light sheet microscopy imaging of light absorption and photosynthesis distribution in plant tissue
TRANSCRIPT
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Short title: Imaging of light absorption and photosynthesis 1
Corresponding authors: 2
Mads Lichtenberg, [email protected]; Michael Kühl, [email protected] 3
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Light sheet microscopy imaging of light absorption and photosynthesis 5
distribution in plant tissue 6
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Mads Lichtenberg1,a,b
, Erik C. L. Trampe1,a
, Thomas C. Vogelmann2 and Michael Kühl
1,3,b 8
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1 Marine Biological Section, Department of Biology, University of Copenhagen, 10
Strandpromenaden 5, 3000 Helsingør Denmark 11
2 Department of Plant Biology, University of Vermont, 12
63 Carrigan Drive, Burlington, VT, USA 13
3 Climate Change Cluster (C3), University of Technology Sydney, 14
15 Broadway, Ultimo, Sydney, NSW 2007, Australia 15
a These authors contributed equally to this work 16
b Corresponding authors 17
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Author contributions: ML, ET, TV and MK designed the research; ML and ET performed 20
experiments; TV and MK contributed new analytical tools; ML, ET, TV and MK analyzed data; 21
ML and ET wrote the article with contributions from TV and MK. 22
23
Funding: This study was supported by a Sapere-Aude Advanced grant from the Danish Council for 24
Independent Research ǀ Natural Sciences (MK), and grants from the Carlsberg Foundation (MK). 25
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One sentence summary: Fine scale characterization of light absorption and photosynthesis across 27
plant tissue sections show that quantum yields of PSII are highly affected by tissue light gradients. 28
Plant Physiology Preview. Published on August 18, 2017, as DOI:10.1104/pp.17.00820
Copyright 2017 by the American Society of Plant Biologists
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Abstract 29
In vivo variable-chlorophyll-fluorescence measurements of PSII quantum yields in optically dense 30
systems are complicated by steep tissue light gradients due to scattering and absorption. 31
Consequently, externally measured effective PSII quantum yields may be composed of signals 32
derived from cells differentially exposed to actinic light, where cells located deeper inside tissues 33
receive lower irradiance than cells closer to the surface, and can display distinct photophysiological 34
status. We demonstrate how measured distributions of PSII quantum yields in plant tissue change 35
under natural tissue light gradients as compared to conventionally measured quantum yields with 36
even exposure to actinic light. This was achieved by applying actinic irradiance perpendicular to 37
one side of thallus cross-sections of the aquatic macrophyte Fucus vesiculosus L. with laser light-38
sheets of defined spectral composition, while imaging variable-chlorophyll-fluorescence from 39
cross-sections with a microscope-mounted pulse-amplitude-modulated (PAM) imaging system. We 40
show that quantum yields are highly affected by light gradients and that traditional surface-based 41
variable-chlorophyll-fluorescence measurements result in substantial under- and/or over-42
estimations, depending on incident actinic irradiance. We present a method for using chlorophyll 43
fluorescence profiles in combination with integrating sphere measurements of reflectance and 44
transmittance to calculate depth-resolved photon absorption profiles, which can be used to correct 45
apparent PSII electron transport rates to photons absorbed by PSII. Absorption profiles of the 46
investigated aquatic macrophyte were different in shape from what is typically observed in 47
terrestrial leaves, and based on this finding we discuss strategies for optimizing photon absorption 48
via modulation of the structural organization of phytoelements according to in situ light 49
environments. 50
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Keywords: absorption profile, chlorophyll fluorescence, laser sheet microscopy, light attenuation, 52
photosynthesis, quantum yield. 53
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Introduction 54
Estimating photosynthetic parameters using variable chlorophyll fluorescence techniques has 55
become increasingly popular due to its ease of use and non-invasive nature. The basic fluorescence 56
signals of ‘open’ or ‘closed’ reaction centers (F and Fm, respectively) change according to actinic 57
