aim is to find fluorescent biosignatures specific to
cyanobacterial species and crude oil.
2. MATERIALS AND METHODS
2.1 Seawater sample preparation
Seawater samples were collected offshore from Askö Island
which is located some 80 km south of Stockholm, on 29^ July
2011. The samples were transported using dry ice and were
received at UCL on 1% August 2011. Subsequently, these
samples were stored in a -80 degC freezer until they were
measured on the 23" August 2011.
From an optical perspective, natural sea water is comprised of
three main components: inorganic suspended material, Coloured
Dissolved Organic Matter (CDOM) and phytoplankton.
Inorganic suspended material is defined as all inorganic
particulates that are not included in the phytoplankton
component CDOM is a group of organic and dissolved
substances, which consists of humic and fulvic acids. Due to
their exponential absorption curve with strong absorption from
UV decreasing towards the red spectral region it is often known
as yellow substance or ge/bstoff. CDOM has a fluorescence
peak at an excitation/emission (ex/em) pairing of around
355/450 nm (Hoge et al, 1993; Nieke et al. 1997) with
fluorescence efficiency linearly correlated with absorption
(Green and Blough, 1994). In addition to CDOM,
photosynthetic pigments of cyanobacterial cells also make a
significant contribution to the fluorescence emission of
seawater. Phycocyanin (PC) is a cyanobacteria specific pigment
receiving and transferring light energy to chlorophyll-a (Chl-a)
for photosynthetic activity. PC has an absorption peak at 620nm
with a fluorescence emission peak at 650 nm. Chl-a absorbs
light at wavelengths of 440 and 680 nm inducing peak
fluorescence at 685nm. Apart from photosynthetic pigments,
Nicotinamide Adenine Dinucleotide Phosphate (NADPH) being
a primary product of photosynthesis, has an absorption peak at
340 nm and a fluorescence emission peak at 460 nm
(Steigenberger et al, 2004). In addition to PC and Chl-a
fluorescence, cyanobacteria also induce fluorescence from the
aromatic amino acid tryptophan (Dartnell et al., 2010 and see
Dartnell et al., 2011 for model EEMs and references therein).
Fluorescence properties of the major constituents of seawater
are listed in Table 1.
Prior to the measurements, the seawater samples (frozen after
sampling for transport) were thawed and centrifuged for 10
minutes to concentrate the cells present. After resuspending the
cell pellet, the samples were pipetted into a 3.0 mL quartz
cuvette and analysed with a Perkin Elmer LS55 Luminescence
spectrometer (Perkin Elmer, Cambridge, UK) The purpose of
the centrifuging was to increase the concentration of each
constituent in the seawater sample and so obtain an improved
signal to noise ratio.
2.2 Crude oil sample preparation
Five different crude oil samples collected from various oil spill
events were also used for the spectro-fluorescence and optical
reflectance measurements. All of these oil samples are the
equivalents of weathered crude oil with diverse consistency in
terms of their thickness.
International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XXXIX-B8, 2012
XXII ISPRS Congress, 25 August — 01 September 2012, Melbourne, Australia
To obtain detailed fluorescence features of PAHs, oil samples
were pipetted into sample well plates and measured with a
fiber-optic attachment on the same Perkin Elmer LS55
spectrometer (PE-LS55).
Before optical reflectance measurements, oil samples were
transferred onto five black plates, and a Solux light source
pointed at the samples with a zenith angle of 45 degrees. In the
mean time, optical fiber probe head was positioned about 3cm
above the oil samples at a zenith angle of 0 degrees (i.e. nadir-
viewing angle). The oil sample reflectance was measured using
an EPP2000 High Resolution spectrometer (Stellarnet, Florida,
US), and reflectance spectra were recorded from 200 nm up to
1129 nm.
2.3 Excitation-emission matrices
An Excitation-Emission Matrix (EEM) contains entire spectral
information of measured samples giving a detailed pattern of
fluorescence intensity of measured samples by covering a broad
range of both excitation and emission wavelengths (an example
is given in figure 1). EEMs record a series of emission spectra
produced by incrementing the excitation wavelengths over a
broad range of the electromagnetic spectrum from UV to near
infrared region. It stacks emission spectra generated by the
excitation into a three-dimensional matrix. Using a contour
landscape to display and visualise such an information rich
dataset is an effective means to demonstrate the characterization
of the fluorescence responding from complex microbial cells
and other chemical samples. To obtain a clearer visualisation, it
is reasonable to flatten the 3D landscape into two-dimensional
planes using colour coding to illustrate the intensity of
fluorescence emission, so that the emission peaks are not
obscured behind one another and represents a clear excitation-
emission map of a sample's fluorescence response (Dartnell et
al., 2010).
3. RESULTS
3.1 Fluorescence properties of cyanobacteria cellular
pigments
For the concentrated seawater samples, emission spectra were
recorded between 300 and 800 nm, with data points logged
every 0.5 nm. The excitation wavelength was incremented in 15
nm steps between 240 and 705 nm, and the result of the spectro-
fluorescence measurements is displayed using EEM in figure 1.
There are three diagonal intense emission stripes exhibited in
figure 1. These are artifacts of the spectrometer instrument due
to diffraction-grating effects of the spectrometer, which is used
for selecting various excitation wavelengths. To be specific, the
first diagonal with the steepest gradient on the EEM of 1 is
caused by Raleigh scattering of the excitation light from
measured sample. The second and third diagonal strips on the
EEM with a gradient of 1/2 and 1/3 are second and third order
harmonic artifacts of excitation light. It is important to know
that only the emission recorded between the first and second
diagonal lines is relevant to fluorescence properties of measured
samples.
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