Optical Properties of Corals Distort Variable Chlorophyll ... · Optical Properties of Corals Distort Variable Chlorophyll Fluorescence Measurements1 Daniel Wangpraseurt,a,b,c,2 Mads

Optical Properties of Corals Distort Variable ChlorophyllFluorescence Measurements1

Daniel Wangpraseurt,a,b,c,2 Mads Lichtenberg,a Steven L. Jacques,d Anthony W. D. Larkum,e andMichael Kühla,e,3

aMarine Biological Section, Department of Biology, University of Copenhagen, DK-3000 Helsingør, DenmarkbDepartment of Chemistry, University of Cambridge, Cambridge CB2 1EW, UKcScripps Institution of Oceanography, University of California San Diego, La Jolla, California 92037dTufts University, Medford, Massachusetts 02155eClimate Change Cluster, University of Technology Sydney, Ultimo, New South Wales 2007, Australia

ORCID IDs: 0000-0003-4834-8981 (D.W.); 0000-0002-0675-4554 (M.L.); 0000-0002-0420-134X (A.W.D.L.); 0000-0002-1792-4790 (M.K.).

Pulse-amplitude–modulated (PAM) fluorimetry is widely used in photobiological studies of corals, as it rapidly providesnumerous photosynthetic parameters to assess coral ecophysiology. Coral optics studies have revealed the presence of lightgradients in corals, which are strongly affected by light scattering in coral tissue and skeleton. We investigated whether coraloptics affects variable chlorophyll (Chl) fluorescence measurements and derived photosynthetic parameters by developingplanar hydrogel slabs with immobilized microalgae and with bulk optical properties similar to those of different types ofcorals. Our results show that PAM-based measurements of photosynthetic parameters differed substantially betweenhydrogels with different degrees of light scattering but identical microalgal density, yielding deviations in apparent maximalelectron transport rates by a factor of 2. Furthermore, system settings such as the measuring light intensity affected F0, Fm, andFv/Fm in hydrogels with identical light absorption but different degrees of light scattering. Likewise, differences in microalgaldensity affected variable Chl fluorescence parameters, where higher algal densities led to greater Fv/Fm values and relativeelectron transport rates. These results have important implications for the use of variable Chl fluorimetry in ecophysiologicalstudies of coral stress and photosynthesis, as well as other optically dense systems such as plant tissue and biofilms.

The ecological success of coral reefs is largely due tothe successful symbiotic relationship between the coralanimal host and its photosymbiotic microalgae be-longing to the genus Symbiodinium. This highly efficientsymbiotic interaction is susceptible to changes in envi-ronmental conditions, such as excess solar radiationand above-average seawater temperatures, which canlead to the breakdown of the coral–algal symbiosis andthe visible paling of the coral colony known as “coralbleaching” (Weis, 2008). Given the importance ofSymbiodinium photosynthesis for coral health, coral

photosynthesis has been studied intensively from mo-lecular to environmental scales (Falkowski et al., 1990;Dubinsky and Falkowski, 2011). Coral photosynthesiscan be studied with techniques quantifying photosyn-thetic O2 production or carbon fixation (Kühl et al.,1995; Hoogenboom et al., 2012; Osinga et al., 2012),but photophysiological measurements based on varia-ble chlorophyll (Chl) a fluorescence are now widelyused in coral research and many other areas of terres-trial and aquatic photosynthesis research (Warner et al.,1996, 2010; Ralph and Gademann, 2005; Szabó et al.,2014). In contrast to gas exchange or C-fixation mea-surements that require significant sample handling,variable Chl fluorescence relies on optical light pulsingschemes that are applied externally with minimalsamplemanipulation or directly in the natural habitat, anda variety of commercial instruments for cuvette-based,fiber-optic or imaging measurements are available(e.g. Schreiber, 2004). In coral reef science, pulse-amplitude–modulated (PAM) Chl a fluorimeters areby far the most commonly used instrument to probephotosynthesis (Warner et al., 2010).

Variable Chl fluorimetry quantifies the fate of absorbedlight energy trapped by the photosynthetic apparatus viachanges in Chl a fluorescence, which tracks the redoxstatus of PSII and the balance between photochemical andnonphotochemical quenching processes. PAM-basedmeasurements employ the so-called “saturation pulse”

1This work was supported by the Carlsberg Foundation (Distin-guished Postdoctoral Fellowship CF15-0582 to D.W. and an Instru-ment grant to M.K.) and the Independent Research Fund Denmark/Natural Sciences (Sapere-Aude Advanced Grant to M.K.).

2Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Daniel Wangpraseurt ([email protected]).

D.W., M.L., A.W.D.L., and M.K. conceived and designed the ex-periments; D.W., M.L., and A.W.D.L. performed experiments; D.W.,S.L.J., and M.K. analyzed and interpreted data; D.W. and S.L.J. de-veloped optical models; D.W. wrote the article with contributionsfrom all authors.

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method (Schreiber, 2004). The PAM technique generatesmultiple photochemical charge separations (multipleturnover) and fully reduces QA via the application of50–1,000-ms multiple turnover flashlets (“fat flashes”; seeKromkamp and Forster, 2003). The fluorescence yieldbefore the saturation pulse indicates the level of fluores-cence when QA is maximally oxidized and PSII reactioncenters are fully open. Such minimum fluorescence yieldis denoted as F0 and F, referring to dark- and light-acclimated samples, respectively. The saturation pulse isassumed to lead to the complete closure of PSII reactioncenters, such that photochemical quenching is fullyinhibited (Schreiber et al., 1993). Consequently, fluores-cence emission ismaximal and this parameter is knownasthe “maximal fluorescence yield,”where Fm and Fm9 referto dark- and light-acclimated samples, respectively(Schreiber, 2004). From these measurements, many de-rived parameters describe the photophysiology of theinvestigated sample.In coral reef science, the most frequently used fluo-