irradiance and are powerful monitors of the status and activity of the photosynthetic apparatus 58
(Baker, 2008). Most measurements of variable chlorophyll fluorescence in complex plant tissues, 59
and in other surface-associated cell assemblages like biofilms and sediments, rely on external 60
measurements with fiber-optic or imaging fluorimeters under the assumptions that i) different cells 61
are subjected to the same amount of measuring light and actinic irradiance, ii) that saturating pulses 62
are indeed saturating all cells, and iii) that the fluorescence detected is emitted equally from all 63
sampled cells (Serodio, 2004). These assumptions are influenced by the optical density of the 64
sample where optical dilute refers to a negligible or only moderate light attenuation through a 65
sample (e.g. a dilute algal suspension or plant tissue with only a few cell layers), while optically 66
dense samples such as algal biofilms and thicker plant tissues absorb all, or most of, the incident 67
light. As a result the assumptions are usually valid in optically dilute samples (Klughammer and 68
Schreiber, 2015), whereas steep light gradients in densely pigmented tissues or algal biofilms will 69
distort the measurements of maximal and effective PSII quantum yields. Cells located deeper inside 70
tissues will receive less actinic irradiance than cells close to the surface. Thus, externally integrated 71
measurements of variable chlorophyll fluorescence contain a complex mixture of signals originating 72
from different layers in the structure exposed to different levels of measuring and actinic light, and 73
the actual operational depth of such measurements remains unknown. This inherent limitation of 74
such measurements can e.g. lead to light-dependent overestimations of effective PSII quantum 75
yields of up to 40% e.g. in microphytobenthic assemblages (Serodio, 2004). 76
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Previous efforts to describe the internal gradients of photosynthetic efficiencies have used 77
microfiber based pulse amplitude modulation (PAM) techniques (Schreiber et al., 1996) revealing 78
distinct differences between such internal and external variable chlorophyll fluorescence 79
measurements (Oguchi et al., 2011). Another challenge is to quantify the internal light gradients to 80
estimate the total actinic light exposure in different tissue layers, i.e. the scalar irradiance. The 81
scalar component becomes increasingly important in deeper tissue layers as light becomes 82
progressively more diffuse due to multiple scattering (Kühl and Jørgensen, 1994). This can be 83
measured with fiber optic scalar irradiance microprobes (Kühl, 2005; Rickelt et al., 2016), which 84
collect light isotropically via a small (30-150 µm wide) spherical tip cast on the end of a tapered 85
optical fiber. Such measurements enabled estimates of internal rates of PSII electron transport 86
corrected for the specific tissue light gradients in corals and plants (Lichtenberg and Kühl, 2015; 87
Lichtenberg et al., 2016). However, to obtain absolute electron transport rates (ETR) through PSII, 88
it is necessary to know the absorption factor, which describes the PSII absorption cross-section and 89
the balance between PSI and PSII photochemistry, and these parameters cannot be calculated from 90
measurements of light availability. In addition, due to the small tip size of fiber optic radiance 91
microprobes (usually <50 µm) used to detect the fluorescence, microfiber-based measurements of 92
variable chlorophyll fluorescence are also prone to reflect the natural heterogeneity of such systems 93
(Lichtenberg and Kühl, 2015; Lichtenberg et al., 2016). A method was recently proposed for 94
calculating absolute electron turnover rates of PSII, but the approach was limited to surface 95
measurements or optically thin systems (Szabó et al., 2014). It is thus of great importance to further 96
explore how steep gradients of light influence photosynthetic efficiencies in complex 97
photosynthetic tissues and surface associated phototrophic communities. 98
Internal gradients of light absorption have been quantified from fluorescence profiles in terrestrial 99
leaves (Takahashi et al., 1994; Vogelmann and Han, 2000; Slattery et al., 2016) and this technique 100
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has been combined with fine scale measurements of CO2 fixation to investigate the relationship 101
