Fluorescence spectroscopy study of bioluminescent insects M. Uherek1, J. Fischerova2 and D. Chorvat jr.1,2 1. International Laser Centre, Bratislava, Slovakia 2. Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovakia, Motivation Bioluminescence is the light emission in living organisms that appears as a result of specific chemical reactions. In Arthropoda the luminescence is due to catalytic reaction of luciferin with luciferase enzyme with 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 animals and can be utilized for characterization of fixed samples such as museum deposits. One of the natural candidates for this application is Luciferin. In addition to its bioluminescent character, Luciferin acts also as a fluorescent pigment, able to absorb UV light and emitting in blue-green spectral region, depending on its conformational state .  N.N. Ugarova and L.Y. Brovko, Luminescence 2002;17:321–330 Lampyris noctiluca source: wikipedia.org The European common glow-worm is a firefly species of the genus Lampyris. The males are smaller than females, had bioluminiscent organ in abdominal part. Females had often twice the size of the males, are larviforme and their wings are missing. This type of signalling serves for communication between sexes during a well-known period around Summer solstice. The particular species are different in intensity of bioluminiscence, length of intervals and length of solitary luminiscence. Using bioluminiscence come into dehydrogenating of luciferine by luciferaze. Pyrophorus noctilucus The click-beetles are species of the genus Pyrophorus (Elateridae), known for specific clicking mechanism that can bounce the beetle into the air, as well as bioluminescence ability. They have two strongly luminescent spots at the posterior corners of the pronotum, and another ventral spot. Pyrophorus (cucujo) live in many areas of tropical and subtropical America. Image: Traces of bioluminescing glow-worms, Zelezna studnicka, Bratislava [ Canon EOS D60, 30s exposition] source: A.E.Brehm, Illustriertes Thierleben Materials and Methods 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 noctiluca and Pyrophorus noctilucus. Samples (Lampyris from own collection, Pyrophorus from museal deposits) were collected in nature and fixed . For spectroscopic characterization of both animals and purified firefly luciferin (used as a reference), we used: • • • • steady-state absorption spectrometer Cary-50 Bio (Varian) fluorescence spectrometer Fluorolog 3-11 (SPEX-HORIBA) fiberoptic spectrometer Maya (Ocean Optics) time-correlated single photon counting system (Becker-Hickl) based on SPC-830 card and pulsed laser diode excitation (BDL-473 and BDL 375). Spatial distribution of fluorescence was imaged by: a) custom macro imaging setup, comprised of Optem fiberoptic halogen source, glass filters (excitation bandpass 390-470nm, emission long-pass 520nm) and Canon D-60 camera with Canon and Sigma lens. b) advanced confocal laser scanning microscope workstation Zeiss LSM 510 META NLO with 458/477/488nm Ar:ion excitation, 473nm picosecond laser diode excitation, or 1048nm femtosecond (2-photon) excitation source with fluorescence lifetime imaging detection (Becker-Hickl TCSPC system with PML-SPEC detector and Oriel spectrograph). For imaging we used PlanNeofluar 2.5x, A-Plan 10x, and PlanApochromat 20x objectives. Results Macroscopic fluorescence imaging of Lampyris noctiluca shows that the spatial distribution of fluorescence surprisingly does not completely collocalize with the location of its well-known luminescent organs: a b Macroscopic fluorescence imaging of Lampyris noctiluca. a) Glow-worm in standard, white-light illumination, b) in fluorescence contrast a b Macroscopic fluorescence imaging of Pyrophorus a) in standard, white-light illumination, b) in fluorescence contrast Following macroscopic observations we investigated fluorescence spectra and fluorescence decays at different locations of the firefly, using multispectral confocal microscopy and TCSPC. We found slight differences of its spectral maximum at different measured regions (bioluminescent spot / eye / legs). a b c 3000 2500 Intensity 2000 abdomen 1500 leg 1000 eye 500 0 450 500 550 600 650 700 Multispectral confocal