rescence parameter is the maximum PSII quantumyield, which is calculated from saturation pulse mea-surements on dark-acclimated samples as follows: Fv/Fm = (Fm-F0)/Fm. The Fv/Fm parameter is considered akey proxy for coral health, and differences in Fv/Fmbetween coral measurements are interpreted as achange in coral fitness (Jones et al., 2000; Wiedenmannet al., 2013). The effective quantum yield of PSII,FPSII =(Fm9-F)/Fm9, is determined via a saturation pulse mea-surement under a known actinic irradiance of photo-synthetic active radiation (PAR; Genty et al., 1989). Anestimate of the relative PSII-derived photosyntheticelectron transport rate is calculated as rETR = FPSII 3PAR (Ralph and Gademann, 2005), whereas determi-nation of the absolute ETR requires information aboutthe PSII absorption cross section (Szabó et al., 2014).When calculated over a range of actinic irradiancelevels, rETR versus irradiance curves (i.e. light curves)can be determined, which enables the calculation of themaximum electron transport rate (ETRmax) and thelight-use efficiency factor (a; i.e. the initial slope ofthe rETR-versus-irradiance curve). Measurement pro-tocols for the application of PAM on corals are well-described (e.g. Warner et al., 2010), and the maximumPSII quantum yield and rETR-versus-irradiance–curveparameters are frequently used to interpret the healthand photo-physiological acclimation state of Symbiodi-nium within the coral host (Ralph et al., 2005).However, the application of variable Chl fluores-

cence is based on the assumptions that all photosyn-thetic entities (cells/chloroplasts) are (1) equallyexposed to the incident actinic light levels, (2) equallyexposed to the measuring light (ML) and emitting flu-orescence equally, and (3) effectively saturated by thesaturation pulse (Schreiber et al., 1996; Serôdio, 2004).In other words, it is assumed that (1) each photosyn-thetic cell has identical fluorescence excitation/emission,and (2) the generated fluorescence from each cell hasequal probability to be detected by the fluorimeter.These assumptions are only met, if the light distribution

within the sample is homogenous, such as in opticallydilute algal cultures (e.g. Ting and Owens, 1992). Incontrast, most photosynthetic tissues exhibit strongscattering and absorption, leading to a heterogenousdistribution of irradiance within the sample (e.g.Serôdio, 2004; Evans, 2009; Oguchi et al., 2011; Szabóet al., 2014; Evans et al., 2017; Lichtenberg et al., 2017).For instance, steep light gradients exist in biofilms (Kühland Jørgensen, 1994), which can lead to an effectiveoverestimation of VPSII and ETR (Serôdio, 2004). Suchgradients can also affect measurements of the Chl a flu-orescence kinetics (Susila et al., 2004). Reabsorption offluorescence emission can pose a challenge for Chl afluorimetry in optically dense tissues (Naus et al., 1994;Bartosková et al., 1999). Amethod for correcting variablefluorescence measurements in optically dense algal me-dia under constant optical geometries (e.g. cuvettes) hasbeen proposed (Klughammer and Schreiber, 2015), butthis approach assumes a simple exponential light atten-uation (Lambert–Beer’s law), which is too simplisticfor light-scattering photosynthetic tissues (Kühl andJørgensen, 1994; Wangpraseurt et al., 2016a).Knowledge of the tissue inherent optical properties

(IOPs) allows us to predict light propagation by solvingthe radiative transfer equation. The IOPs are defined asthe probability of light absorption per infinitesimal pathlength (ma, [mm21]); the probability of light scatteringper infinitesimal path length (ms, [mm21]); and the an-isotropy of scattering (g), i.e. the average cosine ⟨cos u⟩ ofthe scattering angle (u), and the refractive index (n). Instrongly light-scattering samples, ms is combined with gto define the reduced scattering coefficient ms9 = ms $(12g; Jacques, 2013). The reduced scattering coefficientdescribes photon diffusion in a random walk of stepsizes of 1/ms9, where each step involves isotropic scat-tering. Recent progress in coral optics (Wangpraseurtet al., 2014b, 2016a; Swain et al., 2016) revealed thatcoral tissues and skeletons can be strongly light-scattering and that ms9 is variable between coral spe-cies. For instance, ms9 of coral skeletons can vary by oneorder of magnitude (Marcelino et al., 2013), thus sub-stantially affecting the amount of light that is back-scattered into the overlying algal layer (Marcelino et al.,2013; Wangpraseurt et al., 2016a). Thick-tissued coralscan have light-scattering host pigments (e.g. green flu-orescent protein [GFP]) situated on top of the algal layer(Lyndby et al., 2016;Wangpraseurt et al., 2017b). In sucha scenario, light is effectively scattered before it reachesSymbiodinium cells, leading to a strong surface en-hancement of scalar irradiance, E0 (= fluence rate;Lyndby et al., 2016).The absorption coefficient (ma) of corals is largely de-

pendent on Symbiodinium cell density and Chl a contentper cell (Wangpraseurt et al., 2016a). Algal densities incorals vary seasonally (Chen et al., 2005) and in response toenvironmental stress (Glynn, 1996). Symbiodinium densityaffects vertical light attenuation,where densely pigmentedcorals are characterized by steep light gradients, whileless-pigmented corals have a diffusely enhanced tissuelight environment (Wangpraseurt et al., 2017a). The

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photosynthetic yield of Symbiodinium can be verticallystratified within the coral tissue (Lichtenberg et al., 2016;Wangpraseurt et al., 2016b). During coral bleaching,microalgal symbionts experience photoinhibition (Warneret al., 1999) and such damage is likely more prominent inthe light-exposed top layers of coral tissue (Lichtenberget al., 2016; Wangpraseurt et al., 2016b). Corals thus rep-resent a challenging study organism for variable Chlfluorimetry; yet, to our knowledge, no studies have aimedat ground-truthing the central assumptions of PAMmeasurements on corals.

The use of natural coral samples for such studywould be easily contrived by variability of the IOPs ofindividual samples (e.g. changes in ma, ms9). To avoidthis variability, we developed optical analogs to coralsusing a biomedical tissue optics approach. Opticalphantoms are often created to solve problems related to

the propagation of light in scattering tissues, e.g. forcalculating the light dose in photodynamic therapy andcancer treatment (Pogue and Patterson, 2006). The op-tical response of human tissue is mimicked by opticalphantoms consisting of (1) a gel-like planar matrix (e.g.gelatin, agar, or agarose), (2) light-scattering particles(e.g. SiO2, TiO2, polystyrene microspheres), and (3)light absorbers (e.g. intralipid, India ink; Tuchin, 2007).

