Light Sheet Microscopy Imaging of Light Absorption andPhotosynthesis Distribution in Plant Tissue1

Mads Lichtenberg,a,2,3 Erik C.L. Trampe,a,2 Thomas C. Vogelmann,b and Michael Kühla,c,3

aMarine Biological Section, Department of Biology, University of Copenhagen, 3000 Helsingør, DenmarkbDepartment of Plant Biology, University of Vermont, Burlington, Vermont 05405cClimate Change Cluster, University of Technology Sydney, Ultimo New South Wales 2007, Australia

ORCID IDs: 0000-0002-0675-4554 (M.L.); 0000-0003-0697-5397 (T.C.V.); 0000-0002-1792-4790 (M.K.).

In vivo variable chlorophyll fluorescence measurements of photosystem II (PSII) quantum yields in optically dense systems arecomplicated by steep tissue light gradients due to scattering and absorption. Consequently, externally measured effective PSII quantumyields may be composed of signals derived from cells differentially exposed to actinic light, where cells located deeper inside tissuesreceive lower irradiance than cells closer to the surface and can display distinct photophysiological status. We demonstrate howmeasured distributions of PSII quantum yields in plant tissue change under natural tissue light gradients as compared withconventionally measured quantum yields with even exposure to actinic light. This was achieved by applying actinic irradianceperpendicular to one side of thallus cross sections of the aquatic macrophyte Fucus vesiculosuswith laser light sheets of defined spectralcomposition, while imaging variable chlorophyll fluorescence from cross sections with a microscope-mounted pulse amplitude-modulated imaging system. We show that quantum yields are highly affected by light gradients and that traditional surface-basedvariable chlorophyll fluorescence measurements result in substantial underestimations and/or overestimations, depending on incidentactinic irradiance. We present a method for using chlorophyll fluorescence profiles in combination with integrating spheremeasurements of reflectance and transmittance to calculate depth-resolved photon absorption profiles, which can be used to correctapparent PSII electron transport rates to photons absorbed by PSII. Absorption profiles of the investigated aquatic macrophyte weredifferent in shape from what is typically observed in terrestrial leaves, and based on this finding, we discuss strategies for optimizingphoton absorption via modulation of the structural organization of phytoelements according to in situ light environments.

Estimating photosynthetic parameters using variablechlorophyll fluorescence techniques has become in-creasingly popular due to its ease of use and noninva-sive nature. The basic fluorescence signals of open andclosed reaction centers change according to actinic ir-radiance and are powerful monitors of the statusand activity of the photosynthetic apparatus (Baker,2008). Most measurements of variable chlorophyllfluorescence in complex plant tissues, and in othersurface-associated cell assemblages like biofilms andsediments, rely on external measurements with fiber-optic

or imaging fluorimeters under the assumptions that (1)different cells are subjected to the same amount of mea-suring light and actinic irradiance, (2) saturating pulses areindeed saturating all cells, and (3) thefluorescence detectedis emitted equally from all sampled cells (Serodio,2004). These assumptions are influenced by the opticaldensity of the sample, where optical dilute refers to anegligible or only moderate light attenuation througha sample (e.g. a dilute algal suspension or plant tissuewith only a few cell layers), while optically dense samplessuch as algal biofilms and thicker plant tissues absorball, or most, of the incident light. As a result, the as-sumptions are usually valid in optically dilute samples(Klughammer and Schreiber, 2015), whereas steep lightgradients in densely pigmented tissues or algal biofilmswill distort the measurements of maximal and effectivePSII quantum yields. Cells located deeper inside tissueswill receive less actinic irradiance than cells close to thesurface. Thus, externally integrated measurements ofvariable chlorophyll fluorescence contain a complexmixture of signals originating from different layers inthe structure exposed to different levels of measuringand actinic light, and the actual operational depth ofsuch measurements remains unknown. This inherentlimitation of such measurements can lead to light-dependent overestimations of effective PSII quantumyields of up to 40% (e.g. in microphytobenthic as-semblages; Serodio, 2004).

1 This study was supported by a Sapere-Aude Advanced grantfrom the Danish Council for Independent Research/Natural Sciences(M.K.) and grants from the Carlsberg Foundation (M.K.).

2 These authors contributed equally to the article.3 Address correspondence to mads.lichtenberg@bio.ku.dk or

mkuhl@bio.ku.dk.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:Michael Kühl (mkuhl@bio.ku.dk).

M.L., E.C.L.T., T.C.V., and M.K. designed the research; M.L. andE.C.L.T. performed experiments; T.C.V. and M.K. contributed newanalytical tools; M.L., E.C.L.T., T.C.V., and M.K. analyzed data;M.L. and E.C.L.T. wrote the article with contributions fromT.C.V. and M.K.

www.plantphysiol.org/cgi/doi/10.1104/pp.17.00820

Plant Physiology�, October 2017, Vol. 175, pp. 721–733, www.plantphysiol.org � 2017 American Society of Plant Biologists. All Rights Reserved. 721 www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from

Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Previous efforts to describe the internal gradients ofphotosynthetic efficiencies have used microfiber-based pulse amplitude modulation (PAM) techniques(Schreiber et al., 1996), revealing distinct differences be-tween such internal and external variable chlorophyllfluorescence measurements (Oguchi et al., 2011). An-other challenge is to quantify the internal light gradi-ents to estimate the total actinic light exposure indifferent tissue layers (i.e. the scalar irradiance). Thescalar component becomes increasingly important indeeper tissue layers as light becomes progressivelymore diffuse due to multiple scattering (Kühl andJørgensen, 1994). This can be measured with fiber-opticscalar irradiance microprobes (Kühl, 2005; Rickelt et al.,2016), which collect light isotropically via a small (30–150 mm wide) spherical tip cast on the end of a taperedoptical fiber. Such measurements enabled estimates ofinternal rates of PSII electron transport corrected for thespecific tissue light gradients in corals and plants(Lichtenberg and Kühl, 2015; Lichtenberg et al., 2016).However, to obtain absolute electron transport rates(ETRs) through PSII, it is necessary to know the ab-sorption factor, which describes the PSII absorptioncross section and the balance between PSI and PSIIphotochemistry, and these parameters cannot be calcu-lated from measurements of light availability. In addi-tion, due to the small tip size of fiber-optic radiancemicroprobes (usually less than 50 mm) used to detect thefluorescence, microfiber-based measurements of varia-ble chlorophyll fluorescence also are prone to reflect thenatural heterogeneity of such systems (Lichtenberg andKühl, 2015; Lichtenberg et al., 2016). A method was re-cently proposed for calculating absolute electron turno-ver rates of PSII, but the approachwas limited to surfacemeasurements or optically thin systems (Szabó et al.,2014). It is thus of great importance to further explorehow steep gradients of light influence photosyntheticefficiencies in complex photosynthetic tissues andsurface-associated phototrophic communities.

Internal gradients of light absorption have beenquantified from fluorescence profiles in terrestrialleaves (Takahashi et al., 1994; Vogelmann and Han,2000; Slattery et al., 2016), and this technique has beencombinedwith fine-scale measurements of CO2 fixationto investigate the relationship between chlorophyllconcentration, light absorption, and photosynthesis athigh spatial resolution (Vogelmann and Evans, 2002;Evans and Vogelmann, 2003). These studies generallyfound a good correlation between the light absorptionof different spectral ranges and the associated CO2fixation profiles. However, the CO2 fixation rates re-lied on freeze clamping 14CO2-preincubated leaf sam-ples with concomitant paradermal sectioning andmeasurements by scintillation counting, which is alaborious process that is limited in the spatial resolu-tion by the sectioning process to ;40 mm (Vogelmannand Evans, 2002). Here, we present a novel experi-mental approach and show its application for map-ping gradients of light absorption and photosynthesisin aquatic plant tissue.

The lower community photosynthesis often observedin aquatic systems as compared with terrestrial systems(Sand-Jensen and Krause-Jensen, 1997) can be largelyexplained by the inability of aquatic macrophytes toobtain an optimal 3D structural organization in relationto the incident irradiance (Binzer and Sand-Jensen,2002a, 2002b), unlike their terrestrial counterparts,which can regulate leaf inclination to increase can-opy light utilization (McMillen and McClendon, 1979;Myers et al., 1997). In addition, specialized cell/tissuestructures in terrestrial plants can increase photon ab-sorption such as in sun-adapted leaves with well-developed palisade cells that can act as light funnelsdirecting light into the photosynthetically active meso-phyll layer (Vogelmann and Martin, 1993), while someshade-adapted understory plants can alleviate lightlimitation by focusing light in the mesophyll layer viaplanoconvex epidermal cells and intercellular air spaces(Vogelmann et al., 1996; Brodersen and Vogelmann,2007). In contrast, mostmacroalgae are not recognized tohave specialized tissue structures to facilitate the pene-tration of light, although there have been reports of lightguides in some green algae (Ramus, 1978).

