Analysis of spectral excitation for measurements

of fluorescence constituents in natural waters

Alexander Chekalyuk* and Mark Hafez
Lamont Doherty Earth Observatory of Columbia University, 61 Route 9W, Palisades, New York 10964, USA
*
chekaluk@ldeo.columbia.edu

Abstract: Field measurements of chlorophyll-a (Chl), phycoerythrin (PE),

chromophoric dissolved organic matter (CDOM), and variable fluorescence
(Fv/Fm) in diverse waters of the California Current, Mediterranean Sea and
Gulf of Mexico using 375, 405, 510 and 532 nm laser excitation
wavelengths (EW) are analyzed. EW = 375 and 405 nm were found more
suitable for Chl assessment in high-Chl (> 10 μg/l) waters. Both EW = 532
and 510 nm can be used to efficiently stimulate PE fluorescence for
structural characterization of phytoplankton communities. EW = 375 nm
and 405 nm can provide best results for CDOM assessments in offshore
oceanic waters; the green EWs can be also used for CDOM measurements
in fresh and estuarine water types in conjunction with spectral
discrimination between CDOM and PE fluorescence. Both EW = 405 and
510 are suitable for photo-physiological Fv/Fm assessments, though using
EW = 405 nm may result in underestimation of PE-containing
phytoplankton groups present in mixed phytoplankton assemblages.
©2013 Optical Society of America
OCIS codes: (010.4450) Oceanic optics; (280.4788) Optical sensing and sensors; (300.0300)
Spectroscopy; (140.0140) Lasers and laser optics.

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1. Introduction
Actively-stimulated fluorescence of natural waters can provide rich information about aquatic
fluorescence constituents, including phytoplankton, chromophoric dissolved organic matter
(CDOM; see a table of abbreviations in the Appendix), oil and poly-aromatic hydrocarbons
(PAHs). In vivo fluorescence of chlorophyll a (Chl) and accessory phycobiliprotein (PBP)
pigments can be used for characterization of Chl concentration (Cchl) and phytoplankton
biomass (e.g., [1–7]), community composition [4–11], their photo-physiological state and
photosynthetic activity (e.g., [1,5–7,12–25]). The broadband fluorescence of CDOM and
PAHs can be used for assessment and spectral characterization of CDOM (e.g., [26,27]), oil
and oil products [28].
Various light sources (e.g., lasers, light emitting diodes (LEDs), and lamps) can be used to
stimulate fluorescence of aquatic constituents. The unique characteristics of laser emission
provide lasers certain advantages over other excitation sources used in aquatic fluorometry. In
particular, the spectrally narrow laser emission provides for improved selectivity, efficiency
of excitation, and reduces (vs. relatively broadband excitation) the spectral overlap between
the water Raman scattering (R) and fluorescence bands of aquatic constituents. The latter
simplifies spectral deconvolution (SDC) [5] and improves sensitivity of fluorescence
measurements. Low divergence of the laser emission, as well as laser capacity for short-pulse
operation made possible remote laser fluorescence measurements (LIDAR fluorosensing).
Despite these advantages, the relatively large size, cost, power consumption, and limited
number of excitation wavelengths (EWs) of available lasers have limited for a long time the
broad operational use of laser fluorometry in aquatic research and environmental monitoring.
The dye lasers used in the early experimental airborne LIDAR fluorosensors [29–31] allowed
tuning the excitation wavelength to optimize the fluorescence measurements. The XeCl
excimer laser (308 nm) pumping the dye laser [32] provided wavelength-tunable excitation
(320-670 nm) with higher repetition rate and pulse energy. The 532 and 355 nm excitation
with solid-state Nd:YAG lasers was used in many laser fluorosensors for measuring Chl,
phycoerythrin (PE), and CDOM fluorescence, and remote photo-physiological assessments
(e.g., [22,23,33–39]). The nitrogen lasers (EW = 337 nm) were also used in earlier LIDAR
systems [36] and laboratory settings [40] for measurements of phytoplankton variable
fluorescence. The argon laser (EW = 488 nm) was used for assaying photochemical
characteristics of phytoplankton groups [19–21]. A wavelength-tunable optical parametric