In this study, we created multiple planar hydrogelslabs to replicate the bulk optical properties of coralsand characterized their variable Chl fluorescence-derived parameters, including Fv/Fm, Y(II), and rETR(Supplemental Table S1). Specifically, we examined therole of light scattering by replicating three major coralcategories: (1) corals with strongly backscatteringskeletons (“skeleton” hydrogel), (2) corals with low-scattering skeletons (“transparent” hydrogel), and (3)

Figure 1. Coral tissue organization andartificial tissue design. A–C, Basic organi-zation of Scleractinic corals. A, Smallfragment of a faviid coral (scale bar = 1mm). B, Close-up of the cross section, re-vealing thewhite coral skeleton, the brownalgal layer on top of the skeleton, and theGFP like-pigment granules (“GFP”) on thecoral tissue surface (scale bar = 1 mm). C,Close-up of GFP granules (scale bar =200 mm). D–I, Coral-tissue–mimicking hy-drogels. Schematics of three-layer “GFP”(D), Two-layer “skeleton” (E), and two-layer“transparent” (F) designs. G–I, Respectivephotographs of thin cross sections of hy-drogels. J–M, Hydrogels for investigatingthe effect of changes in coral absorption. Jand K, High microalgal-density–tissue de-sign (J) and top-view photograph (K). L andM, Medium microalgal density hydrogel(L) and top-view photograph (M). N–P,Hydrogels for investigating the effect ofcoral stress. N, “Healthy” coral designwith two layers of Rhodomonas sp., O,“Stressed” coral design with one layer ofNannochloropsis sp. on top of one layer ofRhodomonas sp., P, “Bleaching” designwith one layer ofNannochloropsis sp. of areduced cell density on top of R. salina.

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Coral Optics and Variable Chlorophyll Fluorescence


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corals with light scattering in skeleton and tissue(e.g. due to GFP host pigments; “GFP” hydrogel). Wealso assessed the role of light absorption for Chl afluorimetry by creating hydrogels with differentmicroalgal densities and PSII efficiencies. Furthermore,we developed a light-propagation model (chlorophyllfluorescence Monte Carlo [Chf-MC] simulation) thatallows for prediction of the generated fluorescence anddetected fluorescence as a function of tissue opticalproperties. Although we focused on the widely appliedPAM method, it is also relevant for other variable Chlfluorescence methods such as fast repetition rate fluo-rimetry (Gorbunov et al., 2001). The optical phantomapproach (Fig. 1) and numerical models can easily bealtered to address identical questions in other light-scattering samples such as leaves and biofilms.

RESULTS

Effects of ML Settings and Light Scattering on VariableChl Fluorescence

Hydrogels with identical absorber density but dif-ferent light-scattering properties showed up to 5-folddifferences in F0 for the same ML intensity (Fig. 2,A–D). Highest F0 values were achieved for the skeletonhydrogel (F0 = 0.162; Fig. 2, B and D). ML intensity alsoaffected measurements of the maximal fluorescenceyield (Fm) and the calculation of Fv/Fm (Fig. 2, E and F).For ML = 3–4, Fv/Fm values were ;0.74 for all threecoral-mimicking hydrogels, while for ML # 3 and . 5,Fv/Fm values differed by up to 0.1 (Fig. 2F).The in vivo light microenvironment measured with

fiber-optic microsensors differed for the three coral-mimicking hydrogels, and photon E0 (400–700 nm) at

the hydrogel surface reached 109% (60.85 SE; n = 8) of thedownwelling photon irradiance, Ed (Supplemental TableS1) for the transparent hydrogel, 142% Ed (69 SE; n = 8) forthe skeleton hydrogel, and 244%Ed (612.3 SE; n= 8) for theGFP hydrogel (Fig. 3). The steepest light attenuation wasmeasured in the GFP hydrogel, and the lowest, in thetransparent hydrogel (Fig. 3). The top layer (0–750 mm) ofthe light-scattering GFP hydrogel (Fig. 1D) created a sub-surface maximum in E0 (at ;250 mm below the hydrogelsurface); this was followed by rapid light attenuationwithin the light-absorbing algal layer (750–1,500 mm). Forthe skeleton hydrogel, light attenuated to 125% Ed withinthe first 300 mm, after which light scattering by the un-derlying layer caused a subsurfacemaximumof;700mmfrom the hydrogel surface that reached 170% Ed (Fig. 3).Steady-state light curves revealed that the effective

quantum yield of PSII (VPSII) and the derived relativeelectron transport rates (rETR) differed between thethree coral-mimicking hydrogels (Fig. 4), where theshape of the curves depended on the light field pa-rameter used to quantify the actinic light level. Whenplotted as a function of Ed, the VPSII was higher for theGFP hydrogel than for the skeleton and transparenthydrogel, and this difference was larger for rETR cal-culations. For instance, at Ed = 1,000 mmol photons m22

s21, rETR was ;1.5 times higher for the GFP versus theother two hydrogels (Fig. 4C). Correction of ETR forin vivo E0 led to similar patterns between the GFP andskeleton hydrogel, which now both showed higher rETRvalues than the transparent hydrogel (Fig. 4D).

Effects of Light Absorption on Variable Chl Fluorescence

We constructed hydrogels with identical scatteringproperties but different light absorption properties

Figure 2. Effect of light scattering onvariable Chl fluorescence parameters ofdark-acclimated coral-tissue–mimickinghydrogels. A–C, Example images ofminimal fluorescence yields (F0) forML = 4, showing GFP (A), skeleton (B),and transparent (C) hydrogels. The whitecircle shows the area over which F0 wasintegrated. D–F, Effect of measuring lightintensity on F0 (D); Fm, maximal fluores-cence yield (E); and the maximum PSIIquantum yield, Fv/Fm (F). Note thatno measurements are shown at ML . 4(= 0.8 mmol photons m22 s21) andML.8 (= 1.6 mmol photons m22 s21) forskeleton and transparent hydrogels, re-spectively, due to indications of actiniceffects in these hydrogels at higher MLlevels.