Macroalgal members of the Fucales have morphologi-cally differentiated tissues such as the basal thallus, thegrowing sterile frond, and fertile receptacles, while cellsare differentiated into meristoderm, cortex, and medul-lary layers on the tissue scale (Garbary and Kim, 2005).While all cell types contain plastids (Moss, 1983), the outermeristoderm and cortex cells contain more chloroplastsand thylakoids than the medullary filaments. It has beensuggested that the medullary filaments could play a rolein the longitudinal translocation of materials (Moss, 1983;Raven, 2003) and, further, that they may play a structuralrole in providing elasticity in terms of a cushion-like effectprotecting against wave action (Moss, 1983). The medullalayer is surrounded on both sides by anatomically simi-lar layers of cortex, meristoderm, and epidermis cells(henceforth referred to as the cortex), in contrast to bifacialterrestrial plant leaves that display morphologically andphysiologically differentiated abaxial and adaxial domains.In Fucus spp., steep gradients of light and photosyn-thesis have been measured using fiber-optic microprobesand microelectrodes, although this approach is ratherchallenging in such cohesive tissues (Spilling et al., 2010;Lichtenberg and Kühl, 2015).

In this study, we aimed to resolve how photosyn-thetic efficiencies are affected by steep light gradients indifferent spectral regions. This was accomplished bythe use of a novel multicolor laser light sheet micros-copy setup to image the distribution of light absorptionand photosynthetic activity over transverse sections ofan aquatic macrophyte to resolve how photosyntheticefficiencies are affected by steep light gradients in dif-ferent spectral regions. We applied laser light sheets ofdefined spectral composition perpendicular to one sideof thallus cross sections while imaging the distributionof chlorophyll fluorescence and variable chlorophyllfluorescence from the cut surface.We compared suchdatawith measurements obtained with equal illumination of

722 Plant Physiol. Vol. 175, 2017

Lichtenberg et al.

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

the cross section to describe, to our knowledge for the firsttime, how PSII quantum yields are affected by naturallight gradients in optically dense tissues. This novelmethod can resolve such gradients routinely and withhigher resolution as compared with other microscaleapproaches such as mapping with fiber-optic probes(Kühl and Jørgensen, 1994; Lichtenberg et al., 2016).

RESULTS

Cross-Thallus Chlorophyll Fluorescence Profiles

Using a novel microscopic setup (Fig. 1; for details, see“Materials andMethods”),weused both even illuminationof plant tissue cross sections and illumination with lasersheets of defined spectral composition incident perpen-dicularly on tissue cross sections. When illuminatedhomogenously across the algal thallus cross section, bothcortex layers of Fucus vesiculosus displayed equally highamounts of chlorophyll that were 2.5- to 5-fold higherthan in the central medulla (Figs. 2 and 3), assuming thatrelative chlorophyll content can be estimated from flu-orescence using epiillumination (Vogelmann and Evans,2002). The fluorescence profiles under light sheet illu-mination perpendicular to one side of the cross sectionshowed that blue light (425–475 nm) was attenuatedmost strongly in an exponential manner with depth and

decreased to less than 21% of the maximum fluorescence(Fmax) ;250 mm inside the thallus (Fig. 3). Fluorescenceprofiles over the thallus cross section using green (525–575 nm) and red (615–665 nm) light showed similar at-tenuation but decreased to a minimum fluorescencemore than 2 times higher thanwas found for blue light ata similar depth in the thallus. Blue, green, and red light-induced fluorescence profiles all displayed Fmax valuesclose to the thallus surface. When using broadbandwhite light illumination, a peak was located at the sameposition as the Fmax of the blue, green, and red profilesfollowed by an intermittent decrease before reachingFmax ;100 mm inside the thallus (Fig. 3). Common for allprofiles was that the fluorescence showed a peak closeto the illuminated cortex followed by a decrease to-ward the center of the medulla before increasing againtoward the shaded cortex. The relative largest increasetoward the shaded thallus side was in the order blue,red,, green,white. The width of the peaks was ofsimilar size and extended 150 to 200 mm from thesurfaces toward the center of the thallus (Fig. 3).

Absorption Profiles

Integrating sphere measurements of thallus reflec-tance, transmittance, and absorptance displayed typical

Figure 1. Experimental setup for light sheet microscopy in combination with variable chlorophyll fluorescence imaging. A, The sampleholder consisted of a cuvette cut down to 11-mm height (internal dimensions of 103 103 10mm). The sample was mounted in agar inthe bottomof the sample holder,whichwas filledwith seawater and then closedwith a coverslip. B, Spectral composition of the laser lightused for measurements of chlorophyll fluorescence profiles and the actinic light for measurements of variable chlorophyll fluorescence.The laser was adjusted to have the same absolute photon irradiance independent of spectral composition. C, Schematic drawing of theexperimental setup for measuring chlorophyll fluorescence profiles. a, The algal thallus sample positioned in the cuvette; b, microscopeobjective; c, filter cube with long-pass filter; and d, CCD camera. Illumination of the sample was done with a personal computer (PC)-controlled supercontinuum laser connected to a spectral line filter unit and a laser sheet generator. D, Schematic drawing of the exper-imental setup for variable chlorophyll fluorescencemicroscopy. a, Sample fixed in the cuvette; b, microscope objective; c, emission filter;d, dichroic beamsplitter cube; e, dichroic filter; f,mirror (to ocular); and g, CCDcamera.Weaknonactinicmodulatedmeasuring lightwasprovided by a software-controlled red-green-blue (RGB) LED unit. Actinic light was provided perpendicular to one side of the thallussurface by a personal computer-controlled supercontinuum laser connected to a spectral line filter unit and a laser sheet generator.

Plant Physiol. Vol. 175, 2017 723

Imaging of Light Absorption and Photosynthesis

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

characteristics for densely pigmented opaque planttissues (Fig. 4). Reflectance was relatively uniformat;3% of the incident irradiance, although slightly higherin the green/yellow part of the spectrum (around 570 nm).Absorptance spectra (Fig. 4; Supplemental Fig. S1) showedin vivo absorption peaks from major photopigmentspresent in brown macroalgae, such as chlorophyll a(440 and 675 nm; Johnsen et al., 1994) and chlorophyll c(460, 590, and 635 nm; Shibata and Haxo, 1969; Kühlet al., 1995), fucoxanthin (in vitro absorption peaks inhexane at 425, 450, and 475 nm and extending to580 nm in vivo; Govindjee and Braun, 1974), and othercarotenoids (400–540 nm; Govindjee and Braun, 1974).The mean absorptance averaged over photosyntheti-cally active radiation (PAR; 400–700 nm) using broad-band white light was 92% of the incident irradiance.Transmittance was highest (10%–13%) in the green/yellow part (around 570 nm) of the spectrum and wasclose to zero in the blue and red spectral regions, whilethe mean transmittance was 5% of the incident irradi-ance (Fig. 4).

By normalizing the chlorophyll fluorescence profiles(Fig. 3) to the total absorptionmeasured for blue, green,red, and white light with an integrating sphere (Fig. 4),we could calculate the depth of specific photon ab-sorption inside the thallus (Fig. 4; Supplemental Fig.S3). The different thallus regions (cortex/medulla) wereestimated to be, on average, 150 mm in thickness (Fig.3). When illuminating the thallus with the laser sheet,the apparent absorption of photons was always highestin the upper and lower cortex as compared with themedulla, where the fractional absorption was lowest(Fig. 4; Table I).

We modeled the light availability in the F. vesiculosusthallus by using measured scalar irradiance attenuationcoefficients of cortex and medulla layers from theclosely related brown alga Fucus serratus (Lichtenbergand Kühl, 2015), assuming monoexponential attenua-tion of light in the thallus (Fig. 5; see “Materials andMethods”). These modeled curves of light attenuationwere compared with the curves of attenuation due toabsorption found in this study (Fig. 5) to test if thefound absorption profiles were in the same order as theattenuation profiles, as would be expected for suchdensely pigmented systems. We found an average lightattenuation coefficient of 5.64 mm21 (R2 = 0.97) over theentire thallus, with higher attenuation coefficients in thecortex layers (upper cortex = 6 mm21 [R2 = 0.99] andlower cortex = 9.8mm21 [R2 = 0.96]) than in themedullalayer (4.3 mm21 [R2 = 0.99]; Fig. 5). These values were inthe same order as the light attenuation coefficients ofcortex andmedulla layers in F. serratus (6.8 and 3.4mm21

for cortex and medulla, respectively; Lichtenberg andKühl, 2015), suggesting that the distribution of photonabsorption can be found by a combination of chloro-phyll fluorescence profiles and measurements of totalabsorption.