between chlorophyll concentration, light absorption and photosynthesis at high spatial resolution 102
(Vogelmann and Evans, 2002; Evans and Vogelmann, 2003). These studies generally found a good 103
correlation between the light absorption of different spectral ranges, and the associated CO2 fixation 104
profiles. However, the CO2 fixation rates relied on freeze clamping 14
CO2 pre-incubated leaf 105
samples with concomitant paradermal sectioning, and measurements by scintillation counting, 106
which is a laborious process that is limited in the spatial resolution by the sectioning process to ~40 107
µm (Vogelmann and Evans, 2002). Here we present a novel experimental approach and show its 108
application for mapping gradients of light absorption and photosynthesis in aquatic plant tissue. 109
The lower community photosynthesis often observed in aquatic systems as compared to terrestrial 110
systems (Sand-Jensen and Krause-Jensen, 1997) can be largely explained by the inability of aquatic 111
macrophytes to obtain an optimal 3D structural organization in relation to the incident irradiance 112
(Binzer and Sand-Jensen, 2002b, a), unlike their terrestrial counterparts that e.g. can regulate leaf 113
inclination to increase canopy light utilization (McMillen and McClendon, 1979; Myers et al., 114
1997). In addition, specialized cell/tissue structures in terrestrial plants can increase photon 115
absorption, e.g. in sun-adapted leaves with well-developed palisade cells that can act as light 116
funnels directing light into the photosynthetically active mesophyll layer (Vogelmann and Martin, 117
1993), while some shade-adapted understory plants can alleviate light-limitation by focusing light 118
in the mesophyll layer via plano-convex epidermal cells and intercellular air spaces (Vogelmann et 119
al., 1996; Brodersen and Vogelmann, 2007). In contrast, most macroalgae are not recognized to 120
have specialized tissue structures to facilitate penetration of light, although there has been reports of 121
light guides in some green algae (Ramus, 1978). 122
Macroalgal members of the Fucales have morphological differentiated tissues such as the basal 123
thallus, the growing sterile frond, and fertile receptacles, while cells are differentiated into 124
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meristoderm, cortex, and medullary layers on the tissue scale (Garbary and Kim, 2005). While all 125
cell types contain plastids (Moss, 1983), the outer meristoderm and cortex cells contain more 126
chloroplasts and thylakoids than the medullary filaments. It has been suggested that the medullary 127
filaments could play a role in longitudinal translocation of materials (Moss, 1983; Raven, 2003), 128
and further that they may play a structural role in providing elasticity in terms of a ‘cushion-like’ 129
effect protecting against wave action (Moss, 1983). The medulla layer is surrounded on both sides 130
by anatomically similar layers of cortex, meristoderm and epidermis cells (henceforth referred to as 131
cortex), in contrast to e.g. bifacial terrestrial plant leaves that display morphologically and 132
physiologically differentiated abaxial- and adaxial domains. In Fucus, steep gradients of light and 133
photosynthesis have been measured using fiber-optic microprobes and microelectrodes, although 134
this approach is rather challenging in such cohesive tissues (Spilling et al., 2010; Lichtenberg and 135
Kühl, 2015). 136
In this study, we aimed to resolve how photosynthetic efficiencies are affected by steep light 137
gradients in different spectral regions. This was accomplished by the use of a novel multicolor laser 138
light sheet microscopy setup to image the distribution of light absorption and photosynthetic 139
activity over transverse sections of an aquatic macrophyte to resolve how photosynthetic 140
efficiencies are affected by steep light gradients in different spectral regions. We applied laser light-141
sheets of defined spectral composition perpendicular to one side of thallus cross-sections while 142
imaging the distribution of chlorophyll fluorescence and variable-chlorophyll-fluorescence from the 143
cut surface. We compared such data with measurements obtained with equal illumination of the 144
cross-section to describe for the first time how PSII quantum yields are affected by natural light 145
gradients in optically dense tissues. This novel method can resolve such gradients routinely and 146
with higher resolution as compared to other microscale approaches such as mapping with fiber-147