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(Fig. 1, J–M). Surface photon E0 (400–700 nm) in thehydrogel with medium algal density (1.0 3 106 cellscm22) was ;1.4-fold higher than that in the hydrogelwith high algal density (3.53 106 cells cm22; 205% Ed60.27 SE versus 146% Ed 6 0.51 SE; n = 4). This differenceincreased as a function of vertical depth, and mea-surements at depths of .2,000 mm showed up to3-fold enhanced E0 values in the medium- versus high-algal–density hydrogel (Fig. 5A).

Microalgal density had a significant effect on esti-mates of the maximum quantum yield, where hydro-gels with 3.5 3 106 cells cm22 showed ;0.04 unitshigher Fv/Fm values than hydrogels with 1.03 106 cellscm22; Student’s t test: t(6) = 11.25, P , 0.01 (Fig. 5B).Likewise, microalgal density affected rETR, and atEd = 500 mmol photons m22 s21, rETR was ;2.7-fold

higher for the high- versus medium-algal–density hy-drogel (rETR = 41.1 6 0.1 versus 14.7 6 0.8; n = 4;Fig. 5C). Additionally, rETR values were corrected forthe in vivo E0, as determined with E0 microsensors foreach respective photic zone (i.e. the hydrogel layer thatcontained microalgal cells). Correction for in vivo E0slightly improved this discrepancy, but calculations ofrETRmax and the light-use efficiency factor (a) were still;1.35 (rETRmax = 80 versus 59) and 2-fold higher(a = 0.22 and 0.11) for high- versusmedium-algal–densityhydrogels.

Effects of Bio-optical Properties and Simulated CoralBleaching on Variable Chl Fluorescence

Hydrogels mimicking a stressed but not bleachedcoral, i.e. harboring a top layer with high algal densitybut low photosynthetic potential, showed low rETR andonset of photoinhibition (Fig. 6). However, hydrogelsmimicking a partially bleached and stressed tissue, i.e.harboring a top layer with reduced microalgal densityand photosynthetic potential, showed moderate rETRwith rETRmax = 38 (at Ed . 500 mmol photons m22 s21)and no signs of photoinhibition (Fig. 6).

DISCUSSION

Variable Chl fluorimetry is a key tool for probingphotosynthesis in vivo (Baker, 2008). However, the as-sumptions underlying the calculation of variable Chlfluorescence parameters might not be fulfilled whenmeasuring externally on highly stratified and densephotosynthetic tissues, such as corals, biofilms andplant tissues (Serôdio, 2004; Evans, 2009; Szabó et al.,2014). Our results showed that F0, Fm, and Fv/Fm wereaffected by the scattering properties of coral-mimickinghydrogels (Fig. 2). In a first approximation, we candescribe the detected F0 signal by using three simpleterms: (1) the ML intensity incident on an algal cell,

Figure 3. In vivo light microenvironment in coral-tissue–mimickinghydrogels with different scattering properties. Photon E0 of PAR(400–700 nm) was normalized to the Ed of PAR and plotted against thevertical depth (mm) of the coral mimics. The algal layer is distributedbetween depth = 0–750 mm for the transparent (red) and skeleton (blue)mimic, while the algal layer is between 750 and 1,500 mm in the GFP(green) mimic. Four replicate gels were measured at two random spots(total n = 8 6 SE).

Figure 4. Effect of light scattering on the effectivequantum yield VPSII (A and B) and rETR (C and D)of coral-tissue–mimicking hydrogels. MeasuredVPSII and calculated rETR were plotted as a func-tion of the Ed (A and C) and with the correctedin vivo E0 (B and D). Symbols with error barsrepresent means6 SE (n = 5 biological replicates).





i.e. fluorescence excitation; (2) the fluorescence emis-sion per cell, which is governed by the biophysicalproperties of the cell, such as Chl a content and darkacclimation (Warner et al., 2010); and (3) the probabil-ity that such emitted fluorescence is detected bythe imaging instrument (fluorescence escape; seeSupplemental Text). Because algal cells can collect lightfrom all directions, the excitation term is not describedby the downwelling irradiance of the ML, but by the E0(= fluence rate) ofML (Kühl et al., 1995), which in turn isaffected by the tissue optical properties (Jacques, 2013;see optical simulations in Supplemental Text). In thefirst experiment (Fig. 1), we kept ma constant whilemodulatingms9, which created characteristic differencesin the light microenvironment between three differentcoral tissue mimics (Fig. 3). The enhancement of photon

irradiance at the tissue surface of the skeleton hydrogelwas due to the strong backscattering (Fig. 3). In con-trast, light attenuation was described by a simple ex-ponential attenuation for the transparent hydrogel(Fig. 3).The observed differences in the F0 signal between the

skeleton and transparent hydrogels for a given ML in-tensity were due to two mechanisms (Fig. 7). Firstly, thefluence rate of the ML was enhanced in the absorbinglayer (0–750-mm depth) for the skeleton versus thetransparent hydrogel (Fig. 3). The higher fluence rate ledto an increased chance of photon absorption and thushigher levels of fluorescence generation (see opticalsimulations in Supplemental Fig. S2). Secondly, al-though an individual algal cell acts as an isotropic pointsource (i.e. emitting fluorescence equally well in all di-rections; Schreiber, 2004), the detected fluorescence sig-nal depends on the propagation of fluorescent light fromthis point source, through the tissue toward the fluo-rimeter (Welch et al., 1997). Because intact corals aretypically monitored externally using a fiber or camera inbackscattering configuration, the reflectivity of the skel-eton controls the upwelling fluorescence toward thedetector. For the transparent hydrogel, the downwellingfluorescence (Fd) was essentially lost, while backscatter-ing by the skeleton hydrogel led to an effective redirec-tion of the otherwise-lost Fd (Fig. 7).For the GFP hydrogel, the light-scattering elements

were placed on top of the light-absorbing algal layer(Fig. 1). Scattering diffuses the incident light, and diffuselight penetrates less into biological tissue than collimatedlight (Tuchin, 2007; Wangpraseurt and Kühl, 2014;Supplemental Fig. S2). Thus, although intense scatteringin the top tissue layer would enhance the chance of flu-orescence emission and subsequent upwelling of gen-erated fluorescence, it also leads to a steep attenuation ofthe MLwithin the algal layer (Lyndby et al., 2016; Fig. 3;Supplemental Fig. S2). The vertical attenuation of E0(400–700 nm) within the light-absorbing layer was de-scribed according to Lambert–Beer’s law for the