PSII Quantum Yields and PhotosyntheticElectron Transport

In the dark-acclimated state, all thallus layers dis-played a maximal PSII quantum yield of greater than0.6, indicating no major stress factor on photosyn-thetic performance due to cutting or sample handling

Figure 2. Color-codedmaps showing the distribution of chlorophyll a fluorescence (normalized to maximum fluorescence) of anF. vesiculosus thallus cross section illuminated evenly over the cut side with 430-nm light from a xenon lamp (the plot iscomposed of multiple images taken through a 103 objective and stitched together using Adobe Photoshop; A) and thallus crosssections illuminated perpendicular to one side of the thallus surface with a supercontinuum laser sheet of different spectral com-positions of blue light (425–475 nm; B), green light (525–575 nm; C), red light (615–665 nm; D), and white light (400–700 nm; E).

724 Plant Physiol. Vol. 175, 2017

Lichtenberg et al.

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

(Supplemental Fig. S4). When applying actinic irradi-ance homogenously over the cross sections via thebuilt-in LEDs of the microscope PAM system (Fig. 1),the effective PSII quantum yield decreased in all thalluslayers but more so in the medulla as compared with the

cortex layers. The highest decrease was found underhigh incident irradiance, where the effective PSIIquantum yield in the medulla decreased to less than 0.3(Fig. 6). This pattern changed under actinic laser sheetillumination of the cross section from one side. Whilethe PSII quantum yield distribution was apparentlyunaffected by the changed actinic light geometry in thedark-acclimated state and under very low irradiance,the PSII quantum yield decreased rapidly over the il-luminated cortex and reached levels of less than 0.1under the highest irradiance. Due to the strong lightattenuation across the thallus, the PSII quantum yieldsin the medulla and the shaded cortex layers decreasedless than when illuminated homogenously via theimaging PAM actinic light source, and the effective PSIIquantum yield in the shaded cortex remained at levelssimilar to those in dark-acclimated states (greater than0.6) even at the highest irradiance (Fig. 6).

Apparent ETRs through PSII were calculated for theilluminated (upper) cortex, themedulla, and the shaded(lower) cortex, and the rates were corrected for theamount of absorbed photons in the respective tissuelayers. In all cases, the ETRs in the upper and lowercortex layers were very similar when corrected forabsorbed light. The medulla ETRs were similar to thecortex activity on the subsaturated part of the ETRversus irradiance curve but saturated at higher irradi-ance (Fig. 7).

The slope of the ETR versus absorbed light curveunder blue light was lower than for red and white lightbut reached higher ETRs (Fig. 7; Table I). The curvesappeared similar under green and red light, althoughgreen light yielded a lower slope on the subsatu-rated part of the ETR versus absorption curve. TheETR curves measured under broadband white lightappeared qualitatively as a combination of the curvesmeasured under blue, green, and red light, where sat-uration occurred at higher irradiance, similar to the

Figure 3. A and B, Epifluorescence microscopy image (false colors) ofan F. vesiculosus thallus cross section illuminated evenly with blue light(430 nm) from a xenon lamp (A) and the associated fluorescence profile(normalized to maximum fluorescence; B). C and D, Example of a flu-orescence image (false colors) of a thallus fragment irradiated perpen-dicularly to one side of the thallus surface (arrows) with a laser sheet ofred light (615–665 nm) from a supercontinuum laser (C) and chlorophyllfluorescence profiles of cross sections of apical thallus fragments ofF. vesiculosus irradiated perpendicular to the thallus surface with differ-ent spectral bands of blue light (425–475 nm), green light (525–575 nm),red light (615–665 nm), and white light (400–700 nm; D). Data werenormalized to maximum fluorescence, and actual data points are spaced0.8 mm apart. Error bars are not shown for clarity, but mean relative SD

was 67.7% (n = 5).

Figure 4. A, Spectral measurements of reflectance, transmittance, and absorptance of an F. vesiculosus thallus fragment using anintegrating sphere (Supplemental Fig. S1). Data were recorded using either incident blue laser light (425–475 nm; blue lines),green laser light (525–575 nm; green lines), red laser light (615–665 nm; red lines), or white laser light (400–700 nm; black lines).Dashed lines indicate61 SD (n = 3). B, Calculated absorption profiles of cross sections of apical thallus fragments of F. vesiculosusirradiated perpendicular to one side of the thallus surface with different spectral bands of blue laser light (425–475 nm), greenlaser light (525–575 nm), red laser light (615–665 nm), and white laser light (400–700 nm). Absorption was calculated from themeasured chlorophyll fluorescence profiles (Fig. 3D) and was normalized to the bulk absorption measured with an integratingsphere (A). Dashed lines indicate the borders of the cortex and medulla tissue layers. Data points are spaced 0.8 mm apart (n = 5).

Plant Physiol. Vol. 175, 2017 725

Imaging of Light Absorption and Photosynthesis

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

green and red curves, but reaching higher ETR values,probably caused by the blue light component. How-ever, the slopes on the subsaturated part of the ETRversus irradiance curves were significantly differentbetween white light and the average of the blue, green,and red curves in all thallus layers (Table I). The greenand red ETR versus absorbed light curves were similarin appearance, in correspondence with their associatedabsorption profiles, which also were similar (Figs. 4 and7). Surprisingly, the ETR curves under blue light did notreach saturation, and ETRs in the cortex and medullalayers were very similar except at the highest irradi-ance, where a decrease in the medulla layer wasobserved (Fig. 7). Even at the highest irradiance, PSIIquantum yields in the cortex layers remained high,and only a small increase in the nonphotochemicalquenching was observed (Supplemental Fig. S4), whichcould point to better light-processing properties of bluelight compared with green and red light.

DISCUSSION

A novel combination of multicolor light sheet mi-croscopy with variable chlorophyll fluorescence imag-ing enabled the mapping of light absorption andphotosynthetic efficiencies in densely pigmented tis-sues. By combining well-tested methods of integratingsphere measurements and the chlorophyll fluorescenceprofile technique (Takahashi et al., 1994; Vogelmannand Han, 2000; Slattery et al., 2016), we propose amethod for calculating profiles of photon absorptionacross plant tissue sections, which can be combinedwith variable chlorophyll fluorescence imaging ofphotosynthetic efficiency across tissue light gradients.

The conversion of PSII quantum yields measured byvariable chlorophyll fluorescence to absolute rates ofphotosynthetic electron transport activity requiresprecise measurements of (1) mean effective PAR, (2) thePSII absorption cross section, and (3) knowledge aboutthe partitioning between PSI and PSII photochemis-try (Klughammer and Schreiber, 2015). While suchinformation can be obtained in dilute suspensions

of chloroplasts and microalgae (Klughammer andSchreiber, 2015), to measure these parameters in densealgal solutions, plant tissue, and algal biofilms is nottrivial (Szabó et al., 2014; Klughammer and Schreiber,2015). In optically dense systems, light gradients areaffected by both multiple scattering and absorption,and it is important to take diffuse light into accountwhen quantifying actinic light levels (i.e. by measuringthe incident photon flux from all directions with scalarirradiance microprobes; Kühl, 2005). While such sen-sors have tip diameters down to 30 mm (Rickelt et al.,2016), it is difficult to perform scalar irradiance mea-surements in thin, cohesive plant tissues, where mea-surements can be biased by tissue compression due to

Table I. Photosynthetic ETR from PSII versus photon absorption curve parameters and the fractional photon absorption (Abs; in percentage of totalabsorption) calculated for the upper cortex, medulla, and lower cortex under blue (425–475 nm), green (525–575 nm), red (615–665 nm), and white(400–700 nm) irradiance applied perpendicular to one side of the thallus surface of F. vesiculosus

Slopes on the subsaturated part of the ETR versus light curve, maximum ETR values (ETRmax), and the light acclimation index Ek were estimatedfrom curve fitting of the ETR versus photon absorption curves with an exponential function (Webb et al., 1974) using a nonlinear Levenberg-Marquardt fitting algorithm. The RGB values were calculated as average curves of blue, green, and red. Pairwise superscript letters indicate sta-tistically significant differences (one-way ANOVA, P , 0.01; n = 5 for white and n = 4 for RGB).

