The issue of the relative importance of fluorescence to the NIR peak was explored
in a set of observations where blocking filters were used to control the excitation and
emission phenomena associated with fluorescence (Schalles et al., In Prep). The
rationale were: 1) to block incident light in the spectral range known to stimulate
chlorophyll fluorescence and observe whether a decrease in the NIR peak reflectance
occurs; and 2) to block incident light in the red and near-infrared range of the peak and
observe whether an underlying fluorescence pattern was apparent.
In these
observations, light from a quartz halogen projector lamp obliquely illuminated an 8 liter
sample container. Measurements were made with a model SE590 spectroradiometer
(Spectron Engineering) with a 6
o field of view foreoptic placed in nadir view 30 cm
above the water surface. The instrument and foreoptics were calibrated for radiometric
response using an integrating sphere at the NASA-Goddard SFC (Starks et al., 1995).
The spectral composition of incident light was controlled with narrow pass filters
placed over the projector lens. A “green” filter eliminated light below 620 nm and a
red filter blocked light above 540 nm (Corion filters LS600-S-1288 and LL600-SH942). The filters performed properly, but absorbed some light within their
transmission ranges. Corrections for wavelength specific transmittance were applied to
subsequent data measured with the filters. In the example presented here, an algal
culture with a chl a concentration of 410 µg/l and a composition dominated by the
green algae Chlorella and Chlamydomas sp. filled the sample container to a depth of
20 cm. When red and NIR light were excluded using the green narrow pass filter, a
small emission feature, with a peak centered at the in vivo chlorophyll fluorescence
wavelength of 685 nm, was evident (Figure 25). More importantly, the removal of red
and NIR light by the green filter removed almost all of the NIR peak feature, indicating
that most of the energy within the peak is reflectance and not fluorescence. When light
energy below 600 nm, capable of exciting chlorophyll fluorescence, was excluded
using the red filter, the NIR peak was strongly conserved, although the data indicated a
small reduction of energy between about 670 and 720 nm (Figure 25). In other words,
removal of most excitation energy for fluorescence had no significant effect on the
shape or magnitude of the NIR peak.
In further analysis, the corrected radiance values obtained using the red narrow
pass filter were subtracted from the unfiltered radiances, within the wavelength bounds
of the NIR peak. The resultant curve had the same form as the fluorescence emission
curve, but had almost twice the magnitude (see inset). Bandwidth (at 50% of full
maximum) was 28 nm. Note that spectra isolated with both the red and green filters
(inset) had maximum values at 685 nm, in agreement with the accepted wavelength for
chlorophyll fluorescence, and that a shoulder (green filter spectra) or second, smaller
peak (difference spectra) exists to the right of the NIR peak. The latter probably
represents the secondary peak of chl a fluorescence near 730 nm (Butler, 1966).
Although the precise magnitude of the contribution of chlorophyll fluorescence to the
NIR peak remains uncertain in this case, it is clearly small in this example. Based on
integration (data not shown) of the areas between 670 and 720 nm under the curves of
the NIR peak (Figure 25 - unfiltered) and the chlorophyll emission feature (difference
spectra - see upper curve of inset), fluorescence accounted for 7.9% of the energy of the
entire NIR feature. However, at the NIR peak position, located at 709 nm, fluorescence
accounted for only 3.7% of the total energy.
61
Optical Remote Sensing Techniques
in a set of observations where blocking filters were used to control the excitation and
emission phenomena associated with fluorescence (Schalles et al., In Prep). The
rationale were: 1) to block incident light in the spectral range known to stimulate
chlorophyll fluorescence and observe whether a decrease in the NIR peak reflectance
occurs; and 2) to block incident light in the red and near-infrared range of the peak and
observe whether an underlying fluorescence pattern was apparent.
In these
observations, light from a quartz halogen projector lamp obliquely illuminated an 8 liter
sample container. Measurements were made with a model SE590 spectroradiometer
(Spectron Engineering) with a 6
o field of view foreoptic placed in nadir view 30 cm
above the water surface. The instrument and foreoptics were calibrated for radiometric
response using an integrating sphere at the NASA-Goddard SFC (Starks et al., 1995).
The spectral composition of incident light was controlled with narrow pass filters
placed over the projector lens. A “green” filter eliminated light below 620 nm and a
red filter blocked light above 540 nm (Corion filters LS600-S-1288 and LL600-SH942). The filters performed properly, but absorbed some light within their
transmission ranges. Corrections for wavelength specific transmittance were applied to
subsequent data measured with the filters. In the example presented here, an algal
culture with a chl a concentration of 410 µg/l and a composition dominated by the
green algae Chlorella and Chlamydomas sp. filled the sample container to a depth of
20 cm. When red and NIR light were excluded using the green narrow pass filter, a
small emission feature, with a peak centered at the in vivo chlorophyll fluorescence
wavelength of 685 nm, was evident (Figure 25). More importantly, the removal of red
and NIR light by the green filter removed almost all of the NIR peak feature, indicating
that most of the energy within the peak is reflectance and not fluorescence. When light
energy below 600 nm, capable of exciting chlorophyll fluorescence, was excluded
using the red filter, the NIR peak was strongly conserved, although the data indicated a
small reduction of energy between about 670 and 720 nm (Figure 25). In other words,
removal of most excitation energy for fluorescence had no significant effect on the
shape or magnitude of the NIR peak.
In further analysis, the corrected radiance values obtained using the red narrow
pass filter were subtracted from the unfiltered radiances, within the wavelength bounds
of the NIR peak. The resultant curve had the same form as the fluorescence emission
curve, but had almost twice the magnitude (see inset). Bandwidth (at 50% of full
maximum) was 28 nm. Note that spectra isolated with both the red and green filters
(inset) had maximum values at 685 nm, in agreement with the accepted wavelength for
chlorophyll fluorescence, and that a shoulder (green filter spectra) or second, smaller
peak (difference spectra) exists to the right of the NIR peak. The latter probably
represents the secondary peak of chl a fluorescence near 730 nm (Butler, 1966).
Although the precise magnitude of the contribution of chlorophyll fluorescence to the
NIR peak remains uncertain in this case, it is clearly small in this example. Based on
integration (data not shown) of the areas between 670 and 720 nm under the curves of
the NIR peak (Figure 25 - unfiltered) and the chlorophyll emission feature (difference
spectra - see upper curve of inset), fluorescence accounted for 7.9% of the energy of the
entire NIR feature. However, at the NIR peak position, located at 709 nm, fluorescence
accounted for only 3.7% of the total energy.
61
Optical Remote Sensing Techniques