Figure 5. Effect of coral light absorption on thelight microenvironment and variable Chl fluores-cence measurements. Photon E0 (400–700 nm)was normalized to the Ed and plotted against thevertical depth (mm) in the coral mimics. A–D, Thealgal layer is distributed between 0- and 750-mmdepth (A). The maximum quantum yield of PSII,Fv/Fm (B). Relative electron transport rates (rETR)calculated as a function of the Ed (C) and correctedfor in vivo E0 (D). All measurements were per-formed in coral hydrogels mimicking high algaldensity (3.5 3 106 cells cm22) and medium algaldensity (1.03 106 cells cm22). Symbols representmeans6 SE (n = 4 biological replicates) in (A to C).The curve fit shown in (D) was the best fit to theexperimental data (R2 = 0.98 and 0.80) yieldingvalues of ETRmax = 80 and 59, and a = 0.22 and0.11 for the high- andmedium-algal–density coralhydrogels, respectively.

Figure 6. Combined effects of bio-optical properties and simulatedcoral bleaching on measured rETR. Hydrogels mimicking healthy tissuecontained two dense layers of R. salina (43 106 cells cm22); hydrogelsmimicking stressed tissue contained one dense layer of Nanno-chloropsis oculata (23 106 cells cm22) and one dense layer of R. salina(2 3 106 cells cm22); and hydrogels mimicking bleached tissue con-tained one layer ofN. oculata at low density (0.33 106 cells cm22) andone dense layer of R. salina. Data are means 6 SE (n = 2–3 hydrogelreplicates).


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transparent and GFP hydrogels, yielding an attenuationcoefficient that was 1.4-fold higher for the GFP versustransparent hydrogel (1.7 mm21 and 1.2 mm21, respec-tively; data not shown). Together, these results exem-plify that the F0 signal can be strongly affected by thescattering properties of the photosynthetic tissue and thespatial arrangement of light-scattering versus light-absorbing elements in the tissue.

Our results suggest that coral light-scattering mod-ulated (1) the ability of the saturation pulse to fullysaturate PSII, (2) the likelihood for actinic effects duringML probing, and (3) the operational volume that isprobed during Fv/Fm measurements (Fig. 7). Fv/Fmvalues for the skeleton hydrogel were greater than 0.1units (dimensionless) higher than for the transpar-ent and GFP hydrogels when probed with low ML in-tensities (Fig. 2). This likely indicates that skeletonbackscattering facilitated the full saturation of all pho-tosynthetic cells within the tissue volume, creating ahomogenous light environment (Fig. 3; Enriquez et al.,2005). In contrast, the steep light gradient in the GFPhydrogels led to rapid attenuation of the saturationpulse light (Fig. 3), leaving only ;50% of Ed in thelowest layers of the photic zone. In such a scenario, thelikelihood of incomplete PSII saturation increases withvertical tissue depth (Serôdio, 2004; Lichtenberg andKühl, 2015), thus inducing lower Fm values for deepertissue layers. Optical simulations usingChf-MC showedthat increased tissue scattering (from ms9 = 1–10 mm21)reduced the tissue depth for which PSII was fullysaturated by .50% (for ma = 0.1–1 mm21; seeSupplemental Text; Supplemental Fig. S4B). Chf-MCcan serve as an initial point of reference for assessingthe likelihood of incomplete PSII saturation in thesample (Supplemental Fig. S4B). Other approaches,including the multiphase flash method, which uses;1-s–long multiphase flashes to saturate PSII, couldprovide additional instrument improvements that

reduce the likelihood of incorrect Fm9 estimates(Loriaux et al., 2013).

Optical scattering affected the ML intensity withinthe photosynthetic tissue and thus the likelihood of MLinducing actinic effects (Supplemental Fig. S5). The re-lationship between ML used to probe for F and Fm canbe nonlinear at higher light intensity settings, leading toa decrease in the Fm/F and thus a reduction in Fv/Fmvalues (Ting and Owens, 1992). Such nonlinearity iscaused by instrument optics, and although this hasbeen tested only for the PAM 101 (Walz; Ting andOwens, 1992), it is likely that the same artifacts con-tributed to the observed decrease in Fv/Fm for higherML settings when using the I-PAM system (Fig. 2F).

Light scattering affected the operational volume ofthe PAM instrument, i.e. the contribution of fluores-cence from different vertical tissue depths to observedfluorescence from the sample (Supplemental Figs. S2and S4A). For samples containing photosynthetic cellswith variable intrinsic PS II efficiency, differences in theoperational volume could lead to a complex mixture offluorescence signals from different tissue depths(Fig. 6). Such mixed fluorescence signals could theo-retically be decomposed by calculating the depth-specific contribution to observed fluorescence usingChf-MC (Supplemental Text). However, it is a prereq-uisite that the intrinsic properties of PS II efficiency areknown (Klughammer and Schreiber, 2015).

The VPSII and rETR differed between the three light-scattering coral mimics (Fig. 4), and the rETR of the GFPhydrogel was greater than that of the skeleton andtransparent hydrogels when calculated with Ed as ameasure of actinic light (Fig. 4C). Using the average E0within the photic zone as a measure of actinic light re-duced the difference in rETR between the GFP andskeleton hydrogels, and the two light curves wereidentical for E0 , 400 mmol photons m22 s21. Thissuggests that for low actinic light levels, measurements