LightUpper Cortex Medulla Lower Cortex

Slope ETRmax Ek Abs Slope ETRmax Ek Abs Slope ETRmax Ek Abs

% % %Red 0.60 197.09 327.23 44.0 0.59 80.43 135.82 26.0 0.60 192.68 319.05 30.0Green 0.47 225.31 482.46 44.0 0.45 81.33 180.71 26.0 0.47 228.90 489.60 30.0Blue 0.54 878.52 1,620.05 57.0 0.54 254.76 469.67 21.0 0.55 599.82 1,082.76 22.0White 0.60a 378.40 629.79 45.0 0.58b 125.48 216.22 26.0 0.60c 349.31 586.39 29.0RGB 0.54a – – – 0.53b – – – 0.54c – – –

Figure 5. Plots of scalar irradiance attenuation profiles of blue light(420–520 nm) calculated using attenuation coefficients from the cortex(blue triangles) and medulla (red circles) layers (Lichtenberg and Kuhl,2015), and a profile of the attenuation due to the absorption of blue light(425–475 nm) estimated from the observed fluorescence profile (blacksquares).

726 Plant Physiol. Vol. 175, 2017

Lichtenberg et al.

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

the physical impact of the microprobe (Spilling et al.,2010; Lichtenberg and Kühl, 2015). The mean effectivePAR also can be calculated from complex measure-ments of the angular radiance distribution with fieldradiance microprobes (Vogelmann and Björn, 1984;Vogelmann et al., 1989; Kühl and Jørgensen, 1994).Alternatively, information on the cell size distributionand the inherent optical properties (i.e. the scatteringphase function and the scattering and absorption coef-ficients) allows calculations of PAR gradients, but theseparameters are difficult to determine in optically densemedia (Privoznik et al., 1978; Berberoglu et al., 2009;Klughammer and Schreiber, 2015), albeit recent exper-imental and theoretical advances in biomedical opticshave allowed detailed characterization of tissue opticsusing combinations of optical reflection spectroscopy,optical coherence tomography, and Monte Carlo sim-ulations (Wang et al., 1995; Wangpraseurt et al., 2016a,2017).An experimental solution to the above-mentioned

complications relies on measuring the chlorophyll

fluorescence profile, which represents the net outcomeof photon absorption along the actinic light gradient inthe tissue (Takahashi et al., 1994; Vogelmann and Han,2000). By correlating fluorescence profiles to total ab-sorption, we could measure the direct result of ab-sorption, and the values obtained are thus only affectedby the quantum yield of fluorescence and energytransfer between antenna pigment molecules and PSIIand PSI. However, due to the invasive nature of themethod, some actinic light will be lost from the cutsurface, causing some underestimation of the tissueabsorption in situ (Ichiro et al., 2016). Furthermore, thismethod allows estimates of the distribution of photonabsorption, but it does not enable a separation of pos-sible changes in the absorption cross section or balancebetween PSI and PSII absorption in different tissuelayers.

We found total absorption values from integratingsphere measurements that were similar to those of ter-restrial leaves (Gorton et al., 2010), although the absorp-tion of green/yellow light was higher in F. vesiculosus due

Figure 6. Isopleths (A and B) and images (C–H) of effective PSII quantum yield in apical thallus fragments of F. vesiculosus il-luminated evenly on a cross section or perpendicular on one side of the thallus surface. Images were acquired under red lightusing either direct light from the built-in LEDs of the variable chlorophyll fluorescence imaging system (590–650 nm) or lightperpendicular to the thallus surface provided by a supercontinuum laser (615–665 nm) connected to a tunable single-line filterand delivered via a laser sheet generator. The isopleths (A and B) show the influence of actinic irradiance on the effective PSIIquantum yield (in mmol electrons m22 s21 [mmol photons m22 s21]21) as a function of the depth in the tissue when illuminatedeither directly on the cross section (A) or perpendicular to the surface of the thallus (B). Illumination in B was given from left toright. Line profiles (line width = 15 pixels) were taken on thallus parts with similar thickness (;250 mm), with cortex layers alsodisplaying similar thicknesses (;50–75mm). C to H show images of effective PSII quantum yield in darkness, moderate irradiance(5676 18mmol photonsm22 s21), and saturating irradiance (1,0876 30mmol photonsm22 s21) under direct even illumination ofthe cross section (C–E) andwith laser light sheet illumination perpendicular to the thallus surface (F–H). Illumination in F to Hwasgiven from the bottom to the top. Bar = 0.2 mm.

Plant Physiol. Vol. 175, 2017 727

Imaging of Light Absorption and Photosynthesis

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

to the presence of accessory brown algal pigments such asfucoxanthin, which displays a high efficiency of energytransfer to chlorophyll a (;95%; Yukihira et al., 2017). Dueto the low reflection and transmission in the thallus, theabsorption will be in the same order as the light attenu-ation. Comparing the calculated attenuation of light dueto absorption with the light attenuation calculated usingscalar irradiance microprofile measurements from a pre-vious study (Lichtenberg and Kühl, 2015; see “Materialsand Methods”), we found a whole-thallus absorptioncoefficient that was lower than cortex attenuation coeffi-cients and higher than medulla attenuation coefficients(Fig. 5). We predicted identical absorption coefficients inthe cortex layers, since these layers are not anatomicallydifferent and, in addition, displayed similar levels ofchlorophyll fluorescence under uniform epi-illumination.Surprisingly, the absorption coefficient of the shadedcortex was larger than the one found in the illuminatedcortex (Fig. 5). We speculate that the light field angularitycould have caused this difference. Previously, it wasshown that the absorptance of plant tissue can be differentunder collimated versus diffuse light (Brodersen andVogelmann, 2010; Gorton et al., 2010), and here, the in-cident light on the illuminated cortex was collimatedwhile light reaching the shaded cortex had a higher dif-fuse component due to internal scattering. Furthermore,we note that our calculations were based on profiles ofblue light, which was almost completely absorbed, mak-ing the calculations of absorption in the shaded cortexmore prone to errors due to the lower signal-to-noiseratio. Absorption profiles are further complicated due to

the self-absorption of red fluorescence by chlorophyll.Thus, red fluorescence profiles are likely to betterrepresent absorption profiles than profiles of far-redfluorescence (Vogelmann and Han, 2000). Here, weused a long-pass filter (greater than 670 nm) to detectfluorescence; therefore, the resulting profiles comprisedboth red and far-red chlorophyll fluorescence, and fu-ture studies should divide the detected fluorescencesignals into red and far-red fluorescence. We also notethat our laser sheet had a Gaussian beam profile, whichmakes positioning close to the edge of the cut thallussurface difficult and may create a potential spilloverof photons onto the cut side. This limitation could beresolved by shaping the beam (e.g. by the generalizedphase-contrast method; Bañas et al., 2014) to transformthe Gaussian beam profile to a sharp rectangular shape,and such work is now under way.

The shape of the white fluorescence profile across thethallus was slightly different in appearance from theprofiles for blue, green, and red light. Previously, it wasshown that profiles of carbon assimilation and chloro-phyll fluorescence profiles followed each other closelydepending on the spectral quality (Sun et al., 1996;Vogelmann and Han, 2000), and it has been proposedthat profiles of carbon fixation under white light can bedescribed as the mean when using blue, green, and redlight (Sun et al., 1998; Vogelmann andHan, 2000). Here,we show that, for plant tissue harboring a range of ac-cessory pigments such as fucoxanthin, the absorptiveproperties are more complex, resulting in a differentresponse to white light than what can be expected from

Figure 7. Apparent ETRs through PSII cor-rected for absorbed photons (Fig. 4). Mea-surements were performed with 20-sacclimation to each increasing actinicirradiance level of blue light (425–475 nm;A), green light (525–575 nm; B), red light(615–665 nm; C), and white light (400–700 nm; D) as provided by a laser sheetilluminating a thallus fragment perpendic-ular to one side of the thallus surface. Datapoints represent means 6 SE (n = 5, exceptA, where n = 4).

728 Plant Physiol. Vol. 175, 2017

Lichtenberg et al.

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

the combination of measurements made under mono-chromatic light. Furthermore, it was shown that greenlight drives photosynthesis more efficiently deeper interrestrial plant tissue than blue and red light, due to alarger penetration depth of green light in leaf tissues(Sun et al., 1998; Terashima et al., 2009). In contrast, thepresence of fucoxanthin and carotenes in Fucus spp.caused the green light (525–575 nm) to be absorbedequally effectively as red light. However, not allwavelengths were absorbed equally effectively, and thespectral region around 570 to 605 nm displayed re-duced absorption as compared with the other spectralregions of PAR. Since only the white light treatmentcovered this part of the spectrum, it is possible that il-lumination with broadband white light caused thedifferently shaped absorption profile. This might beconfirmed by measuring additional chlorophyll fluo-rescence profiles using yellow light (e.g. 570–605 nm) tovalidate if this would result in fluorescence profileswith Fmax located deeper in the thallus, similar to fluo-rescence profiles in terrestrial leaves illuminated withgreen light (Vogelmann and Han, 2000).

