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Time-gated fluorescence microscopy in cellular and molecular biology
H Schneckenburger1, M H Gschwend, R Sailer
1Institut für Lasertechnologien in der Medizin und Messtechnik an der Universität Ulm, Germany.
Cellular and Molecular Biology (Noisy-Le-Grand, France)
|October 9, 1998
Summary
Time-gated fluorescence spectroscopy enables selective detection of cellular fluorophores like NADH and flavins in living cells. This technique also aids in assessing mitochondrial function and intracellular conditions like calcium levels and temperature.
Area of Science:
- Biophysics
- Cellular Biology
- Spectroscopy
Background:
- Time-gated fluorescence spectroscopy offers enhanced sensitivity and specificity compared to conventional methods.
- Intrinsic cellular fluorophores and specific markers are crucial for understanding cellular processes.
Purpose of the Study:
- To describe an experimental setup for time-gated fluorescence spectroscopy and microscopy.
- To demonstrate its applications in cellular and molecular biology, focusing on selective fluorophore detection and probing cellular functions.
Main Methods:
- Utilized time-gated fluorescence spectroscopy and microscopy.
- Investigated intrinsic fluorophores (nicotinamide adenine dinucleotide (NADH), flavins) and extrinsic probes (rhodamine 123, calcium orange, laurdan).
- Evaluated non-radiative energy transfer, fluorescence lifetimes, and spectral changes under varying conditions.
Main Results:
- Achieved selective detection of NADH and flavins in living cells.
- Quantified mitochondrial malfunction via energy transfer efficacy changes.
- Demonstrated ratio fluorometry for calcium concentration using calcium orange.
- Showed improved intracellular temperature measurements with laurdan.
- Successfully discriminated porphyrins from chlorophyll background in plants.
Conclusions:
- Time-gated fluorescence spectroscopy is a powerful tool for sensitive and selective analysis in biological systems.
- The technique provides novel insights into mitochondrial function, ion concentrations, and temperature.
- Its application extends to detecting specific molecules in complex biological environments.