Figure 7. Propagation of PAM-based ML in biological tissues with different scattering coefficients. A and B, Excitation (blue) andChl fluorescence (red) for transparent hydrogel (A) and skeleton hydrogel (B). The optical properties of the light absorbing top layer(layer 1) are constant (i.e. identical algal density and biophysical properties of an algal cell) for both gels, but layer 2 is eithertransparent or light-scattering. For the transparent hydrogel, ML absorption by an algal cell (s) is a function of the primary incidentbeam (solid blue line), while indirect light (dotted blue lines) is lost through the transparent layer. For the skeleton hydrogel,indirect light is redirected via backscattering by layer 2. Such scattering enhances the chance of ML absorption and thus leads togreater fluorescence emission (bold red arrows). Fluorescence emission is an isotropic process but the propagation of fluorescentlight is affected by tissue optical properties. For the transparent hydrogel, only primary upwelling fluorescence (Fu1) contributes tothe detected fluorescence signal, while for the skeleton hydrogel the Fd is redirected and adds to the upwelling fluorescence (Fu2).C, A steep light gradient (green line) leads to an underrepresentation of fluorescence detection from lower cell layers compared toan homogenous light environment (black line), given that the operational volume (dotted lines) from which fluorescence iscollected is a function of the theoretical instrument detection limit (Elimit), which is modulated by the in vivo photon E0.












of the average in vivo E0 within the entire photic zonecan, to some extent, correct for rETR estimates fromcorals with different degrees of light scattering(Marcelino et al., 2013). For higher actinic irradiance,such correction was not successful, and rETR wasgreater for the GFP hydrogel than for the skeleton hy-drogel (Fig. 4D). The enhancement of rETR in the GFPscenario was likely due to the presence of the steep lightgradient (Fig. 3), ensuring that the irradiance incidenton lower cell layers enabled optimal conditions forphotosynthesis.We found that both Fv/Fm and rETR were reduced for

hydrogels with lower microalgal cell density (Fig. 5,B–D). Lower microalgal cell density decreased lightabsorption, which enhanced the internal light micro-environment (Fig. 5A) and consequently loweredVPSII.Given that rETR is calculated by multiplying VPSII withPAR, differences in the internal light microenvironmentcan lead to substantial artifacts in the calculation of rETR(Fig. 4C). Additionally, correct calculations of absoluteETR require knowledge of the absorption factor, whichin itself is affected by optical scattering (SupplementalFig. S6; Szabó et al., 2014; Wangpraseurt et al., 2014c).The Fv/Fm measurements were performed for hydro-

gels with different absorber densities (at ML = 2), which

yielded the same F0 but higher Fm values for the hydrogelwith enhanced microalgal cell densities. Although theexact mechanisms underlying these differences are un-clear, these first measurements have important implica-tions for coral science, given thatmicroalgal cell density ishighly variable between coral species (Drew, 1972) andwithin a species due to factors such as differences in lightacclimation (Falkowski and Dubinsky, 1981), seasonalfluctuations (Chen et al., 2005), and environmental stress(e.g. coral bleaching; Weis, 2008). Bleached corals canexhibit an approximate doubling of the fluence ratewithin coral tissues compared to healthy corals (Swainet al., 2016; Wangpraseurt et al., 2017a), and such changein the internal light microenvironment was successfullymimicked with our hydrogels with different absorberdensities (Fig. 5A). Thus, differences in Fv/Fm and rETRbetween coral individuals with different algal cell den-sities should be interpreted with caution, and might, tosome extent, reflect different optical properties as well asdifferences in photophysiological status.PAM-based measurements are often used to assess

changes in photochemical efficiency during coralbleaching (Jones et al., 2000; Rodriguez-Roman et al.,2006). During such environmental stress, both the op-tical properties of the coral and VPSII of the algal

Table 1. Material properties of coral-tissue–mimicking hydrogels

The algal species was R. salina unless indicated otherwise. The / indicates that no hydrogel layer was fabricated.

Experiment Hydrogel Properties Top Layer Mid Layer Base Layer

Coral scattering GFP Matrix ASW+ 1% agarose ASW+ 1% agarose ASW+ 1% agaroseSiO2 4% 0% 5%Algae 0 cells cm22 2.5 3 106 cells cm22 0 cells cm22

Thickness 0.75 mm 0.75 mm 2.5 mmSkeleton Matrix / ASW+ 1% agarose ASW+ 1% agarose

SiO2 / 0% 15%Algae / 2.5 3 106 cells cm22 0 cells cm22

Thickness / 0.75 mm 2.5 mmTransparent Matrix / ASW+ 1% agarose ASW+ 1% agarose


Thickness / 0.75 mm 2.5 mmCoral absorption High Matrix / ASW+ 1% agarose ASW+ 1% agarose


Thickness / 0.75 mm 2.5 mmMedium Matrix / ASW+ 1% agarose ASW+ 1% agarose

SiO2 / 1% 5%Algae / 1 3 106 cells cm22 0 cells cm22

Thickness / 0.75 mm 2.5 mmCoral stress Stressed Matrix ASW+ 1% agarose ASW+ 1% agarose ASW+ 1% agarose

SiO2 0% 0% 5%Algae 2 3 106 cells cm22 (N. oculata) 2 3 106 cells cm22 0 cells cm22

Thickness 0.75 mm 0.75 mm 2.5 mmStressed and bleached Matrix ASW+ 1% agarose ASW+ 1% agarose ASW+ 1% agarose

SiO2 0% 0% 5%Algae 0.3 3 106 cells cm22 (N. oculata) 2 3 106 cells cm22 0 cells cm22

Thickness 0.75 mm 0.75 mm 2.5 mmHealthy Matrix ASW+ 1% agarose ASW+ 1% agarose ASW+ 1% agarose

SiO2 0% 0% 5%Algae 2 3 106 cells cm22 2 3 106 cells cm22 0 cells cm22

Thickness 0.75 mm 0.75 mm 2.5 mm


Wangpraseurt et al.





symbiont undergo changes over time (Iglesias-Prietoet al., 1992; Wangpraseurt et al., 2017a). Cells fromtop layers exposed to supra-optimal irradiance arelikely to be stressed to a greater extent than cells fromdeeper layers (Lichtenberg et al., 2016; Wangpraseurtet al., 2016b). We found that changes in cell density andspatial differences inVPSII lead to amisinterpretation ofvariable fluorescence signals. For instance, hydrogelsmimicking stressed corals by containing a top layerwith normal algal cell density but with reduced pho-tochemical efficiency, showed much lower rETR thanhydrogelsmimicking bleached corals (see Fig. 1; Table 1for hydrogel configurations). The high density of thelow-performing cells in the top hydrogel layer limitedthe operational volume in the stressed-coral scenario. Incontrast, a reduction in the cell density of top layersenhanced the operational volume to measurements ofwell-performing lower cell layers, effectively enhancingrETR (Fig. 6).