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29257
oscillator was evaluated for remote identification of dominant and sub-dominant
phytoplankton groups [41]. Most of the LIDAR fluorosensor applications for oil detection
involved excimer lasers excitation at 308 nm [28].
The recent developments in optics and electronics have provided new opportunities for the
laser fluorescence measurements. New compact, low power and affordable lasers provide an
array of wavelengths to stimulate various fluorescence constituents; new miniature, yet
sensitive spectrometers and photosensors are available for florescence detection. The
Advanced Laser Fluorometry (ALF) has been recently developed [5] on this basis. The ALF
technique can provide rich information about phytoplankton biomass, pigments, community
structure, photo-physiological state, photochemical characteristics, and CDOM content. The
ALF technique has been extensively used in the field studies, including coastal and offshore
areas of the Pacific and Atlantic Oceans, Mediterranean, Arabian, and Bering Seas,
Chesapeake, Delaware, and Monterey Bays, Hudson and York Rivers [5–7,42,43]. A
commercial version of the ALF instrument, the Aquatic Laser Fluorescence Analyzer (ALFA)
was developed in collaboration with Western Environmental Technologies Laboratories, Inc
[44]. The next generation ALF-T instrument [45] incorporates improved optical design and
swappable sample compartments for various types of measurements. It can be configured
with either single laser (EW = 510 nm) or several excitation lasers (EW = 375, 405, and 510
nm) to extend the ALF analytical capabilities.
The original ALF fluorometer [5] is a compact benchtop field instrument that combines
spectral and temporal measurements of laser-stimulated emission (LSE) using 405 and 532
nm laser excitation. The EW = 405 nm is used for CDOM and Chl fluorescence
measurements. Though not optimal for stimulating their fluorescence (the CDOM and Chl
absorption peaks are located in UV and at 440 nm, respectively), the 405 nm excitation has
been proven to be suitable for field measurements of both CDOM and Chl fluorescence [5–7].
The 405 nm laser is also used for measurements of variable fluorescence, Fv/Fm. The 532 nm
laser serves in the ALF instrument for stimulation of PE fluorescence used for detection and
identification of PBP-containing phytoplankton groups and Chl fluorescence measurements
(the 405 nm excitation is not efficient for PE fluorescence excitation).
The new ALF-T and ALFIS instruments build upon the use of new 375 and 510 nm diode
lasers. The former was incorporated in the ALF-T design [45] to provide potential for
detection and spectral discrimination between oil/PAH and CDOM background fluorescence.
The 510 nm excitation allows assessment of the key aquatic fluorescence characteristics, such
as Chl and PBP pigments, as well as variable fluorescence [45] (the latter was not possible
with 532 nm laser due to technological limitations [5]). The use of both 510 nm and 405
lasers in the ALF-T instrument provided new analytical capabilities, such as Fv/Fm
measurements with alternate blue/green excitation for more representative photo-
physiological assessments of various phytoplankton groups.
While both 375 and 510 nm lasers were successfully tested in the laboratory [45,46], it
was important to evaluate the new excitation sources in the field conditions. Our recent
deployments of several ALF instrument modifications [5,44,45] provided field fluorescence
measurements in diverse waters with four excitation wavelengths: 375, 405, 510 and 532 nm.
In this article, we analyze this data to evaluate the efficiency of new and earlier used
excitation sources for analysis of fluorescence constituents. Some results can be also used for
optimizing the design and measurement protocols of LED- and lamp-based fluorometers.
2. Field measurements
A significant amount of data was measured during the P1208 process cruise (R/V Melville) in
the California Current conducted by the California Current Ecosystem Long Term Ecological
Research (CCE-LTER) program in July-August 2012. Two new ALF instruments, ALFA and

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29258
 Fig. 1. A map of the ALF underway transect measurements during the CCE LTER process
cruise in the California Current, July-August 2012. Dots indicate locations of the underway
sampling used for correlation analysis. “Fig 2A” and “Fig 2B” mark locations of spectral
measurements displayed in Fig. 2.

