Instrumentation For Biological/Physical Microstructure
by Tim Cowles and Russ Desiderio
Laboratory studies of zooplankton feeding mechanisms, selectivity, and
swimming behavior over the past two decades have demonstrated the need
to understand the conditions experienced by planktonic organisms in
their natural habitat, on the time and space scales relevant to the
organism. At the same time, advances in oceanic microstructure
measurement technology by physical oceanographers have provided
temperature, salinity, density, and turbulent kinetic energy shear data
on centimeter vertical scales. These data have led to new insights into
the intermittent nature of turbulent vertical mixing in the upper ocean.
Given these data, what is the appropriate scale for evaluation of the
organism's behavioral and physiological response to the mean and
fluctuating components of the environment?
Our approach to answering this question has been to integrate new
biooptical instrumentation with existing physical oceanographic
instruments to obtain coincident biological/physical data over small
spatial scales and short time scales. We now have two different
instrument packages which can begin to address small-scale biological
variability in the field, and its connection to physical microstructure.
Laser/Fiber Optic Micro-structure Fluorometer
Our first development project was a free-falling, retrievable
microstructure profiler that simultaneously measures fluorescence
emission spectra of photosynthetic pigments, temperature, conductivity
and horizontal velocity shear. (This system was developed in
collaboration with Dr. Jim Moum, a physical oceanographer at Oregon
State). A free-fall design was used to eliminate the effects of ship
motion on microstructure data. A shipboard Argon laser provides 488 or
514 nm excitation energy through a 200 m optical fiber which is
connected to a custom-designed, optical sensor in the nosecone of a
well-tested microstructure profiler (Caldwell et al., 1985). The
excitation light illuminates a flow-through sample volume (approximately
0.25 ml) in the optical sensor. The fluorescence emitted by the
phyto-plankton cells in the sample volume is collected by a second
optical fiber attached to a shipboard multichannel diode array detector
system. The detection system collects 30 fluorescence emission spectra
(550nm-750nm) per second, yielding approximately 2 cm vertical
resolution of the water column at the 0.50 m s-1 drop rate of the
instrument package. Microstructure temperature, conductivity and
horizontal velocity shear data are acquired simultaneously. Following
data collection, fluorescence emission spectra from each 2 cm layer are
individually smoothed with a 7 nm wide filter and corrected for baseline
shifts and fiber fluorescence. The peak value for chlorophyll (Chl)
emission is found between 680-688 nm while the emission maximum for
phycoerythrin (PE) is found between 565-595 nm. We use the Raman
scattering of water as an internal standard to correct for fluctuations
in laser power output and attenuation due to flexing of the optical
fiber. Detailed vertical profiles (2 cm vertical resolution) of pigment
emission are obtained by integrating the Raman-scaled bands of
wavelengths (PE: 565-595 nm and Chl: 680-688 nm) for each of the
approximately 5000 emission spectra per 100 m profile (see Desiderio et
al., 1993 for instrumentation details). The data for Chl and PE then
are merged with the microstructure physical data to provide
high-resolution depth profiles. Figure 1 shows a profile collected off
the Oregon coast which contains numerous "thin layers", i.e.,
chlorophyll fluorescence features less than 0.5 m thick, between 30 and
60 meters. Most profiles have several thin layers within and below the
thermocline, and successive profiles often reveal a few thin layers
which persist for 40-60 mins. One thin layer persisted within a narrow
isopycnal band for 6 hours. The fluorescence emission spectra collected
with this system can detect differences in photosynthetic pigment
composition between assemblages located at different depths within the
euphotic zone (Cowles et al., 1993), thus providing in situ taxonomic
information. These data on thin layers and composition of the
autotrophic assemblage provide a starting point for the evaluation of
the microhabitat of the individual planktonic organism.Although this
profiling microstructure instrumentation has opened a new window on
small-scale biological/physical interactions, this instrument is a
prototype device. High spatial resolution spectroscopy is possible by
using optical fibers for analog optical transmission of in situ
fluorescence, but the use of optical fibers also limits deployment since
manual retrieval (hand-over-hand) is required after every free-fall
profile.