This experimental study has shown that coral opticalproperties can contrive the interpretation of PAM-basedfluorescence measurements. The next step is to developtheoretical models that predict the likelihood of opticalartifacts in a sample and ideally correct for such artifacts.We have taken first steps by developing aChf-MCmodelfor photosynthetic tissues that allows for predicting thelikelihood of PSII saturation, the actinic effects of ML,and the operational volume anddepthdistribution of thecollected fluorescence (see Supplemental Text). Thesimulations can be adjusted to account for absorption ofemitted fluorescence. The optical model is limited to aone-layer system, which is best applicable to structurallysimple photosynthetic tissues. Future efforts should in-clude the modeling of Chl a fluorescence in multipletissue layers and in a 3D architecture (Fang, 2010). Theuse of tissue phantomswith defined optical properties isa promising approach to examine and qualify the pre-cision of variable Chl fluorimetry in plant tissues andbiofilms. Quantification of inherent optical parameters inphotosynthetic tissues might furthermore lay the ex-perimental basis for better light-propagation models(Jacques, 1998; Mycek and Pogue, 2003; Swartling et al.,2003), whichwill enable optimal measurement protocolsand instrument configurations.

MATERIALS AND METHODS

Experimental Approach

Experiment 1 was aimed at understanding how differences in coral lightscattering affect variable Chl fluorescence measurements in identical algalpopulations, i.e. the same algal culture at identical cell densities. Thick-tissuedfaviid corals often have light-scattering GFP granules on top of the light-absorbing algal layer, which is also subject to light scattering from the coralskeleton (Lyndby et al., 2016; Wangpraseurt et al., 2017b). The “GFP” hydrogelconsisted of a three-layer system with a thin (750 mm) light-scattering upperlayer (mimicking GFP scattering), a light-absorbing layer (algae), and a thicklight-scattering base layer (skeleton; Fig. 1; Table 1). However, not all coralsfollow this tissue arrangement, and other corals do not have light-scatteringGFP granules. In thin-tissued corals, light scattering can be dominated by thebackscattering properties of the skeleton (Enriquez et al., 2005). To mimic thisoptical configuration, the “skeleton hydrogel” was prepared to be identical to

the GFP hydrogel but without the top GFP layer. In the “transparent” hydrogel,the light-scattering skeleton layer was replaced with a 1% agarose gel layer (seeschematics in Fig. 1).

In Experiment 2, we examined how changes in coral light absorption mightaffect variable Chl fluorescence measurements. In healthy corals, algal densitiescan vary seasonally from ;1.5–6 3 106 cells cm22 (Chen et al., 2005). Wetherefore created hydrogels with microalgal densities of 3.5 3 106 cells cm22

(“high density”) and 106 cells cm22 (“medium density”; Fig. 1; Table 1).Experiment 3mimicked a coral stress scenario to explore systematically, how

combined changes in microalgal density and photophysiology affect variableChl fluorescence measurements in corals. We mimicked a “stressed coral,”where top algal layers have reduced photosynthetic quantum yields, whilelower layers are operating with high yields. For this, we created a light-absorbing algal layer with Nannochloropsis sp. (thickness = 750 mm, algaldensity = 2 3 106 cells cm22) on top of a Rhodomonas salina layer(thickness = 750mm, algal density = 23 106 cells cm22), exhibiting a Fv/Fm= 0.7.The double layer was placed on top of the light-scattering skeleton hydrogel.We also created a hydrogel mimicking a “stressed and bleached coral” by usinga reduced density of Nannochloropsis (3 3 105 cells cm22) in the top layer(Table 1). The “healthy coral” consisted of two layers of R. salina (each750-mm–thick andwith an algal density of 23 106 cells cm22) on top of the high-backscatter hydrogel.

Hydrogel Fabrication

To develop a hydrogel with light-scattering properties similar to those ofcorals, we used a protocol developed for human tissues (Wagnières et al., 1997),where hydrogels with tissue-like properties for visible light were constructedwith a reduced scattering coefficient of ms9 = 1.5–3.4 cm21 between 400 and 450nm (Wagnières et al., 1997). The reduced scattering coefficient of coral skeletonsis highly variable, with ms9 ranging between 3 and 140 cm21 (Marcelino et al.,2013; Swain et al., 2016), and the optical properties of living coral tissue ap-parently exhibit a similar variability (Wangpraseurt et al., 2016a). Given thevariability in coral scattering, we did not aim to quantify in detail the reducedscattering coefficient of our hydrogel, but rather to develop a hydrogel that fallswithin the bulk part of light scattering observed in corals.

The developed hydrogels were composed of (1) a gel-like matrix, (2) light-scattering particles, and (3) light-absorbing algae. We used a 1% agarose (Ul-trapure low-melting-point agarose; Thermo Fisher Scientific) solution in filtered(0.2 mm) seawater, which is rather optically clear in the visible part (Wagnièreset al., 1997). The agarose was prepared by heating the agarose–seawater mix-ture in a microwave, ensuring that the solution was clear and free of gas bub-bles. The low agarose concentration ensured that the hydrogel wasmechanically similar to soft tissues such as coral tissue and exhibited gas dif-fusion properties similar to seawater. Light scattering was achieved by mixingthe hydrogel with defined concentrations of silicon dioxide particles (sizefraction: 99% between 0.5 and 10 mm and 80% between 1 and 5 mm; SigmaAldrich) to achieve the desired scattering (see Table 1; Wagnières et al., 1997).Such silicon dioxide particles are nontoxic to microalgae and cyanobacteria(Dickson and Ely, 2013) and exhibit a good broadband scattering of white lightat the chosen particle size distribution (Wagnières et al., 1997).