ALF-T, were used for underway transect measurements of laser-stimulated emission (LSE)
and discrete sample analysis (Fig. 1). The former was configured similarly to the original
ALF [5] and uses laser excitation at 405 and 532 nm; the latter provided laser fluorescence
excitation at 375, 405, and 510 nm. The Chl and PE underway fluorescence measurements
were analyzed to evaluate the efficiency of assaying phytoplankton pigments, photo-
physiology, and community composition using EW = 375, 405, 510, and 532 nm. The local
CDOM concentration was too low to achieve an acceptable S/N ratio in CDOM fluorescence
measurements with EW = 510 nm. More reliable ALF-T CDOM fluorescence data from the
Gulf of Mexico (ECOGIG EN527 cruise; July 2013) were analyzed to evaluate the CDOM
assessments with EW = 375, 405, and 510 nm. The regression analysis of Fv/Fm measured
with EW = 405 and 510 nm was conducted using ALFA measurements in the Ligurian Sea
(Mediterranean) on the LLOMEX13 cruise (NATO Undersea Research Center; March 2013).
The seawater LSE spectra shown in Fig. 2 were measured with EW = 375, 405, 510 and
532 nm at locations marked as “Fig. 2A” and “Fig. 2B,” respectively, in Fig. 1. They illustrate
typical relationships between the fluorescence bands of Chl (Fchl), CDOM (FCDOM), water
Raman, and elastic scattering (ES). The superscript indices here and below indicate the
excitation wavelengths in nm; the spectra are normalized to their maxima and displayed in
arbitrary units. The actual ES intensity was decreased in the spectra by several orders of
magnitude due to measuring LSE via appropriately selected long-pass or 514 nm notch filters
used in the ALF-T optical design [45]. The ~20 nm gap centered at 514 nm is evident in the
LSE spectra.
Fluorescence intensities of aquatic constituents in natural waters are comparable to the
intensity of water Raman band (Fig. 2; see [5] for more examples) and almost equally
affected by the excitation and other instrument characteristics, measurement geometry and
aquatic optical properties (e.g., [31]). Therefore, fluorescence intensity normalized to water
Raman (F/R) is a convenient, instrument-independent parameter for assaying the aquatic
constituents [31,33,36]. The ALF SDC analysis allows accurate quantification of constituent
emission bands in a broad intensity range despite their spectral overlap [5]. Nonetheless, the
latter may result in less accurate SDC retrievals of the F/R ratio if F/R >> 1 or F/R << 1.

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29259
 Fig. 2. Example of LSE spectra measured with EW = 375, 405, 510 and 532 nm in Chl-rich
(A; Cchl = 4.31 μg L−1) and low-Chl (B; Cchl = 0.14 μg L−1) in seawater during the underway
survey in the California Current (Aug. 2012; the sampling locations are shown in Fig. 1).

The examples in Fig. 2 illustrate that the relationship between intensities of water Raman
and constituent fluorescence measured in a given water sample can vary a great deal
depending on EW. Both F and R intensities are highly dependent on EW (F depends on the
EW closeness to the fluorescence excitation peak; R ~EW−4). Therefore, the resulting F/R
ratio strongly depends on the excitation wavelength. This affects the selection of the
excitation sources for measuring the fluorescence constituent of interest and optimizing the
instrument configuration for measurements in fresh, estuarine, coastal, or offshore waters.
3. Chlorophyll fluorescence measurements
Chl concentration, Cchl, is a key parameter broadly used for assessment of phytoplankton
biomass and biological productivity in natural waters (e.g., [2,4]). Though the in vivo Chl
fluorescence per Cchl unit is highly variable [1,4], the ALF measurement protocols and
analytical algorithms have been optimized [6] to provide accurate Cchl assessment [5–7].
Figure 3 displays the regression relationships between Fchl/R values derived by the SDC
analysis of LSE spectral measurements at locations marked with dots in Fig. 1 and
independent fluorometric Cchl measurements in pigment extracts from water samples taken at
these locations. The high magnitudes of determination coefficients suggest that LSE
measurements with each of four EWs can be used for reasonably accurate assessments of Chl
concentration. Despite the spectral overlap between the R532 and Fchl bands at high Chl
concentrations (for example, Fig. 2(a)) that could potentially compromise the accuracy of
SDC-derived R532 values, the ALF measurements with EW = 532 nm have shown the highest
correlation between Fchl/R and Cchl among four EWs, consistently with our earlier
observations in the California Current [7]. The R2 values appeared to be slightly lower for
EW = 510 nm, further decreasing for EW = 405 and 375 nm. The higher scatter in plots Figs.
3(a) and 3(b) vs. 3(c) and 3(d) may be associated with higher variability in Fchl per unit of Cchl
for the former data sets. Indeed, the UV or blue light does not efficiently stimulate Fchl in
marine cyanobacteria due to low blue/UV absorption of their PBP photosynthetic pigments,
while the green light provides efficient Fchl excitation due to its high absorption by PEs, their
main accessory pigments ([5]). Thus, the green excitation appears to be more suitable for
fluorescence assessment of cyanobacteria ([24,25]) that may constitute significant and
variable fraction of total phytoplankton biomass in the natural waters (e.g., [7]).