Photosynthesis

We demonstrate that PSII quantum yields and de-rived apparent ETR across the thallus cross sectionsstrongly differed between homogenous actinic light il-lumination of the cross section and unidirectional ac-tinic light illumination on one side of the thallus with alaser light sheet. Under low incident irradiance, theyields were very similar in all layers and betweenmeasurements. However, as incident light directly onthe cut side increased, the yields decreased across thetissue, with the highest decreases found in the medulla(Fig. 6). This was not the case under laser light sheetillumination perpendicular to one side of the thallussurface, where we found strongly reduced yields in theilluminated cortex, while yields in the shaded cortexwere unaffected (Fig. 6), even at the highest incidentphoton irradiance (1,108 mmol photons m22 s21). Thus,when applying actinic light directly on a cross section,our data show that it is possible to both underestimateand overestimate PSII quantum yields as comparedwith yields found under natural light gradients. Using amultilayer leaf model, Evans (2009) found that, as ir-radiance increased on the adaxial side, the quantumyields were reduced progressively deeper into the me-sophyll. However, quantum yields at the abaxial sidewere unaltered even under high blue irradiance, whichis in good agreement with the findings of this study.Evans (2009) showed that surface-based measurementsof fPSII to estimate ETR were only valid in some casesbut overestimated ETR when the leaf was inverted.Similarly, mesophyll conductance measurements couldbe influenced when estimated using surface-based fPSIIvalues (Evans, 2009). With our new method, fPSIIvalues can now be measured under natural tissue lightgradients and can be corrected for photon absorption,thus making it possible to get more detailed insights

into mesophyll conductance heterogeneities (e.g. byusing depth-resolved fPSII data in combination withgas-exchange measurements; Evans, 2009; Pons et al.,2009).

The differences in PSII quantum yield measuredunder illumination directly on the cross section orperpendicular to the side of the thallus surface indicatethe difference between the photosynthetic potentialunder equal illumination and the realized photosyn-thesis under tissue light gradients. We show here that,even under high incident irradiance, photosyntheticelectron transport in the lower cortexwas not saturated;therefore, an even illumination of tissue cross sectionswill underestimate the PSII quantum yield as comparedwith shaded parts during high unidirectional illumi-nation. Apparent ETRs in the cortex layers were verysimilar when corrected for absorption, while the me-dulla layer displayed saturation and lower ETRs at in-creasing irradiance, indicating a lower photosyntheticcapacity, probably due to lower pigment content (Figs.3 and 7). The slope of the initial part of the ETR curves,which is a measure of the light utilization efficiency atsubsaturating photon flux, was similar in all thalluslayers, albeit consistently slightly lower in the medulla(Table I). In a recent study, absolute ETRs of thin-tissued corals were calculated (Szabó et al., 2014), andthe rates found in this study were of similar magnitude.Szabó et al. (2014) also found initial slopes of the sub-saturated part of the ETR versus irradiance curves thatwere slightly higher, probably reflecting differences inphotochemical acclimatization that have been shown tobe tightly linked to the optical properties of coral tissues(Lichtenberg et al., 2016; Wangpraseurt et al., 2016a,2016b).

Fucus spp. are often found in the intertidal zone and,thus, on a daily basis, experiences a variable light en-vironment as a function of water depth and concen-tration of organic and inorganic particles as well asdissolved organic matter attenuating solar irradiance.Therefore, air-exposed plant parts will be subjected tothe full solar spectrum, while the incident light field onalgae situated in deeper oligotrophicwaters will be blueshifted due to the absorption of red light by water. Incontrast, the incident light field on algae in shalloweutrophic waters will be green shifted due to the ab-sorption of blue and red wavelengths by suspendedphytoplankton. On a tissue scale, the medulla layer ofFucus spp. will, on average, experience the lowestphoton irradiance compared with the cortex layers(Lichtenberg et al., 2016) and, therefore, could bethought of as a shade-adapted compartment in the algalthallus. Shade-acclimatized phytoelements will nor-mally display higher light use efficiencies (i.e. steeperinitial slope on the photosynthesis-irradiance curve)but lower photosynthetic capacity (Pmax) due to in-creased pigment content and biochemical regulationsin the photosynthetic machinery and/or ultrastructuralchanges in the chloroplasts (Lichtenthaler et al., 1981,2007; Lichtenthaler and Babani, 2004; Sarijeva et al.,2007). However, as the initial slopes of the ETR versus

Plant Physiol. Vol. 175, 2017 729

Imaging of Light Absorption and Photosynthesis

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

absorbed light curves in the medulla were both lowerand displayed saturation at lower irradiances, medullalayers cannot be described as photosynthetically shadeadapted in conventional terms. Conversely, it appearsthat the structural organization of the thallus layerscould be adapted to maximize photon absorption in theouter cortex layers while having a relatively translucentcentral medulla with low absorptive properties. This isin contrast to terrestrial leaves, where the even illumi-nation of tissue layers is achieved by increased internalscattering due to intercellular airspaces, with the con-comitant absorption profiles following an exponentialattenuation with depth (Vogelmann and Han, 2000).This fundamental difference is in good agreementwhenconsidering their respective positions in terrestrial andaquatic habitats, as terrestrial leaves can organize theirpositions according to the angle of solar radiation,whereas aquatic macrophytes are limited in theirstructural organization by strong drag and shearingforces imposed by waves and currents, randomly ex-posing both sides of the thallus to direct light. Bymaximizing absorption in the outer layers and having atranslucent central layer, F. vesiculosus can maximizelight harvesting by allowing photons not absorbed inthe illuminated thallus to propagate to tissue layerswith unused photosynthetic potential, thereby ensur-ing a more efficient resource distribution.

The ETR versus absorbed light curves measured indifferent tissue layers under laser light sheet illumina-tion were similar to what was found previously inFucus spp. (Lichtenberg and Kühl, 2015), althoughthese microfiber PAM-based measurements were as-sociated with high SD values due to the small mea-surement volume of the fiber-optic microprobe, whichmakes such measurements prone to microscale tissueheterogeneity effects. With the experimental approachpresented in this study, it is now possible to integratephotosynthetic responses from specific tissue layersmuch more precisely and, in addition, to correct themfor the amount of photons absorbed by that given tissuelayer. Here, we used a 103microscope objective, but inprinciple, such measurements could be performed ateven higher magnification (e.g. to investigate single cellgradients of light) and photosynthetic efficiencies (e.g.in large algal cells). The combination of multicolor lightsheet microscopy with variable chlorophyll fluores-cence imaging on plant tissue cross sections provides analternative to more destructive methods such as con-structing profiles of CO2 fixation from paradermalsectioning (Evans and Vogelmann, 2003) or nanoscalesecondary ion mass spectroscopy (Kilburn et al., 2010;Wangpraseurt et al., 2016b) and allows sequentialmeasurements on the same sample (e.g. under differentlevels of actinic irradiance) or comparisons of diffuseversus collimated light fields (Brodersen et al., 2008).Here, we demonstrated the application on aquaticmacrophyte tissue, but the technique is readily appli-cable to many other types of plant tissues, includingterrestrial leaves as well as photosynthetic biofilms andsymbioses.

CONCLUSION

The combination ofmulticolor light sheet microscopyand variable chlorophyll fluorescence imaging is apowerful technique that enables fine-scale characteriza-tion of light absorption and PSII quantum yields acrossplant tissue sections. Furthermore, quantification ofphoton absorption from light sheet-induced cross-tissuefluorescence profiles can be used in concert with variablechlorophyll fluorescence imaging, enabling calculationsof ETRs that otherwise require knowledge of the ab-sorption cross section and the mean effective PAR.

The spectral flexibility of a white supercontinuumlaser source allows this method to be used in other pho-tosynthetic systems with different anatomical structuresand pigmentation. In this manner, the role of specific ac-cessory pigments in light propagation and photosynthesiscan be investigated further.

MATERIALS AND METHODS

Sample Collection and Preparation

Stands of the brownmacroalga Fucus vesiculosuswere collected in the littoralzone at various locations around Helsingør, Denmark, during late summer andwere maintained in 10-L buckets continuously flushed with 0.2 mm of filteredaerated seawater (temperature = 16°C, salinity = 32) for up to 1 week prior toexperiments. Samples were kept under a 12:12-h light:dark cycle under aphoton irradiance of ;50 mmol photons m22 s21 (PAR, 400–700 nm) as pro-vided by a fluorescent tube (Philips Master TL-D90, 18 W; Philips).