After adding the silicon dioxide particles, the agarose solution was vor-texed for ;30 s, ensuring a homogenous distribution of the light-scatteringparticles. The solution was cooled down to ;30°C, after which the micro-algae were added at defined concentrations (see Fig. 1). We selected twotypes of light-absorbing microalgae, Symbiodinium sp. and Rhodomonas sal-ina. Preliminary experiments were performed with Symbiodinium sp., whilethe main experiments were performed with R. salina, which was similar incell size (8–10 mm) and was easier to grow and maintain in a healthy state.For both algal species, PAM-relevant blue light absorption is dominated byChl a along with additional contributions by Chl c (Ka�na et al., 2013;Wangpraseurt et al., 2014a). However, for the purpose of this study, the typeof algal strain is largely irrelevant, as we investigated the effect of basic light-scattering mechanisms on variable Chl a fluorescence measurements. Thesolution of agarose, SiO2 and microalgae was transferred rapidly into petridishes (diameter 35 mm, height 10 mm), where they were left to cure for atleast 30 min.

Apparent Optical Properties of Tissue Hydrogels

The light microenvironment of coral mimics was measured in vivo using E0

microsensors (Rickelt et al., 2016) as described inKühl (2005) andWangpraseurt






et al. (2012). Briefly, E0 probes were constructed with a spherical isotropic light-collecting tip of ;80 mm (Rickelt et al., 2016). The probe was mounted on amotorized micromanipulator (PyroScience) controlled by a PC running dedi-cated software (Profix; PyroScience) and oriented at an angle of;45° relative tothe vertically incident light. Measurements of spectral E0 were performed fromthe surface of the hydrogel in vertical step sizes of 80 mm. The spectral E0 wasthen integrated over the spectral range of PAR (400–700 nm) and expressed inpercentage of the incident downwelling irradiance (Ed; Kühl, 2005).

Variable Chl Fluorescence Imaging

We used a variable Chl fluorescence imaging system (Mini I-PAM, Walz;Ralph et al., 2005). The I-PAM was equipped with blue light-emitting diodes(460 nm) and delivered a maximum saturation pulse intensity (SP = 10) of.2,700 mmol photons m22 s21. The ML intensity was calibrated for ML1–12 atfrequency 1 using a fast data logger (ULM-500; Walz) connected to cosine-corrected PAR sensor, yielding an average photon irradiance output of 0.3,0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2.1, 2.3, and 2.6 mmol photons m22 s21. TheI-PAM system was mounted on a heavy stand and the hydrogels illuminatedvertically from above. Initial measurements were performed to calibrate thefocal distance and aperture settings between the camera head and hydrogelsamples, after which, focus and aperture were fixed. All measurements wereperformed with the hydrogels within petri dishes that were placed on topof a black light-absorbing surface. Measurements were performed in adarkened room.

Measurements were performed with dark-acclimated samples (after;30 min in darkness) to examine differences in the background fluorescencebetween the “transparent,” “skeleton,” and “GFP” hydrogels. For each mea-surement, the PAM settings were fixed (ML = 4, gain = 1, damping = 2, fre-quency = 1). Measurements were also performed to examine the effects ofchanges in ML intensity on F0, Fm, and the calculated Fv/Fm. Each sample wasmeasured at a range of ML intensities (ML settings ranging between 1 and 12),beginning with the lowest ML intensity setting. For each ML intensity, a satu-ration pulse was applied for 720 ms (intensity setting = 10, yielding 2,700 mmolphotons m22 s21), and a resting period of 1 min was used between saturationpulses.

We also examined the effects of light scattering on samples illuminated withdefined actinic irradiance levels, i.e. light-acclimated samples. Steady-state lightcurves of rETR versus photon irradiance were measured over a range of actinicincident photon irradiances of PAR (400–700 nm) ranging from 0 to;1,600 mmol photons m22 s21. For each light curve, the sample was dark-acclimated for ;15 min before a light curve was measured using an exposuretime of 5 min at each irradiance level. Before starting the steady-state lightcurves, theML intensity was adjusted such that F0 for the different coral mimicsyielded comparable values (i.e. F0 = 0.08). Likewise, ML intensity was adjustedsuch that F0 = 0.08 for Experiments 2 and 3 (Fig. 1).

Data Analysis

The effective photosynthetic quantum yield of PSII was calculated asVPSII =(Fm9-F)/Fm9 and relative photosynthetic PSII electron transport rates were cal-culated as rETR = PAR 3 VPSII (Baker, 2008). Calculated rETR versus photonirradiance curves were fitted with an exponential function (Webb et al., 1974) toestimate the maximal relative PSII electron transport rates (rETRmax) and thelight-use efficiency factor, a. Nonlinear curve fitting was performed in Ori-ginPro (9.3; OriginLabs) using a Levenberg-Marquart least-squares fittingalgorithm.

Optical Simulations

A probability light distributionmodel was developed to calculate the depth-dependent generation and escape of Chl fluorescence (Chf-MC). Details of themodel and optical simulations can be found in Supplemental Text,Supplemental Figures S1 to S6, and Supplemental Table S2.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Schematic of photon energy flow in PAM-basedvariable Chl fluorescence measurements.

Supplemental Figure S2. Chf-MC model simulating the penetration of ML.

Supplemental Figure S3. Chf-MC model investigating the effect of reab-sorption on observed fluorescence.

Supplemental Figure S4. Chf-MC model calculating the penetration depthof the generated fluorescence.

Supplemental Figure S5. Chf-MC model calculating the percent of photo-synthetic tissue overexposed by ML.

Supplemental Figure S6. Two-layer Monte Carlo simulation of light ab-sorption by microalgal cells for the skeleton hydrogel showing the effectskeletal backscattering on the tissue absorption factor.

Supplemental Table S1. Abbreviations.

Supplemental Table S2. The optical properties assumed for the excitationand fluorescence wavelengths..

Supplemental Text. Chf-MC model to simulate the effect of changes inlight scattering and pigment density on calculations of the PSII maxi-mum quantum yield (Fv/Fm).

ACKNOWLEDGMENTS

We thank Sofie Jakobsen for excellent technical assistance, and Lars Rickeltfor manufacturing scalar irradiance microprobes.

Received October 15, 2018; accepted January 16, 2019; published January 28,2019.

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Optical Properties of Corals Distort Variable Chlorophyll ... · Optical Properties of Corals Distort Variable Chlorophyll Fluorescence Measurements1 Daniel Wangpraseurt,a,b,c,2 Mads