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29260
 Fig. 3. Correlation between Chl concentration and Chl fluorescence normalized to water
Raman scattering measured with EW = 375 (A), 405 (B), 510 (C), and 532 (D) nm (see a map
of the ALF measurements in Fig. 1).

The results of this regression analysis are summarized in Table 1. As evident from the
slope values of regression plots in Fig. 3 (row 2 in Table 1), the use of green EWs results in
greater (vs. blue/UV excitation) magnitudes of the Fchl/R ratio per unit of Chl, which may
help to improve the accuracy of measurements in low-Chl waters, where Fchl/R<<1 (for
example,

Fig. 4. Correlation between Chl fluorescence normalized to water Raman measured with
various excitation wavelengths (same data set as in Fig. 3).

Figure 2(b)). Quantitatively, the slope magnitudes in Fig. 4 indicate (row 3 in Table 1) that
excitation at 510 nm resulted in 8.1, 6.1, and 1.1 times higher values of Fchl/R ratio vs. Fchl/R
magnitudes obtained using 375, 405, and 532 nm excitation, respectively. Such significant,
almost one order of magnitude range of Fchl/R variability with excitation wavelength can be
explained by the EW-dependence of both fluorescence and Raman components of the ratio
(section 2). Assuming REW ~EW−4, the relative (vs. 510 nm) changes in Raman intensity for
the analyzed EWs are displayed in row 4 of Table 1. The relative efficiency of Chl
fluorescence excitation for EW = 375, 405, 510, and 532 nm (row 5) can be calculated for
each wavelength via division of values from row 3 by the respective values from row 4. Thus,
the 510 nm excitation wavelength used for fluorescence measurements in the new ALF
instrument design [45] appeared to be the most efficient for Chl fluorescence measurements
among four EWs analyzed. It also results in the highest Fchl/R values used for Cchl assessment
in laser fluorometry (e.g., [5–7]).

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29261
 Table 1. Summary of Regression Analysis in Figs. 4 and 5.

1 Excitation wavelength (EW), nm 375 405 510 532

2 Cchl:(FchlEW/REW) {Fig. 3} 3.44 2.58 0.42 0.47

510 510 EW EW
3 (F chl /R ):(F chl /R ) {Fig. 4} 8.14 6.12 1.00 1.08
EW 510
4 (R ):(R ) 3.42 2.51 1.00 0.84

5 (Fchl510):(FchlEW) 2.38 2.43 1.00 1.28

4. Phycoerythrin fluorescence measurements

The regression relationships between three spectral types of PE fluorescence obtained for the
ALF underway LSE measurements using 510 and 532 nm excitation at the locations marked
with dots in Fig. 1 are displayed in Fig. 5. The PE1-containing blue-water cyanobacteria [5]

Fig. 5. Regression relationships between group-specific spectral types of PE fluorescence

measured with EW = 510 and 532 nm during SearSoar 1 and 2 underway surveys in the
California Current (Aug. 2012, see a map in Fig. 1).

were most abundant among the PBP-containing phytoplankton groups in this offshore area. It
resulted in greater FPE1/R magnitudes vs. FPE2/R and FPE3/R values (Figs. 5(a), 5(b), and 5(c),
respectively, higher S/N ratio of the FPE1/R retrievals, and respectively higher correlation
Table 2. Comparative Analysis of Excitation Efficiency for Three Spectral Types of
Phycoerythrin Fluorescence Using 532 and 510 nm Excitation

1 PE spectral fluorescence band PE1 PE2 PE3

2 PE peak wavelength, nm 565 578 590
532 532 510 510
3 (FPE /R ):(FPE /R ) 2.05 1.42 1.36
532 510
4 FPE :F PE 1.72 1.19 1.14

