M. Uherek1, J. Fischnerova2 and D. Chorvat jr.1,2
1. International Laser Centre, Bratislava, Slovakia
2. Faculty of Matematics, Physics and Informatics, Comenius University, Bratislava, Slovakia,
Bioluminescence in insects is the light emission that occurs as a result of catalytic reaction of luciferin with different luciferase enzymes in the presence of ATP and oxygen. In contrast to bioluminescent reactions, fluorescence use photons as the energy source, thus is not dependent on the presence of balanced homeostasis of enzymes in living animal and can be utilized for characterization of fixed samples such as museum deposits. One of the natural candidates for this application is luciferin, which in addition to its bioluminescent character acts also as a fluorescent pigment.
In this study we utilized an advanced microscopy, spectroscopy and fluorescence lifetime detection to characterize spatial and spectral properties of luciferin in samples of genus Lampyris and Pyrophorus. Spatial distribution of fluorescence was imaged either by custom macro-imaging setup, or by advanced confocal laser scanning microscope with fluorescence lifetime imaging detection. For spectroscopic characterization of both animals and purified firefly luciferin, used as a reference, we used steady-state absorption and fluorescence spectrometry and time-correlated single photon counting with pulsed laser diode excitation.
We observed that both emission spectra and fluorescence decay characteristics are similar in both examined species of Lampyris and Pyrophorus. Fluorescence spectrum of isolated luciferin correspond to the spectrum shape recorded from animals, while its time-resolved fluorescence decay show different kinetics. The origin of this feature was investigated by using different molecular environments (water, organic solvents and polymer matrices) used as solvents. Our results indicate that spatial distribution of luciferin in insects is not completely constrained to their luminescent organs. In summary, we demonstrated that spectrally-resolved fluorescence lifetime microscopy provides a synergic approach with potential for detailed characterization of photophysical properties of luciferin inside intact insect samples, helping to understand the properties of their bioluminescent system.
Vršanský P. et al., Naturwissenschaften, in press (PMID: 22864963).
Authors acknowledge collaboration with Dr. Peter Vršanský and funding from the project NanoNet2 (ITMS 26240120018) under the OP Research and Development of the European EFRD fund.
Mr. Martin Uherek
International Laser Centre, Ph.D. Student
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