Prior to measurements, an apical thallus fragment was cut ;1 cm from thethallus tip with a razor blade, and the cut side was rinsed in seawater with atransfer pipette to wash away pigments leaking from cut chloroplasts. Thesample holder (Fig. 1) consisted of a standard plastic cuvette, cut down to aheight of 11 mm to allow insertion on the microscope (internal size = 103 10310 mm). To fix the sample in the cuvette, ;300 mL of 20 g L21 seawater agar(Sigma-Aldrich) was transferred to the cuvette and allowed to cool to 20°C,after which a slit was cut parallel to the cuvette window to allow insertion of thethallus fragment. The thallus was inserted flush with the edge of the cuvette,filled with seawater (16°C, salinity = 32), and closed with a coverslip.

Chlorophyll Fluorescence Profiles

Profiles of absorbed light as estimated from chlorophyllfluorescence profilesacross tissue sections were imagedwith a customizedmicroscope setup (Fig. 1).The sample cuvette (see above) was mounted on an inverted microscope (IX81;Olympus) with a 103 objective (UPlanSApo 103/0.40; Olympus). The samplewas illuminated perpendicular to one side of the thallus surface by a super-continuum laser (SuperK Extreme, EX-B; NKT Photonics). The laser was con-nected to a tunable single-line filter module (SuperK Varia; NKT Photonics) viaa single-mode fiberwith a collimated output. The tunable single-line filter couldbe tuned from 400 to 840 nm to produce bandwidths from 1 to 400 nm. Lightfrom the single-line filter module was delivered, via an alignment tool (SuperKConnect; NKT Photonics), to an endlessly single-mode large-mode-areaphotonic crystal fiber (FD7; NKT Photonics) with a collimated output. Thecollimated output was connected to a cylindrical laser sheet generator (NKTPhotonics) with a 14° light sheet half angle, yielding a 5-cm longitudinal line at10-cm distance. The generated laser sheet had a Gaussian beam profile of;1 mm on the latitudinal axis. The output laser optics was mounted on amanual micromanipulator (MM33; Märzhäuser) that allowed easy positioningof the laser sheet in the focal plane of the microscope.

The samplewas positionedwith the cut side facing themicroscope objective,and the laser sheet was adjusted to hit as close to the edge of the cut as possiblewithout illumination spillover to the cut side. After positioning, the sample wasallowed to dark adapt for 15 min.

Images of chlorophyll fluorescence from the cross section were taken with asensitiveCCDcamera (iXon) using afixed exposure time of 70ms.Chlorophyllfluorescence was detected by placing an ultra-steep long-pass edge filter

730 Plant Physiol. Vol. 175, 2017

Lichtenberg et al.

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

(BLP01-664R; Semrock) in the light path between the objective and the camerawith a transmission close to 100% at wavelengths greater than 670 nm andclose to 0% at wavelengths less than 670 nm. Illumination in different spectralbands was achieved by control of the laser and the spectral filtering modulewith the manufacturer’s software (NKTP Control; NKT Photonics). Fourdifferent spectral illumination bands were composed (Fig. 1) and adjusted tothe same photon irradiance (1,190.3 6 1.9 mmol photons m22 s21) as mea-sured with a microquantum sensor (MC-MQS; Walz) connected to a cali-brated quantum irradiance meter (ULM-500; Walz). We used broadbandillumination (50 nm or greater) to ensure the excitation of both PSII and PSI,thus avoiding eventual red-drop effects.

Illumination of the sample during individual image acquisitions was limitedto less than 2 s per image, and the spectral sequence was randomized betweenreplicates. The effect of irradiance on the emitted fluorescence was tested bytreatment with 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (Supplemental Fig.S2). Images were analyzed in ImageJ (version 1.50B), where gray values wereextracted either by the line profile tool or by extracting all gray values. Theimages had a spatial resolution of 0.8 3 0.8 mm pixel21. Maximum chlorophyllfluorescence was normalized to 1 in all images to allow comparison betweenimages. Plots were made in OriginPro (version 9.3; OriginLab). The thickness ofthe thallus varied, both between samples and depending on the location in thecross section (Fig. 2). Therefore, profiles of fluorescence were taken at thallusthicknesses of;450 mm to allow comparison of thallus light gradients over thesame tissue thickness.

Integrating Sphere Measurements of Reflectanceand Transmittance

Thallus reflectance and transmittance were measured using an integratingsphere (diameter = 10 cm, port diameters = 2.5 cm; Labsphere Instruments).The sphere had three port openings: two located opposite each other and oneorthogonal to the two other openings (Supplemental Fig. S1). The incident lightfrom the supercontinuum laser was tuned to different spectral ranges eachwiththe same photon irradiance (see above and Fig. 1). Light was measured with acalibrated spectral irradiance meter (MSC15; Gigahertz Optik) connected to theorthogonally located port on the integrating sphere. For transmittance mea-surements, a thallus fragment was mounted in front of the entrance port be-tween the light source and the integrating sphere, and the port opposite to theentrance was covered with a white reflecting plate. For reflectance measure-ments, a thallus fragment was placed in the port opening opposite the incidentlaser beam at an angle of 5° to 10° to capture both the specular and diffusereflections. Total absorptance (A) by the thallus was estimated as:

A ¼Z ln

li

ðI2R2TÞI

ð1Þ

where I is the incident photon irradiance, R is the reflectance, and T is thetransmittance, all integrated over the spectral region of interest (li 2 ln).

Modeling of Light Attenuation

Light attenuation profiles were modeled using attenuation coefficients, a, ofblue scalar irradiance (420–520 nm) in the cortex and medulla layers measuredin a previous study (Lichtenberg and Kühl, 2015). The model assumed mono-exponential attenuation of incident irradiance, I0, over tissue depth intervals Dz,and the light availability in different tissue depths was then calculated as:

Iz ¼ I0$e2a$Dz ð2ÞThese data were compared with the estimated attenuation due to absorptionquantified as induced fluorescence and corrected for total absorption(Supplemental Fig. S3) across the thallus under blue irradiance (425–475 nm) inthis study. The attenuation due to absorption was calculated by subtracting thecumulative absorption (Fig. 4), integrated in 50-mm increments, from the inci-dent irradiance.

Variable Chlorophyll Fluorescence Imaging

PAM variable chlorophyll fluorescence imaging (Imaging-PAM) with thesaturation pulse method (Schreiber et al., 1995; Schreiber, 2004; Kühl andPolerecky, 2008) was used to assess the photosynthetic performance over crosssections of the algal thallus mounted in cuvettes as described above.

Measurements were performed with a microscope Imaging-PAM system (Fig.1) fitted with a RGB-LED excitation lamp (IMAG-RGB; Heinz Walz) as de-scribed in detail elsewhere (Trampe et al., 2011). Themicroscopewas fitted witha high numerical aperture objective (103/NA0.8, Plan-Apochromat; CarlZeiss). Fast measurements of the effective PSII quantum yield under increasingphoton irradiance (each irradiance step was applied for 20 s) were used tomeasure so-called rapid light curves (White and Critchley, 1999) of relative PSIIelectron transport versus irradiance curves. Two different approaches wereapplied: (1) using increasing actinic irradiances of red light (590–650 nm) asprovided by the system-default internal RGB-LED lamp for equal excitation ofthe exposed cross-section of the thallus from above, in combination with thecustomizable automated light curve function of the software provided by thesystem software (ImagingWin; HeinzWalz); and (2) using the supercontinuumlaser setup as described above as an external actinic light source, illuminatingthe thallus with a light sheet perpendicular to the thallus surface. The actiniclight sheet was controlled manually in stepwise increments in sync with thecustom-defined automated light curve function of the ImagingWin software,facilitating a semiautomated acquisition of rapid light curves, while the system-default internal actinic RGB-LED light source was disconnected. Rapid lightcurves with the laser light sheet were obtained with increasing actinic irradi-ance of blue (425–475 nm), green (525–575 nm), red (615–665 nm), or white(400–700 nm) light. Nonactinic modulated blue measuring light was providedby the built-in LEDs of the Imaging-PAM system during both approaches. Thetwo setups were calibrated using a photon irradiance meter connected to acosine-corrected mini quantum PAR irradiance sensor (ULM-500, MQS-B;Walz). All samples were allowed to dark adapt for 15 min before the mea-surements started. When in the dark-acclimated state, all reactions centers ofPSII were open, enabling imaging of the minimal fluorescence yield (F0). Uponexposure to a high-intensity saturation pulse, all PSII reaction centers closed,permitting imaging of themaximal fluorescence yield (Fm) over the thallus crosssection. From these images, the maximum PSII quantum yield could be calcu-lated as described by Schreiber (2004):