optic probes (Kühl and Jørgensen, 1994; Lichtenberg et al., 2016). 148
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149
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Results 150
Cross-thallus chlorophyll fluorescence profiles 151
Using a novel microscopic setup (Fig. 1; see further details in the Methods section), we used both 152
even illumination of plant tissue cross-sections and illumination with a laser-sheets of defined 153
spectral composition incident perpendicularly on tissue cross sections. When illuminated 154
homogeneously across the algal thallus cross-section, both cortex layers of F. vesiculosus displayed 155
an equally high amount of chlorophyll that was 2.5-5 fold higher than in the central medulla (Fig. 2 156
and Fig. 3), assuming that relative chlorophyll content can be estimated from fluorescence using 157
epi-illumination (Vogelmann and Evans, 2002). The fluorescence profiles under light sheet 158
illumination perpendicular to one side of the cross-section showed, that blue light (425-475 nm) 159
was attenuated strongest in an exponential manner with depth and decreased to <21% of the 160
maximum fluorescence (F(max)) ~250 µm inside the thallus (Fig. 3). Fluorescence profiles over the 161
thallus cross-section using green (525-575 nm), and red (615-665 nm) light showed similar 162
attenuation but decreased to a minimum fluorescence >2 times higher than was found for blue light 163
at a similar depth in the thallus. Blue, green and red light induced fluorescence profiles all displayed 164
F(max) values close to the thallus surface. When using broadband white light illumination, a peak 165
was located at the same position as the F(max) of the blue, green and red profiles followed by an 166
intermittent decrease before reaching F(max) ~100 µm inside the thallus (Fig. 3). Common for all 167
profiles was that the fluorescence showed a peak close to the illuminated cortex followed by a 168
decrease towards the center of the medulla before increasing again towards the shaded cortex. The 169
relative largest increase towards the shaded thallus side was in the order of blue < red < < green < 170
white. The width of the peaks was of similar size and extended 150-200 µm from the surfaces 171
towards the center of the thallus (Fig. 3). 172
173
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174
Absorption profiles 175
Integrating sphere measurements of thallus reflectance, transmittance and absorptance displayed 176
typical characteristics for densely pigmented opaque plant tissues (Fig. 4). Reflectance was 177
relatively uniform at ~3% of the incident irradiance, although slightly higher in the green/yellow 178
part of the spectrum (around 570 nm). Absorptance spectra (Fig. 4 and S1) showed in vivo 179
absorption peaks from major photopigments present in brown macroalgae, e.g., Chl a (440 and 675 180
nm (Johnsen et al., 1994)), Chl c (460; 590; 635 nm (Shibata and Haxo, 1969; Kühl et al., 1995)), 181
fucoxanthin (in vitro absorption peaks in hexane at 425, 450, 475 nm; extends to 580nm in vivo 182
(Govindjee and Braun, 1974)) and other carotenoids (400-540 nm (Govindjee and Braun, 1974)). 183
The mean absorptance averaged over PAR (400-700 nm) using broadband white light was 92% of 184
the incident irradiance. Transmittance was highest (10-13%) in the green/yellow part (around 570 185
nm) of the spectrum and was close to zero in the blue and red spectral regions, while the mean 186
transmittance was 5% of the incident irradiance (Fig. 4). 187
By normalizing the chlorophyll fluorescence profiles (Fig. 3) to the total absorption measured for 188
blue, green, red, and white light with an integrating sphere (Fig. 4) we could calculate the depth of 189
specific photon absorption inside the thallus (Fig. 4; see also Fig. S3). The different thallus regions 190
(cortex/medulla) were estimated to be on average 150 µm in thickness (Fig. 3). When illuminating 191
the thallus with the laser sheet, the apparent absorption of photons was always highest in the upper 192
and lower cortex as compared to the medulla, where the fractional absorption was lowest (Fig. 4; 193
Table 1). 194
We modelled the light availability in the F. vesiculosus thallus by using measured scalar irradiance 195
attenuation coefficients of cortex and medulla layers from a closely related brown alga F. serratus 196
(Lichtenberg and Kühl, 2015) assuming monoexponential attenuation of light in the thallus (Fig. 5; 197
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see text in Materials and Methods). These modelled curves of light attenuation were compared with 198
