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 [1].
[1] 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 [2].
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.
[2] 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 [4].
[3] Y. Oba, T. Shintani, T. Nakamura, M. Ojika, S. Inouye: Biosci. Biotechnol. Biochem., 72 (5),
1384–1387, 2008
[4] 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).