between the fluorescence measurements with 510 and 532 nm excitation (R2 = 0.96 vs. 0.60
and 0.75 for FPE2/R and FPE3/R, respectively). The higher scatter in Figs. 5(b) and 5(c) vs.
Figure 5(a) was caused by the lower S/N ratio in the FPE2 and FPE3 signals due to low offshore
concentration of green-water cyanobacteria and cryptophytes ([7]; note relatively low FPE2/R
and FPE3/R magnitudes in Figs. 5(b) and 5(c) vs. FPE1/R values in 5(a)).
The regression slope values in panels (a), (b), and (c) in Fig. 5 suggest that the 510 nm
excitation has resulted in 2.05, 1.42 and 1.36 lower FPE/R values vs. EW = 532 nm for the
PE1, PE2, and PE3 spectral fluorescence, respectively. The FPE/R dependence on EW was
determined by the EW dependences of both FPE and R components of the ratio. The relative
efficiency of FPE excitation using EW = 532 nm vs. 510 nm, FPE532:FPE510 can be calculated for
PE1, PE2, and PE3 via multiplying the respective values of (FPE532/R532):(FPE510/R510) (row 3,
Table 2) by R532:R510 = 0.84 (row 4, Table 1). Thus, the 510 nm excitation wavelength used in
the new ALF-T instrument [45] appeared to be less efficient for PE fluorescence excitation
than EW = 532 nm used in the original ALF instrument [5] and the earlier laser fluorometers.

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29262
The difference is relatively small for PE2 and PE3 fluorescence measurements, but almost
two-fold for measuring the PE1 fluorescence.
5. Fv/Fm measurements using 405 and 510 nm laser excitation
The initial field deployments of the new ALF instruments [45] have demonstrated the
potential of measuring variable fluorescence with multi-spectral excitation. Both the next
generation ALF-T [45] and commercial ALFA [44] instruments can be configured for Fv/Fm
measurements using alternate 405 and 510 nm excitation. An example of continuous high-
resolution underway measurements of variable fluorescence using alternate 405 and 510 nm
excitation in the Ligurian Sea (Mediterranean) is displayed in Fig. 6. The measurements were

Fig. 6. A transect map of continuous ALF underway Fv/Fm measurements with 405 and 510 nm
excitation in the Ligurian Sea (Mediterranean; March 2013).

conducted using the ALFA-X-405/510 instrument in collaboration with the NATO Undersea
Research Center. The high correlation (R2 = 0.98; Fig. 6(b)) was found between the Fv/Fm405
and Fv/Fm510 values over most of the cruise track. The slope value shows that variable
fluorescence measurements with EW = 405 nm yielded 13% lower Fv/Fm values than those
measured using EW = 510 nm over most of the track. Despite this difference, both Fv/Fm405
and Fv/Fm510 measurements have shown broad range of variability (0.1-0.5), which seems
reasonable assuming nutrient-rich conditions of spring bloom and potential down-regulation
of the phytoplankton photochemical efficiency by the solar-induced non-photochemical
quenching [6,47]. There are several groups of points in Fig. 6(b) that show deviation from the
trend line due to lower magnitude of the (Fv/Fm405): (Fv/Fm510) ratio. Our preliminary analysis
of the concurrent ALF spectral data suggests that such deviations were observed in the areas
of relative abundance of blue-water cyanobacteria in the phytoplankton community. Since the
green excitation is more efficient for stimulating cyanobacterial fluorescence than the blue
light, this may be interpreted as an indication that cyanobacteria had higher photochemical
efficiency than eukaryotic phytoplankton. Such assumption is consistent with the earlier field
observations showing that cyanobacteria may indeed show higher photochemical efficiency
than eukaryotes in natural phytoplankton assemblages [21].
6. CDOM fluorescence measurements with 375, 405, and 510 nm laser excitation
The relative efficiency of 375, 405, and 510 nm excitation for CDOM fluorescence
measurements was evaluated using the underway flow-through measurements with the ALF-
T-375/405/510 instrument [45] in the Gulf of Mexico (ECOGIG cruise; R/V Endeavor, July

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29263
 Fig. 7. Regression relationships between CDOM fluorescence normalized to water Raman
measured with laser excitation at 375, 405, and 510 nm excitation for the underway ALF
measurements in the Gulf of Mexico (July 2013). The transect map is displayed in panel A.

2013). Overall, 1,384 LSE measurements were conducted in the offshore and coastal waters
(Fig. 7(a)). The SDC analysis of the LSE measurements [5,45] was used to derive the FCDOM
and R values. The linear regression relationships between the FCDOM/R values measured with
375, 405, and 510 nm excitation are displayed in Fig. 7. The data shows that each excitation
wavelength, including 510 nm, could be used for reasonably accurate assessment of CDOM
content in the CDOM-rich Gulf waters. The slope values of the regression equations (row 2 in
Table 3) suggest that EW = 375 nm resulted in 2.7 and 4.9 higher FCDOM/R values vs. 405 and
Table 3. Relative Efficiency of CDOM Fluorescence Excitation for Various Wavelengths
1 EW1/EW2 375/405 405/510 375/510
2 (FCDOMEW1/REW1):(FCDOMEW2 /REW2) 2.68 1.81 4.85
3 REW1: REW2 1.36 2.51 3.42
4 FCDOMEW1: FCDOMEW2 3.64 4.53 16.73