FV=Fm ¼ ðFm 2 F0Þ=Fm ð3ÞFrom imaging of the fluorescence yield, F, while the sample was illuminatedwith a predefined level of actinic light (PAR, in mmol photons m22 s21), and themaximum fluorescence yield during a saturation pulse, Fm9, images of the ef-fective PSII quantum yield could be calculated as:

fPSII ¼ ðFm’2 FÞ=Fm’ ð4ÞFrom these values, the relative photosynthetic electron transport rate (rETR) isusually derived using the equation:

rETR ¼ fPSII 3PAR3AF ð5Þwhere PAR is the incident photon irradiance and AF (the absorption factor) is aconstant set to 0.42, assuming that 84% of the incident light is absorbed(Björkman and Demmig, 1987) and an even distribution of absorbed photonsbetween PSII and PSI (Schreiber et al., 2012). In our study, we estimated internalETR rates (in units of mmol electrons m22 s21) in different regions of the thallus(cortex/medulla) by multiplying average fPSII values (in units of mmol elec-trons m22 s21 [mmol photons m22 s21]21) for a given area with the total amountof absorbed photons (in units of mmol photons m22 s21) in that area. Thewavelength-dependent absorption was calculated by normalizing the chloro-phyll fluorescence profiles to the total absorption (Supplemental Fig. S3). Thisessentially gives an estimate of thewavelength-dependent photon absorption atany given depth in the thallus, assuming that 1 unit of fluorescence was thedirect result of 1 unit of absorption. In reality, however, this is affected by factorssuch as the quantum yield of fluorescence and energy transfer between antennapigment molecules and PSII/PSI. Therefore, the true photon absorption will varyslightly, and the calculated photon absorptions are only estimates.

The light saturation coefficient (i.e. the photon scalar irradiance at the onset oflight saturation of photosynthesis, Ek) was calculated as Ek ¼ ETRmax=a, whereETRmax is the maximum activity and a is the initial slope of the ETR versusphoton absorption curve; both parameters were obtained by curve fitting of ETRmeasurements using an exponential function (Webb et al., 1974) by means of anonlinear Levenberg-Marquardt fitting algorithm (OriginPro 2015; OriginLab).

Statistics

One-way ANOVAs were applied to test differences in the slopes of the ETRcurves between different light treatments. Data were first tested for normality

Plant Physiol. Vol. 175, 2017 731

Imaging of Light Absorption and Photosynthesis

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

(Shapiro-Wilk) and for equal variance. Statistical analysis was performed inSigmaPlot (version 12.5), and the significance level was set to P , 0.01.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Schematic drawing of integrating sphere mea-surements of transmittance and reflectance.

Supplemental Figure S2. Chlorophyll fluorescence profiles over cross sec-tions of apical thallus fragments of F. vesiculosus.

Supplemental Figure S3. Concept of the calculations of photon absorptionprofiles across plant tissue cross sections.

Supplemental Figure S4. PSII quantum yield and nonphotochemicalquenching as a function of absorbed photons in the upper cortex, me-dulla, and lower cortex of F. vesiculosus.

ACKNOWLEDGMENTS

We thankNKT Photonics for generous loan of the supercontinuum laser andfor excellent technical support.

Received June 19, 2017; accepted August 14, 2017; published August 18, 2017.

LITERATURE CITED

Baker NR (2008) Chlorophyll fluorescence: a probe of photosynthesisin vivo. Annu Rev Plant Biol 59: 89–113

Bañas A, Palima D, Villangca M, Aabo T, Glückstad J (2014) GPC lightshaper for speckle-free one- and two-photon contiguous pattern exci-tation. Opt Express 22: 5299–5311

Berberoglu H, Gomez PS, Pilon L (2009) Radiation characteristics ofBotryococcus braunii, Chlorococcum littorale, and Chlorella sp. used for CO2fixation and biofuel production. J Quant Spectrosc Radiat Transf 110:1879–1893

Binzer T, Sand-Jensen K (2002a) Importance of structure and density ofmacroalgae communities (Fucus serratus) for photosynthetic productionand light utilisation. Mar Ecol Prog Ser 235: 53–62

Binzer T, Sand-Jensen K (2002b) Production in aquatic macrophyte com-munities: a theoretical and empirical study of the influence of spatiallight distribution. Limnol Oceanogr 47: 1742–1750

Björkman O, Demmig B (1987) Photon yield of O2 evolution and chloro-phyll fluorescence characteristics at 77 K among vascular plants of di-verse origins. Planta 170: 489–504

Brodersen CR, Vogelmann TC (2007) Do epidermal lens cells facilitate theabsorptance of diffuse light? Am J Bot 94: 1061–1066

Brodersen CR, Vogelmann TC (2010) Do changes in light direction affectabsorption profiles in leaves? Funct Plant Biol 37: 403–412

Brodersen CR, Vogelmann TC, Williams WE, Gorton HL (2008) A newparadigm in leaf-level photosynthesis: direct and diffuse lights are notequal. Plant Cell Environ 31: 159–164

Evans JR (2009) Potential errors in electron transport rates calculated fromchlorophyll fluorescence as revealed by a multilayer leaf model. PlantCell Physiol 50: 698–706

Evans JR, Vogelmann TC (2003) Profiles of C-14 fixation through spinachleaves in relation to light absorption and photosynthetic capacity. PlantCell Environ 26: 547–560

Garbary DJ, Kim KY (2005) Anatomical differentiation and photosyntheticadaptation in brown algae. Algae 20: 233–238

Gorton HL, Brodersen CR, Williams WE, Vogelmann TC (2010) Mea-surement of the optical properties of leaves under diffuse light. Photo-chem Photobiol 86: 1076–1083

Govindjee, Braun BZ (1974) Light absorption emission and photosynthe-sis. In WDP Stewart, ed, Botanical Monographs: Algal Physiology andBiochemistry, Vol 10. University of California Press, Berkeley, CA, pp346–390

Ichiro T, Hiroki O, Takashi F, Riichi O (2016) Light environment within aleaf. II. Progress in the past one-third century. J Plant Res 129: 353–363

Johnsen G, Samset O, Granskog L, Sakshaug E (1994) In vivo absorptioncharacteristics in 10 classes of bloom-forming phytoplankton: taxonomic

characteristics and responses to photoadaptation by means of discrim-inant and HPLC analysis. Mar Ecol Prog Ser 105: 149–157

Kilburn MR, Jones DL, Clode PL, Cliff JB, Stockdale EA, Herrmann AM,Murphy DV (2010) Application of nanoscale secondary ion mass spec-trometry to plant cell research. Plant Signal Behav 5: 760–762

Klughammer C, Schreiber U (2015) Apparent PS II absorption cross-section and estimation of mean PAR in optically thin and dense sus-pensions of Chlorella. Photosynth Res 123: 77–92

Kühl M (2005) Optical microsensors for analysis of microbial communities.Methods Enzymol 397: 166–199

Kühl M, Cohen Y, Dalsgaard T, Jørgensen BB, Revsbech NP (1995)Microenvironment and photosynthesis of zooxanthellae in scleractiniancorals studied with microsensors for O2, pH and light. Mar Ecol Prog Ser117: 159–172

Kühl M, Jørgensen BB (1994) The light-field of microbenthic communities:radiance distribution and microscale optics of sandy coastal sediments.Limnol Oceanogr 39: 1368–1398

Kühl M, Polerecky L (2008) Functional and structural imaging of photo-trophic microbial communities and symbioses. Aquat Microb Ecol 53:99–118

Lichtenberg M, Kühl M (2015) Pronounced gradients of light, photosyn-thesis and O2 consumption in the tissue of the brown alga Fucus serratus.New Phytol 207: 559–569

Lichtenberg M, Larkum AWD, Kühl M (2016) Photosynthetic acclimationof Symbiodinium in hospite depends on vertical position in the tissue ofthe scleractinian coral Montastrea curta. Front Microbiol 7: 230

Lichtenthaler HK, Ac A, Marek MV, Kalina J, Urban O (2007) Differencesin pigment composition, photosynthetic rates and chlorophyll fluores-cence images of sun and shade leaves of four tree species. Plant PhysiolBiochem 45: 577–588

