56
spectra and chromatographic retention properties. However, with over 80 currently
recognized chemically distinct photosynthetic pigments, high operational costs, and
low sample throughput, HPLC remains a research tool. Such laborious extractive
techniques are in general unsuitable for operational ocean observing but are required
for calibration of the bio-optical instruments described below.
Chlorophylls exhibit fluorescence in the visible band: when Chl a extract is
irradiated with blue light (460–470 nm), blood red fluorescence (685–695 nm) is
visually observed. Fluorescence analysis can be over two orders of magnitude more
sensitive than spectroscopic analysis and is much more robust to confounding
suspended material in the sample. Given its greater versatility, fluorescent analysis
is favored over the spectrophotometric approach and has been exploited for Chl a
estimation in oceanography since suitable instruments were developed in the 1950s.
Fluorescence analysis is similar to the photometric procedure; a clarified acetone
extract is poured in an appropriate cuvette and placed in the instrument. In bench
top instruments, a light source is conditioned with a blue narrow band (colored)
filter and aimed to excite the sample. A photomultiplier, usually mounted at right
angles to the light source and shielded by a narrow-band red filter receives the emitted light and rejects residual excitation light. Both Chl b and Chl c also fluoresce but
appropriate choice of the narrow band filter removes this interference. Carotenoids
do not fluoresce. Phycoerythrin fluoresces yellow (575 nm) under green excitation
(525 nm) and is widely used to discriminate between the eukaryotic phytoplankton
and the blue-green cyanobacteria. However, phycoerythrin is a water-soluble pigment and must be subject to aqueous extraction.
Extractive analysis as described above is not easily adapted to autonomous measurement given the necessity for sample disruption, organic extraction, and clarification of the extract. In vivo fluorescence analysis is on the other hand feasible and
widely used despite its many shortcomings. Photosynthesis transforms light energy
into chemical bonds. Thus, any light absorbed but then reemitted by a live cell under
illumination is light not absorbed in the photosynthetic process. In nutrient-rich
near-surface environments under high irradiance, highly productive cells in full
growth fluoresce less. On the contrary, the photosynthetic process of nutrient starved
cells is inefficient and, in these cells, light not used is fluoresced. In stratified waters,
cells in the deep chlorophyll maximum at depths between 30 and 150 m are strategically situated close to the nutracline ensuring an ample nutrient source but are light
limited so they tend to have a high Chl a:C ratio. These cells fluoresce proportionally less when excited than near-surface cells do. Moreover, while extractive photometric analysis deals with a homogeneously dissolved pigment in an optically clear
solution, photosynthetic pigments in vivo are sequestered in individual cells, some
in filamentous colonies, and, in eukaryotes at least, are further packaged within cell
chloroplasts. The medium is optically turbid and self-shading is substantial, both
within and between cells. Chloroplast structure moreover changes throughout the
day, expanding and contracting with the irradiance cycle. These variables further
contribute to the uncertainty of in vivo Chl a measurements.
Despite the shortcomings described, a large number of submersible chlorophyll
fluorometers are in use today and a wide selection of instruments is available.
2 Electronic Sensors and Instruments for Coastal Ocean Observing
spectra and chromatographic retention properties. However, with over 80 currently
recognized chemically distinct photosynthetic pigments, high operational costs, and
low sample throughput, HPLC remains a research tool. Such laborious extractive
techniques are in general unsuitable for operational ocean observing but are required
for calibration of the bio-optical instruments described below.
Chlorophylls exhibit fluorescence in the visible band: when Chl a extract is
irradiated with blue light (460–470 nm), blood red fluorescence (685–695 nm) is
visually observed. Fluorescence analysis can be over two orders of magnitude more
sensitive than spectroscopic analysis and is much more robust to confounding
suspended material in the sample. Given its greater versatility, fluorescent analysis
is favored over the spectrophotometric approach and has been exploited for Chl a
estimation in oceanography since suitable instruments were developed in the 1950s.
Fluorescence analysis is similar to the photometric procedure; a clarified acetone
extract is poured in an appropriate cuvette and placed in the instrument. In bench
top instruments, a light source is conditioned with a blue narrow band (colored)
filter and aimed to excite the sample. A photomultiplier, usually mounted at right
angles to the light source and shielded by a narrow-band red filter receives the emitted light and rejects residual excitation light. Both Chl b and Chl c also fluoresce but
appropriate choice of the narrow band filter removes this interference. Carotenoids
do not fluoresce. Phycoerythrin fluoresces yellow (575 nm) under green excitation
(525 nm) and is widely used to discriminate between the eukaryotic phytoplankton
and the blue-green cyanobacteria. However, phycoerythrin is a water-soluble pigment and must be subject to aqueous extraction.
Extractive analysis as described above is not easily adapted to autonomous measurement given the necessity for sample disruption, organic extraction, and clarification of the extract. In vivo fluorescence analysis is on the other hand feasible and
widely used despite its many shortcomings. Photosynthesis transforms light energy
into chemical bonds. Thus, any light absorbed but then reemitted by a live cell under
illumination is light not absorbed in the photosynthetic process. In nutrient-rich
near-surface environments under high irradiance, highly productive cells in full
growth fluoresce less. On the contrary, the photosynthetic process of nutrient starved
cells is inefficient and, in these cells, light not used is fluoresced. In stratified waters,
cells in the deep chlorophyll maximum at depths between 30 and 150 m are strategically situated close to the nutracline ensuring an ample nutrient source but are light
limited so they tend to have a high Chl a:C ratio. These cells fluoresce proportionally less when excited than near-surface cells do. Moreover, while extractive photometric analysis deals with a homogeneously dissolved pigment in an optically clear
solution, photosynthetic pigments in vivo are sequestered in individual cells, some
in filamentous colonies, and, in eukaryotes at least, are further packaged within cell
chloroplasts. The medium is optically turbid and self-shading is substantial, both
within and between cells. Chloroplast structure moreover changes throughout the
day, expanding and contracting with the irradiance cycle. These variables further
contribute to the uncertainty of in vivo Chl a measurements.
Despite the shortcomings described, a large number of submersible chlorophyll
fluorometers are in use today and a wide selection of instruments is available.
2 Electronic Sensors and Instruments for Coastal Ocean Observing