Multi-Excitation Spectral Fluorometer
In June and August 1993, we deployed a multi-excitation spectral
fluorometer on SeaSoar during the ONR-sponsored Eastern Boundary Current
project off Northern California. This instrument package uses a
tungsten-halogen lamp and a rotating filter wheel to provide three
colors of excitation light (violet, blue, green) to the sample volume;
30 fluorescence emission spectra (550nm-750nm) are collected per second
by twenty pixels of a diode array detector. We thus obtain ten spectra
per second from each excitation color. We can resolve phycoerythrin
fluorescence emission as well as chlorophyll emission. The primary
objective is to exploit the differences in chlorophyll emission
intensity as a function of the three different excitation wavebands to
extract information on the relative contribution of accessory pigments
to the total chlorophyll fluorescence. The lamp, filter wheel assembly,
optical path, sample volume, and monochrometer/detector are contained in
a 5.8" diam X 22" long pressure case which just fits within the SeaSoar
body. We had to implement a custom communications hardware/software
package because our data rate exceeded the normal bandwidth of the
SeaSoar cable. In the SeaSoar use of this instrument, we can obtain 20
cm vertical resolution of fluorescence emission spectra if the vertical
velocity of the SeaSoar is about 1 ms-1. Undulations of the SeaSoar
through the upper 300m yields 1-2 km horizontal resolution for the
instruments on the vehicle.
In October 1993, in East Sound, WA, our multi-excitation
spectral fluorometer was combined with a nine-wavelength absorption/beam
attenuation meter and a CTD on a retrievable, free-fall package in a
collaborative project with Ron Zaneveld (OSU), Percy Donaghay (URI),
Casey Moore (WET Labs, Inc) and Mike Linse (Alpha Omega, Inc). This
integrated package can resolve centimeter-scale spectral absorption,
attenuation, and fluorescence in conjunction with temperature, salinity,
and pressure. This type of integrated, in situ sensor package will
complement the high-resolution microstructure profiling systems, and
provides a slightly larger platform for other newly developed
instruments which have not yet been miniaturized.
Future Developments and Important Technologies
Long-term gains in our understanding of small-scale bio/physical dynamics
will require the incorporation of bio-optical, bio-acoustical, imaging,
and physical measurement systems into integrated towed, profiling,
moored and remote vehicle packages. We expect to see the commercial
development and miniaturization of solid-state lasers with blue
excitation, along with small, msec response CCD detectors. It will be
critical to incorporate this technology into instrumentation "suites"
which include video and holographic observation systems, chemical
detectors, and discrete sampling capabilities. Resolution of
small-scale turbulent mixing, as well as the resolution of the
small-scale velocity shear field will play an integral role in our
understanding of bio/physical interactions, and we need to work closely
with physical oceanographers to integrate these measurement systems into
new instrument packages. Finally, it is important to stress that
compelling scientific questions are the motivating forces for innovative
instrumentation development. We have no lack of compelling questions in
oceanography. (Tim Cowles and Russ Desiderio are with the College of
Oceanic and Atmospheric Sciences at Oregon State University. They thank
NSF and ONR for supporting development of these instruments.)
References
Caldwell, D.R., T.M. Dillon, and J.N. Moum. 1985. The rapid-sampling
vertical profiler: an evaluation. J. Atmos. Ocean. Technol., 2,
615-625.
Cowles, T.J., R.A. Desiderio, and S. Neuer. 1993. In situ
characterization of phytoplankton from vertical profiles of fluorescence
emission spectra. Mar. Biol., 115, 217-222.
Desiderio, R.A., T.J. Cowles, J.N. Moum, and M. Myrick. 1993.
Microstructure profiles of laser-induced chlorophyll fluorescence
spectra: evaluation of backscatter and forward-scatter fiber-optic
sensors. J. Atmos. Ocean. Technol., 10, 209-224.