510 nm excitation, respectively. In turn, the 405 nm excitation resulted on 1.8 greater
FCDOM/R values vs. 510 nm excitation. This can be explained by more efficient stimulation of
both CDOM fluorescence and water Raman scattering with shorter excitation wavelengths.
The relative efficiency of R excitation for various EW combinations were calculated (row 3 in
Table 3) assuming REW ~EW−4. The estimates of relative efficiency of CDOM fluorescence
excitation for various EW combinations are displayed in row 4 of Table 3. They were
obtained by multiplying the respective values in rows 2 and 3. This analysis suggests that
FCDOM excitation at EW = 375 nm is 3.6 and 16.7 times more efficient vs. 405 and 510 nm
excitation, respectively. In turn, EW = 405 nm 4.5-fold more efficiently stimulates FCDOM as
compared to EW = 510 nm. This may be useful for designing fluorometers based on
lamp/LED excitation. The FCDOM/R data in row 2 of Table 3 can be used for optimizing the
laser fluorometers.

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29264
7. An example of ALF oceanographic applications
The above analysis was used for optimization and analysis of our recent field measurements
with the new ALF instruments in the California Current, Bering and Mediterranean Seas,
Chesapeake Bay, and Amazon River Plume, and the acquired data. For example, Fig. 8 shows

Fig. 8. Surface distributions of the key bio-environmental variables measured with ALF-T
instrument across the frontal zone in the California Current in Aug. 2012.

high-resolution surface transect distributions of the key bio-environmental variables built

using ALF-T [45] fluorescence measurements in the California Current in Aug. 2012 (see the
red transect line in Fig. 1). The concurrent underway measurements of shipboard sensors
showed sharp changes of sea-surface salinity and temperature (SSS and SST in Fig. 8(a),
respectively) in the middle of the transect, indicating crossing the oceanic front [7]. The
transect distribution of Cchl, an index of phytoplankton biomass, was calculated from the ALF
measurements of Fchl510/R510 using the regression equation displayed in Fig. 3(c). It shows
several abrupt changes in Cchl (in 20-fold range, 0.1 to 2.2 μg L−1) associated with the frontal
features. The FCDOM375/R375 distribution generally followed the Chl patterns, indicating
biological origin of organic matter in the surveyed offshore area, but showed smaller range of
variability. Interestingly, we found a substantial, two-fold divergence between the Fv/Fm405
and Fv/Fm510 values in the warm low-Chl oligotrophic waters on the western side of the front,
while these variables closely followed each other to the east of the front. The frontal
variability in the Fv/Fm405 vs. Fv/Fm510 patterns could be associated with the respective frontal
changes in the phytoplankton community composition, which is evident from comparison of
Fchl and FPE spatial patterns across the front (Fig. 8(b)). The Fchl and FPE data were converted
in carbon biomass of total phytoplankton (ACF) and blue-water cyanobacteria Synechococcus