Lichtenthaler HK, Babani F (2004) Light adaptation and senescence of thephotosynthetic apparatus: changes in pigment composition, chlorophyllfluorescence parameters and photosynthetic activity. In GC Papageorgiou,Govindjee, eds, Chlorophyll a Fluorescence: A Signature of Photosynthesis.Springer, Dordrecht, The Netherlands, pp 713–736

Lichtenthaler HK, Buschmann C, Döll M, Fietz HJ, Bach T, Kozel U,Meier D, Rahmsdorf U (1981) Photosynthetic activity, chloroplast ul-trastructure, and leaf characteristics of high-light and low-light plantsand of sun and shade leaves. Photosynth Res 2: 115–141

McMillen GG, McClendon JH (1979) Leaf angle: an adaptive feature ofsun and shade leaves. Bot Gaz 140: 437–442

Moss BL (1983) Sieve elements in the Fucales. New Phytol 93: 433–437Myers DA, Jordan DN, Vogelmann TC (1997) Inclination of sun and shade

leaves influences chloroplast light harvesting and utilization. PhysiolPlant 99: 395–404

Oguchi R, Douwstra P, Fujita T, Chow WS, Terashima I (2011) Intra-leafgradients of photoinhibition induced by different color lights: implica-tions for the dual mechanisms of photoinhibition and for the applicationof conventional chlorophyll fluorometers. New Phytol 191: 146–159

Pons TL, Flexas J, von Caemmerer S, Evans JR, Genty B, Ribas-Carbo M,Brugnoli E (2009) Estimating mesophyll conductance to CO2: metho-dology, potential errors, and recommendations. J Exp Bot 60: 2217–2234

Privoznik KG, Daniel KJ, Incropera FP (1978) Absorption, extinction andphase function measurements for algal suspensions of Chlorella pyr-enoidosa. J Quant Spectrosc Radiat Transf 20: 345–352

Ramus J (1978) Seaweed anatomy and photosynthetic performance: eco-logical significance of light guides, heterogeneous absorption and mul-tiple scatter. J Phycol 14: 352–362

Raven JA (2003) Long-distance transport in non-vascular plants. Plant CellEnviron 26: 73–85

Rickelt LF, Lichtenberg M, Trampe ECL, Kühl M (2016) Fiber-optic probesfor small-scale measurements of scalar irradiance. Photochem Photobiol92: 331–342

Sand-Jensen K, Krause-Jensen D (1997) Broad-scale comparison of pho-tosynthesis in terrestrial and aquatic plant communities. Oikos 80: 203–208

Sarijeva G, Knapp M, Lichtenthaler HK (2007) Differences in photosyn-thetic activity, chlorophyll and carotenoid levels, and in chlorophyllfluorescence parameters in green sun and shade leaves of Ginkgo andFagus. J Plant Physiol 164: 950–955

Schreiber U (2004) Pulse-amplitude-modulation (PAM) fluorometry andsaturation pulse method: an overview. In GC Papageorgiou, Govindjee,

732 Plant Physiol. Vol. 175, 2017

Lichtenberg et al.

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

eds, Chlorophyll a Fluorescence: A Signature of Photosynthesis. Springer,Dordrecht, The Netherlands, pp 279–319

Schreiber U, Bilger W, Neubauer C (1995) Chlorophyll fluorescence as anonintrusive indicator for rapid assessment of in vivo photosynthesis. InED Schulze, MM Caldwell, eds, Ecophysiology of Photosynthesis.Springer, Berlin, pp 49–70

Schreiber U, Klughammer C, Kolbowski J (2012) Assessment of wavelength-dependent parameters of photosynthetic electron transport with a newtype of multi-color PAM chlorophyll fluorometer. Photosynth Res 113:127–144

Schreiber U, Kühl M, Klimant I, Reising H (1996) Measurement of chlo-rophyll fluorescence within leaves using a modified PAM fluorometerwith a fiber-optic microprobe. Photosynth Res 47: 103–109

Serodio J (2004) Analysis of variable chlorophyll fluorescence in micro-phytobenthos assemblages: implications of the use of depth-integratedmeasurements. Aquat Microb Ecol 36: 137–152

Shibata K, Haxo FT (1969) Light transmission and spectral distribution throughepi- and endozoic algal layers in the brain coral, Favia. Biol Bull 136: 461–468

Slattery RA, Grennan AK, Sivaguru M, Sozzani R, Ort DR (2016) Lightsheet microscopy reveals more gradual light attenuation in light-greenversus dark-green soybean leaves. J Exp Bot 67: 4697–4709

Spilling K, Titelman J, Greve TM, Kühl M (2010) Microsensor measure-ments of the external and internal microenvironment of Fucus vesiculosus(Phaeophyceae). J Phycol 46: 1350–1355

Sun J, Nishio JN, Vogelmann TC (1996) High light alters photosyntheticcarbon fixation gradients across sun and shade leaves. Plant Cell Envi-ron 19: 1261–1271

Sun J, Nishio JN, Vogelmann TC (1998) Green light drives CO2 fixationdeep within leaves. Plant Cell Physiol 39: 1020–1026

Szabó M, Wangpraseurt D, Tamburic B, Larkum AW, Schreiber U,Suggett DJ, Kühl M, Ralph PJ (2014) Effective light absorption andabsolute electron transport rates in the coral Pocillopora damicornis. PlantPhysiol Biochem 83: 159–167

Takahashi K, Mineuchi K, Nakamura T, Koizumi M, Kano H (1994) Asystem for imaging transverse-distribution of scattered-light and chlo-rophyll fluorescence in intact rice leaves. Plant Cell Environ 17: 105–110

Terashima I, Fujita T, Inoue T, Chow WS, Oguchi R (2009) Green lightdrives leaf photosynthesis more efficiently than red light in strong whitelight: revisiting the enigmatic question of why leaves are green. PlantCell Physiol 50: 684–697

Trampe E, Kolbowski J, Schreiber U, Kühl M (2011) Rapid assessment ofdifferent oxygenic phototrophs and single-cell photosynthesis with

multicolour variable chlorophyll fluorescence imaging. Mar Biol 158:1667–1675

Vogelmann TC, Björn LO (1984) Measurement of light gradients andspectral regime in plant-tissue with a fiber optic probe. Physiol Plant 60:361–368

Vogelmann TC, Bornman JF, Josserand S (1989) Photosynthetic lightgradients and spectral regime within leaves of Medicago sativa. PhilosTrans R Soc Lond B Biol Sci 323: 411–421

Vogelmann TC, Bornman JF, Yates DJ (1996) Focusing of light by leafepidermal cells. Physiol Plant 98: 43–56

Vogelmann TC, Evans JR (2002) Profiles of light absorption and chloro-phyll within spinach leaves from chlorophyll fluorescence. Plant CellEnviron 25: 1313–1323

Vogelmann TC, Han T (2000) Measurement of gradients of absorbed lightin spinach leaves from chlorophyll fluorescence profiles. Plant Cell En-viron 23: 1303–1311

Vogelmann TC, Martin G (1993) The functional-significance of palisadetissue: penetration of directional versus diffuse light. Plant Cell Environ16: 65–72

Wang L, Jacques SL, Zheng L (1995) MCML: Monte Carlo modeling oflight transport in multi-layered tissues. Comput Methods ProgramsBiomed 47: 131–146

Wangpraseurt D, Jacques SL, Petrie T, Kühl M (2016a) Monte Carlomodeling of photon propagation reveals highly scattering coral tissue.Front Plant Sci 7: 1404

Wangpraseurt D, Pernice M, Guagliardo P, Kilburn MR, Clode PL,Polerecky L, Kühl M (2016b) Light microenvironment and single-cellgradients of carbon fixation in tissues of symbiont-bearing corals. ISME J10: 788–792

Wangpraseurt D, Wentzel C, Jacques SL, Wagner M, Kühl M (2017) Invivo imaging of coral tissue and skeleton with optical coherence to-mography. J R Soc Interface 14: 20161003

Webb WL, Newton M, Starr D (1974) Carbon dioxide exchange of Alnusrubra: a mathematical model. Oecologia 17: 281–291

White AJ, Critchley C (1999) Rapid light curves: a new fluorescencemethod to assess the state of the photosynthetic apparatus. PhotosynthRes 59: 63–72

Yukihira N, Sugai Y, Fujiwara M, Kosumi D, Iha M, Sakaguchi K,Katsumura S, Gardiner AT, Cogdell RJ, Hashimoto H (2017) Strate-gies to enhance the excitation energy-transfer efficiency in a light-harvesting system using the intra-molecular charge transfer characterof carotenoids. Faraday Discuss 198: 59–71

Plant Physiol. Vol. 175, 2017 733

Imaging of Light Absorption and Photosynthesis

www.plantphysiol.orgon September 11, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.