cellular processes.
- Multiparameter detection is crucial for understanding spatial and temporal interactions of intracellular parameters.
- Time-correlated single-photon counting (TCSPC) is a valuable technique for lifetime imaging.
Purpose of the Study:
- To evaluate a new TCSPC board for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM).
- To investigate metabolic activity in insect salivary glands using FLIM/PLIM.
- To correlate changes in fluorescence and phosphorescence lifetimes with cellular metabolic state.
Main Methods:
- Utilized a TCSPC board for simultaneous FLIM/PLIM.
- Recorded ns-decaying fluorescence (oxidized flavin adenine dinucleotide - FAD) and μs-decaying phosphorescence (ruthenium complex Kr341).
- Applied dopamine stimulation and metabolic drugs (antimycin, FCCP) to insect salivary glands.
Main Results:
- Dopamine stimulation increased Kr341 phosphorescence decay time, indicating increased oxygen consumption.
- FAD fluorescence decay time decreased upon dopamine stimulation, suggesting fluorescence quenching.
- Drug applications confirmed the mitochondrial origin of the observed signals.
- Simultaneous FLIM/PLIM successfully captured dopamine-induced metabolic changes.
Conclusions:
- Simultaneous FLIM/PLIM is a powerful tool for real-time monitoring of cellular metabolic activity.
- Changes in FAD fluorescence and oxygen-sensitive phosphorescence lifetimes provide insights into mitochondrial function.
- This technique offers a comprehensive approach to understanding intracellular homeostasis.
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