M Uherek 1 J Fischerova 2 and D Chorvat jr 12 1 International Laser Centre Bratislava Slovakia 2 Faculty of Mathematics Physics and Informatics Comenius University ID: 296528
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Slide1
Fluorescence spectroscopy study of bioluminescent insects
M.
Uherek
1
, J.
Fischerova
2
and
D.
Chorvat
jr
.
1,2
1. International Laser Centre, Bratislava, Slovakia
2. Faculty of Mathematics, Physics and Informatics, Comenius University,
Bratislava,
Slovakia, Slide2
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;17:321–330Slide3
Lampyris noctiluca
Image: Traces of
bioluminescing
glow-worms,
Zelezna studnicka, Bratislava [ Canon EOS D60, 30s exposition]
T
he 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.
source: wikipedia.org
Pyrophorus
noctiluc
us
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.
source: A.E.Brehm,
Illustriertes ThierlebenSlide4
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). Slide5
Spatial
distribution of fluorescence
was imaged by: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.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.Slide6
Macroscopic
f
luorescence imaging of Lampyris noctiluca.
a) Glow-worm in standard, white-light illumination, b) in fluorescence contrastResultsMacroscopic 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
bSlide7
Macroscopic
f
luorescence imaging of Pyrophorus
a) in standard, white-light illumination, b) in fluorescence contrast
a
bSlide8
Following
macroscopic
observation
s
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).
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 ].Multi-wavelength time-resolved fluorescence decay, recorded from the bioluminescent spot (d). [ Excitation 375nm ]abc
dSlide9
3D
laser-scanning
confocal microscopy
with nonlinear excitation
3D scan with fluorescence (yellow)
and SHG (cyan)
detail
glow-worm eye
2-photon excitationSlide10
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].
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.
a
bSlide11
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).
Fluorescence excitation and emission spectra of purified Luciferin
(Invitrogen) in various chemical environments: a) DMSO,
b) Etanol, c) Water.
a
bcSlide12
d
)
relative amplitude
a2 (in %)
b
) relative amplitude a1 (in
%)
c) fluorescence lifetime t1 (ps)
e) fluorescence lifetime t2 (ps)
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.Slide13
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: 339–344Slide14
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. AcknowledgementsAuthors 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).