A diverse group of coral reef organisms, representing several phyla, possess fluorescent pigments. We investigated the potential of using the characteristic fluorescence emission spectra of these ...pigments to enable unsupervised, optical classification of coral reef habitats. We compiled a library of characteristic fluorescence spectra through in situ and laboratory measurements from a variety of specimens throughout the Caribbean. Because fluorescent pigments are not species-specific, the spectral library is organized in terms of 15 functional groups. We investigated the spectral separability of the functional groups in terms of the number of wavebands required to distinguish between them, using the similarity measures Spectral Angle Mapper (SAM), Spectral Information Divergence (SID), SID-SAM mixed measure, and Mahalanobis distance. This set of measures represents geometric, stochastic, joint geometric-stochastic, and statistical approaches to classifying spectra. Our hyperspectral fluorescence data were used to generate sets of 4-, 6-, and 8-waveband spectra, including random variations in relative signal amplitude, spectral peak shifts, and water-column attenuation. Each set consisted of 2 different band definitions: 'optimally-picked' and 'evenly-spaced.' The optimally-picked wavebands were chosen to coincide with as many peaks as possible in the functional group spectra. Reference libraries were formed from half of the spectra in each set and used for training purposes. Average classification accuracies ranged from 76.3% for SAM with 4 evenly-spaced wavebands to 93.8% for Mahalanobis distance with 8 evenly-spaced wavebands. The Mahalanobis distance consistently outperformed the other measures. In a second test, empirically-measured spectra were classified using the same reference libraries and the Mahalanobis distance for just the 8 evenly-spaced waveband case. Average classification accuracies were 84% and 87%, corresponding to the extremes in modeled water-column attenuation. The classification results from both tests indicate that a high degree of separability among the 15 fluorescent-spectra functional groups is possible using only a modest number of spectral bands.
Colonies of the Caribbean coral Montastraea cavernosa exhibit a solar-stimulated orange-red fluorescence that is spectrally similar to a variety of fluorescent proteins expressed by corals. The ...source of this fluorescence is phycoerythrin in unicellular, nonheterocystis, symbiotic cyanobacteria within the host cells of the coral. The cyanobacteria coexist with the symbiotic dinoflagellates (zooxanthellae) of the coral and express the nitrogen-fixing enzyme nitrogenase. The presence of this prokaryotic symbiont in a nitrogen-limited zooxanthellate coral suggests that nitrogen fixation may be an important source of this limiting element for the symbiotic association.
A colorful variety of fluorescent proteins (FPs) from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques ...drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP)-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that approximately 15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37 degrees C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.
Solar-induced fluorescence from pigments in the host tissues of reef corals can make a significant contribution to their spectral signature and can affect their apparent color as perceived by a human ...observer. The relative magnitudes of the reflectance and fluorescence contributions to the spectrum can vary as a function of illumination conditions. We have combined measured coral reflectance and fluorescence spectra with modeled downwelling spectral irradiances to investigate the contribution of fluorescence to coral spectra and color. Variations in depth, fluorescence efficiency, and solar zenith angle were modeled. Fluorescence enhancement is greatest when the coral absorbs light at wavelengths that are transmitted well by seawater and emits light efficiently at wavelengths that are moderately attenuated. The methodology provides a means of predicting the combined reflectance and fluorescence spectral signatures and perceived colors of fluorescent corals under arbitrary illumination conditions.
The spectral signatures of coral reefs and related scenes, as they would be measured above the Earth's atmosphere, are calculated using a coupled atmosphere-ocean discrete ordinates radiative ...transfer model. Actual measured reflectance spectra from field work are used as input data. Four coral species are considered, to survey the natural range of coral reflectance:
Montastrea cavernosa,
Acropora palmata,
Dichocoenia stokesii, and
Siderastrea siderea. Four noncoral objects associated with reefs are also considered: sand, coralline algae, green macroalgae, and algal turf. The reflectance spectra as would be measured at the top of the atmosphere are substantially different from the
in situ spectra, due to differential attenuation by the water column and, most importantly, by atmospheric Rayleigh scattering. The result is that many of the spectral features that can be used to distinguish coral species from their surroundings or from one another, which have been used successfully with surface or aircraft data, would be obscured in spectral measurements from a spacecraft. However, above the atmosphere, the radiance contrasts between most coral species and most brighter noncoral objects remain noticeable for water column depths up to 20 m. Over many spectral intervals, the reflectance from dark coral under shallow water is smaller than that of deep water. The maximum top-of-atmosphere radiances, and maximum contrasts between scene types, occur between 400 nm and 600 nm. This study supports the conclusions of recent satellite reef mapping exercises, suggesting that coral reef identification should be feasible using satellite remote sensing, but that detailed reef mapping (e.g., species identification) may be more difficult.