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29265
(SYNF), respectively, using the conversion equations obtained for this area [7]. As evident
from Fig. 8(b), the cyanobacteria, almost non-existent in the warm low-salinity waters in the
western part of the transect, exhibited sharp frontal increase in their biomass (cyan line in Fig.
8(b)) and constituted almost 100% of total phytoplankton biomass (green line in Fig. 8(b)) in
the eastern part of the surveyed area. Thus, the high-resolution underway ALF measurements
can provide rich real-time information about spatial variability of phytoplankton and CDOM
in response to sharp frontal gradients in physical and chemical properties of water masses.
This example illustrates utility of the ALF technique for aquatic research and environmental
monitoring.
8. Discussion
Excitation sources that are spectrally close to the Chl blue absorption peak at 440 nm are
often used for measuring in vivo Chl fluorescence. Though there are diode lasers to provide
440 nm fluorescence excitation, other excitation wavelengths may appear more suitable for
concurrent measurements of several aquatic fluorescence constituents, including Chl. Our
analysis of Chl fluorescence measurements with EW = 375, 405, 510, and 532 nm (section 3)
shows that each of these excitation wavelengths can be used for accurate assessment of Chl
biomass in coastal and offshore oceanic waters. The strong Fchl/R dependence on the spectral
excitation can be used for optimizing Chl fluorescence measurements in different water types.
For example, green excitation can provide accurate Chl assessments in a broad Chl
concentrations range (0.003 – 30 μg L−1) and can be used for representative sampling of
diverse phytoplankton communities, including cyanobacteria-dominant populations (e.g., [7]).
On the other hand, at high Chl concentration (> 50 μg L−1) the high-efficient green excitation
and spectral overlap of Raman and Chl fluorescence bands (for example, Fig. 2(a)) may
decrease the accuracy of SDC quantification of Raman intensity in LSE532 spectra,
compromising in turn Chl fluorescence assessment using Fchl/R ratio. Our field measurements
show that 375 or 405 nm excitation wavelengths can provide comparable to green EWs
efficiency of Fchl excitation (Table 1). These EWs may appear to be more suitable for Chl
measurements in Chl-rich (> 50 μg L−1) fresh or estuarine environments because of
significant spectral separation of water Raman and Chl fluorescence bands (Fig. 2) that
ensures lack of their spectral overlap even at very high Chl concentration.
The 532 nm solid-state lasers have been used as excitation sources for measurements of
phycoerythrin fluorescence of PBP-containing phytoplankton groups along with Chl
fluorescence of mixed phytoplankton assemblages in various laser fluorometers [9,33–36,38],
including the original ALF instrument [5]. The pulsed 532 nm lasers can be also used for
pump-and-probe photo-physiological assessments of phytoplankton variable fluorescence,
including remote sensing applications [22,23]. Unfortunately, the small CW solid-state 532
nm lasers, though most suitable for compact field fluorometers (e.g., [5]), do not provide the
fast enough optical rise time (<1 μs) needed for Fv/Fm measurements at single-turnover time
scale (~100 μs) [5,14,19]. To ensure both Fv/Fm photo-physiological assaying and assessment
of the PBP-containing phytoplankton groups, we had to use two lasers in the original ALF
instrument [5]. The new 510 nm diode laser technology, though slightly less efficient vs. 532
nm lasers for excitation of PE fluorescence (Table 2), can be also used for measuring variable
fluorescence ([45]; section 5; Fig. 8). It also appeared to be the most efficient (among four
analyzed EWs) for Chl fluorescence excitation (Table 1), and suitable for CDOM
fluorescence measurements at moderate and high CDOM concentrations (Fig. 7; Table 3;
using SDC [5] is critical to separate the overlapped CDOM and PE fluorescence). Thus, it can
be used in multi-laser fluorometric settings, or in compact and affordable single-laser
fluorometers (e.g., [45,46]) to provide quite comprehensive characterization of fluorescence
constituents in a broad range of water types.
Our initial field tests of measuring variable fluorescence with alternate 405 and 510 nm
laser excitation have shown that the new 510 nm diode laser can be used for Fv/Fm

