Tech Forum-Microstructure

## 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.