Fluorescence photography is traditionally done in dark conditions owing to the relatively low level of the fluorescence emission compared to ambient light levels. Working in the dark can be awkward, ...especially underwater, and the ability to do the photography during the day would be an advantage. Recent experiments have demonstrated that with the appropriate off‐the‐shelf camera and lighting technology it is possible to make high‐quality fluorescence images in the presence of moderate levels of ambient light. The factors that affect this technique are the ambient light and the specifics of the equipment being used. For the ambient light, the important factors are the inherent optical properties of the water body, depth at which the photography is being done, time of day, cloud cover, and subject orientation and location (insofar as they create shadowing). For the equipment, the important factors are the camera's flash synchronization speed, flash intensity, flash duration, detector sensitivity, and the spectral characteristics of fluorescence barrier filters. The range of conditions under which this technique can be used was determined by modeling. Results are presented for the relatively clear waters of a tropical reef (Bahamas) and the relatively turbid waters of New England. This method of fluorescence photography allows collection of fluorescence images under daylight conditions, rather than at night or with enclosures to create artificial darkness, and thus is much more practical and applicable in the field.
Fluorescent pigments in several Indo-Pacific and Caribbean anthozoans have recently been identified as proteins related to the green-fluorescent protein (GFP) of the hydromedusa Aequorea victoria. ...Here we show that GFP is widely distributed in many Caribbean species. The fluorescence excitation and emission spectra for the pigment are similar to those reported elsewhere for coral and noncoral GFP and the fluorescence quantum yield is estimated to be 35%. Spectral and molecular characterization of the isolated protein clearly show it to be GFP, and laboratory and in situ fluorescence measurements and Western blot analysis show that it is widespread. Bathymetric studies of GFP content using Western blots for the ecologically important congeneric corals Montastraea faveolata and Montastraea cavernosa show that there is no significant correlation between depth and GFP concentration. Nucleotide sequence data of GFP from M. faveolata and M. cavernosa show 88.2% sequence homology with each other and 46.4% homology with A. victoria GFP, whereas the percent homology with A. victoria at the amino acid level was 31.1 and 28.4% for M. cavernosa and M. faveolata, respectively, and 82.7% with each other. Measurements of reflectance and of the excitation spectrum for chlorophyll fluorescence in GFP-containing corals indicate that GFP absorption, emission, and reflection have negligible impact on the level of solar radiation reaching the zooxanthellae and therefore play no role in coral photosynthesis by either addition or removal of photons.
A diverse group of coral reef organisms, representing several phyla, possess fluorescent pigments. We investigated the potential of using the characteristic fluorescence emission spectra of these ...pigments to enable unsupervised, optical classification of coral reef habitats. We compiled a library of characteristic fluorescence spectra through in situ and laboratory measurements from a variety of specimens throughout the Caribbean. Because fluorescent pigments are not species-specific, the spectral library is organized in terms of 15 functional groups. We investigated the spectral separability of the functional groups in terms of the number of wavebands required to distinguish between them, using the similarity measures Spectral Angle Mapper (SAM), Spectral Information Divergence (SID), SID-SAM mixed measure, and Mahalanobis distance. This set of measures represents geometric, stochastic, joint geometric-stochastic, and statistical approaches to classifying spectra. Our hyperspectral fluorescence data were used to generate sets of 4-, 6-, and 8-waveband spectra, including random variations in relative signal amplitude, spectral peak shifts, and water-column attenuation. Each set consisted of 2 different band definitions: 'optimally-picked' and 'evenly-spaced.' The optimally-picked wavebands were chosen to coincide with as many peaks as possible in the functional group spectra. Reference libraries were formed from half of the spectra in each set and used for training purposes. Average classification accuracies ranged from 76.3% for SAM with 4 evenly-spaced wavebands to 93.8% for Mahalanobis distance with 8 evenly-spaced wavebands. The Mahalanobis distance consistently outperformed the other measures. In a second test, empirically-measured spectra were classified using the same reference libraries and the Mahalanobis distance for just the 8 evenly-spaced waveband case. Average classification accuracies were 84% and 87%, corresponding to the extremes in modeled water-column attenuation. The classification results from both tests indicate that a high degree of separability among the 15 fluorescent-spectra functional groups is possible using only a modest number of spectral bands.
Fluorescence effects in cnidarian host tissues have been investigated only sporadically and have largely been described qualitatively. In past studies specimens were selected for investigation based ...on their apparent fluorescence under either daylight or ultraviolet (UV-A or UV-B) illumination, thereby missing relationships between the 2 effects. In this study samples of a variety of Caribbean cnidarian species were selected based on their in situ responses to daylight and/or UV-A illumination. Fluorescence emission spectra (excitation wavelength 365 or 405 nm) were measured for sources in the host tissues. A sorting of specimens by similarity in spectral shape resulted in groupings containing members of different species. Emission spectra measured from separate specimens of a single species exhibited clear differences in some cases. There was no correlation between the spectral shape and the relative fluorescence intensity observed under illumination by ultraviolet light (UV-A, emission maximum ≈ 360 nm). Emission maxima fell in the range of 480 to 590 nm. The only source of fluorescence at wavelengths greater than 650 nm was chlorophyll in zooxanthellae. Some specimens contained material with an emission spectrum identical to that of phycoerythrin (peak wavelength ≈ 575 nm). The results are of interest for studies of photoecology and for applications of coral reef optical remote sensing.
PDF