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29266
assessments in diverse water types (Fig. 6). The dual-laser Fv/Fm measurements across the
frontal zones in the California Current ecosystem have revealed more complex patterns
(sections 5, 7), indicating potential for using multi-wavelength excitation for fluorescence
photo-physiological assessment of distinct phytoplankton groups present in natural
phytoplankton communities. Though these initial results need additional analysis and better
interpretation, it may provide new analytical capabilities of laser fluorometry for
characterization of natural aquatic environments.
We originally intended to use the 375 nm diode laser in the ALF-T instrument [45] mainly
for fluorescence detection of oil/PAH products and, in conjunction with 405 nm excitation,
their spectral discrimination vs. CDOM (the latter provides broadband fluorescence
background, always present in natural waters and spectrally overlapped with the oil/PAH
fluorescence). While we have already proven feasibility of this during our measurements in
the Gulf of Mexico, the field data indicate that Chl fluorescence excitation at 375 nm is
almost as efficient as the 405 nm excitation (Table 1) used for Chl and Fv/Fm measurements in
the original ALF instrument [5,7]). On the other hand, EW = 375 nm is more efficient (vs.
EW = 405 nm) for excitation of CDOM fluorescence. This may be advantageous for CDOM
measurements with EW = 375 nm in low-CDOM offshore oceanic environments, while EW =
405 or 532 may be beneficial for CDOM assessments in the CDOM-rich estuarine and fresh
waters (see examples of such spectra in [5]). It remains to be tested if the 375 diode laser can
be used for Fv/Fm measurements. In case of positive results, the 375 nm excitation may be
used instead of (or in conjunction with) 405 nm laser in some instrument configurations. For
example, a dual-laser ALF-375/405 system focused on environmental applications (oil/PAHs,
CDOM, Chl, and, optionally, Fv/Fm) could be built on this basis.
9. Conclusions
Our analysis of the ALF field measurements of various fluorescence constituents with UV,
blue and green laser excitation provided useful information for optimization of fluorescence
measurements and instrument configurations. Our field measurements of Chl, phycoerythrin,
CDOM, and variable fluorescence in diverse waters of the California Current, Mediterranean
Sea and Gulf of Mexico using 375, 405, 510 and 532 nm laser excitation have shown that
each of these excitation wavelengths can be used for accurate assessment of Chl
concentration in coastal and offshore oceanic waters. EW = 375 and 405 nm may appear to be
more suitable for Chl assessment in high-Chl fresh and estuarine waters. Both EW = 532 and
510 nm can be also used for stimulation of PE fluorescence. EW = 375 nm may provide best
results for CDOM assessments in low-CDOM offshore oceanic waters, while EW = 405 nm
is more suitable for CDOM measurements in high-CDOM fresh and estuarine waters. The
green laser excitation can also provide CDOM assessments in the latter environments if the
instrument provides spectral discrimination of CDOM vs. overlapped PBP fluorescence. Both
EW = 405 and 510 nm are suitable for photo-physiological assessments of Fv/Fm in a broad
range of aquatic environments, though using the blue excitation may result in underestimation
of PBP-containing phytoplankton groups. Some results of the above analysis have been
already used in practice. For example, a new laser fluorometer for in situ measurements, ALF
In Situ (ALFIS) is been currently developed based on the 510 nm laser excitation, fiber-probe
optical design, and miniature low-power electronic components [46]. The ALFIS will be
deployable on CTD sampling rosettes, towed and vertical profilers, autonomous
underwater/surface vehicles, and gliders. Building upon the ALF methods and algorithms, it
is feasible to develop a compact LIDAR-fluorosensor to conduct the ALF measurements from
aircrafts, including unmanned airborne vehicles.

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29267
Appendix
Abbreviations Used in Text
Abbreviation Term
ACF Fluorescence estimate of total autotrophic carbon
biomass, μg L−1
ALF Advanced Laser Fluorometry/Fluorometer
ALFA Aquatic Laser Fluorescence Analyzer
CDOM Chromophoric dissolved organic matter (substance)
Chl Chlorophyll a (pigment)
Cchl Chl a concentration, μg L−1
ES Elastic scattering
EW Excitation wavelength, nm
FCDOM CDOM fluorescence
Fchl Chlorophyll fluorescence
FPE Phycoerythrin fluorescence
Fv/Fm Variable fluorescence
LED Light emitting diodes
LSE Laser-stimulated emission
N Number of data points involved in the regression
analysis
PAHs Poly-aromatic hydrocarbons
PBP Phycobiliprotein (pigments)
PE Phycoerythrin (pigment)
R Water Raman scattering
R2 Coefficient of determination
S/N Signal/noise (ratio)
SDC Spectral deconvolution (method, technique)
SSS Sea surface salinity, dimensionless
SST Sea surface temperature, °C
SYNF Fluorescence estimate of Synechococcus-specific
cyanobacterial carbon biomass, μg L−1

Acknowledgments
This work was sponsored by the National Science Foundation and National Oceanographic
Partnership Program (Award Number: N000141010205; administrated by the Office of Naval
Research). We thank the California Current Ecosystem Long Term Ecological Research
(CCE LTER) and Ecosystem Impacts of Oil and Gas Inputs to the Gulf of Mexico (ECOGIG)
programs, Charles Trees and the NATO Undersea Research Center for the field opportunities,
Fethi Bengil for his operation of the ALFA instrument on the LLMOEX13 cruise, and Ralf
Goericke and Megan Roadman for their chlorophyll data used for the correlation analysis.
Our special thanks to the captain, crew, and technicians of the R/V Melville and Endeavor on
the CCE LTER and ECOGIG cruises.

#198878 - $15.00 USD Received 4 Oct 2013; revised 4 Nov 2013; accepted 5 Nov 2013; published 18 Nov 2013
(C) 2013 OSA 2 December 2013 | Vol. 21, No. 24 | DOI:10.1364/OE.21.029255 | OPTICS EXPRESS 29268