Analysis of spectral excitation for measurements of ... · Analysis of spectral excitation for...

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

*[email protected]

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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#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 29255

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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


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


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.


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]).


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]).


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

3 (Fchl510/R510):(Fchl

EW/REW) {Fig. 4} 8.14 6.12 1.00 1.08

4 (REW):(R510) 3.42 2.51 1.00 0.84

5 (Fchl510):(Fchl

EW) 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

3 (FPE532/R532):(FPE

510/R510) 2.05 1.42 1.36

4 FPE532:FPE

510 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, FPE

532:FPE510 can be calculated for

PE1, PE2, and PE3 via multiplying the respective values of (FPE532/R532):(FPE

510/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.


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/Fm

405 and Fv/Fm

510 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/Fm

405 and Fv/Fm

510 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/Fm

405): (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


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 (FCDOM

EW1/REW1):(FCDOMEW2 /REW2) 2.68 1.81 4.85

3 REW1: REW2 1.36 2.51 3.42 4 FCDOM

EW1: 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.


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 Fchl

510/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 FCDOM

375/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/Fm

405 and Fv/Fm

510 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/Fm

405 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


(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


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.


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.


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