fluorescence image of glow worm head (a) and bioluminiscent organ (b) using multispectral detector Zeiss META. Mean fluorescence spectra from the representative regions of interests are shown at graph (c). [ Excitation 458nm, emission 488-648nm ]. [ Excitation 375nm ] 1,0 0,5 00,0 2 4 ] [ns lay de Multi-wavelength time-resolved fluorescence decay, recorded from the bioluminescent spot (d). d fluorescence intensity [a.u.] Emission wavelenght / nm 6 8 450 600 550 500 [n th m] waveleng 650 3D laser-scanning confocal microscopy with nonlinear excitation glow-worm eye 2-photon excitation 3D scan with fluorescence (yellow) and SHG (cyan) detail Spectroscopy We observed that both emission spectra (a) and fluorescence decays (b) of the bioluminescent spots are similar in both examined species of Lampyris and Pyrophorus . b Normalized fluorescence intensity 1.5 Pyrophorus noctilucus Lampyris noctiluca firefly luciferin 1.0 0.5 0.0 500 550 600 Wavelength (nm) 650 Fluorescence intensity (counts) a 10 5 10 4 10 3 Lampyris Pyrophorus firefly luciferin in H2O 5 10 15 20 Time (ns) Fluorescence spectrum of isolated luciferin in water correspond qualitatively to the spectrum shape recorded from animals, while its time-resolved fluorescence decay show different kinetics. All spectra were recorded upon excitation by 473nm picosecond laser.  P. Vršanský, D. Chorvát, I. Fritzsche, M. Hain and R. Ševčík, Light-mimicking cockroaches indicate Tertiary origin of recent terrestrial luminescence, Naturwissenschaften, Vol 99, No 9 (2012), 739-749. The origin of this feature was investigated by using different molecular environments used as solvents. We characterized the excitation and fluorescence spectra (this slide) and the time-resolved fluorescence decays (next slide) of purified Luciferin in solutions of water, ethanol and DMSO, as well as in solid phase represented by poly-methyl methacrylate block (PMMA). The results were in accordance with previously published data, but surprisingly show that water environment is the only one that mimics the spectral behavior observed in the animal fluorescence (green emission around 500-550nm). a b c Fluorescence excitation and emission spectra of purified Luciferin (Invitrogen) in various chemical environments: a) DMSO, b) Etanol, c) Water. Parameters of the exponential components of the time-resolved fluorescence decays of purified Luciferin. Notation: 1-PMMA, 2-H2O, 3-H2O with LP 500nm emission filter, 4-Ethanol, 5 - DMSO. Concentration of luciferin was 5x10-7 mol/l in all cases. t1 a1 80 70 60 50 40 30 20 10 0 1200 1000 800 600 400 200 0 1 2 3 4 5 1 4 5 t2 5000 100 3 c) fluorescence lifetime t1 (ps) b) relative amplitude a1 (in %) a2 2 4000 80 3000 60 40 2000 20 1000 0 1 2 3 4 5 d) relative amplitude a2 (in %) 0 1 2 3 4 5 e) fluorescence lifetime t2 (ps) Discussion Our results indicate that spatial distribution of luciferin in insects is not completely constrained to their luminescent organs. We hypothesize that on of the possible explanations of this fact could be that luciferin (as a molecular substrate) is present through the whole body of the animal (although in different concentrations), while the bioluminescence occurs only in parts where the Luciferaze enzyme is expressed in controlled manner. These facts can not clearly prove present luciferin through the whole body of the animal because fluorescence ability have many other substances. This problem will be subject of our following research. Although more detailed spectroscopic characterization is needed to decipher its photophysical behaviour, fluorescence spectroscopy seems a promising tool for understand the details, and in perspective also the evolutionary foundation of the firefly bioluminescent system .  Y. Oba, T. Shintani, T. Nakamura, M. Ojika, S. Inouye: Biosci. Biotechnol. Biochem., 72 (5), 1384–1387, 2008  M. Dubuisson. C. Marchand and J-F. Rees, Luminescence 2004; 19: 339–344 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. Acknowledgements Authors acknowledge collaboration with Dr. Peter Vršanský (Geological Institute of SAS, Bratislava), and funding from the projects NanoNet2 (ITMS 26240120018 under the R&D OP of the EFRD fund) and Laserlab Europe III (7FP, EC, contract No. 284464).