curves of attenuation due to absorption found in this study (Fig. 5) to test if the found absorption 199
profiles were in the same order as the attenuation profiles, as would be expected for such densely 200
pigmented systems. We found an average light attenuation coefficient of 5.64 mm-1
(R2=0.97) over 201
the entire thallus, with higher attenuation coefficients in the cortex layers (upper cortex = 6.0 mm-1
202
(R2=0.99); lower cortex = 9.8 mm
-1 (R
2=0.96)) than in the medulla layer (4.3 mm
-1 (R
2=0.99)) (Fig. 203
5). These values were in the same order as the light attenuation coefficients of cortex and medulla 204
layers in F. serratus (6.8 mm-1
and 3.4 mm-1
for cortex and medulla, respectively (Lichtenberg and 205
Kühl, 2015)), suggesting that the distribution of photon absorption can be found by combination of 206
chlorophyll fluorescence profiles and measurements of total absorption. 207
208
PSII quantum yields and photosynthetic electron transport 209
In the dark-acclimated state, all thallus layers displayed a maximal PSII quantum yield of >0.6 210
indicating no major stress factor on photosynthetic performance due to cutting or sample handing 211
(Fig. S4). When applying actinic irradiance homogeneously over the cross-sections via the build in 212
LEDs of the microscope PAM system (Fig. 1), the effective PSII quantum yield decreased in all 213
thallus layers but more so in the medulla as compared to the cortex layers. The highest decrease was 214
found under high incident irradiance, where the effective PSII quantum yield in the medulla 215
decreased to <0.3 (Fig. 6). This pattern changed under actinic laser sheet illumination of the cross-216
section from one side. While the PSII quantum yield distribution was apparently unaffected by the 217
changed actinic light geometry in the dark-acclimated state and under very low irradiance, the PSII 218
quantum yield decreased rapidly over the illuminated cortex and reached levels of <0.1 under the 219
highest irradiance. Due to the strong light attenuation across the thallus, the PSII quantum yields in 220
the medulla and the shaded cortex layers decreased less than when illuminated homogeneously via 221
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the Imaging PAM actinic light source, and the effective PSII quantum yield in the shaded cortex 222
remained at levels similar to dark-acclimated states (>0.6) even at the highest irradiance (Fig. 6). 223
Apparent electron transport rates (ETR) through PSII were calculated for the illuminated (upper) 224
cortex, the medulla, and the shaded (lower) cortex, and the rates were corrected for the amount of 225
absorbed photons in the respective tissue layers. In all cases, the ETR rates in the upper- and lower 226
cortex layers were very similar when corrected for absorbed light. The medulla ETR rates were 227
similar to the cortex activity on the sub-saturated part of the ETR vs. irradiance curve but saturated 228
at higher irradiance (Fig. 7). 229
The slope of the ETR vs. absorbed light curve under blue light was lower than for red and white 230
light but reached higher ETR rates (Fig. 7; Table 1). The curves appeared similar under green and 231
red light, although green light yielded a lower slope on the sub-saturated part of the ETR vs. 232
absorption curve. The ETR curves measured under broadband white light appeared qualitatively as 233
a combination of the curves measured under blue, green and red light, where saturation occurred at 234
higher irradiance, similar to the green and red curves, but reaching higher ETR values probably 235
caused by the blue light component. However, the slopes on the subsaturated part of the ETR vs. 236
irradiance curves were significantly different between white light and the average of the blue, green 237
and red curves in all thallus layers (Table 1). The green and red ETR vs. absorbed light curves were 238
similar in appearance in correspondence with their associated absorption profiles that were also 239
similar (Fig. 4 and Fig. 7). Surprisingly, the ETR curves under blue light did not reach saturation 240
and ETR rates in the cortex and medulla layers were very similar except at the highest irradiance, 241
where a decrease in the medulla layer was observed (Fig. 7). Even at the highest irradiance, PSII 242
quantum yields in the cortex layers remained high, and only a small increase in the NPQ was 243
observed (Fig. S4), which could point to better light processing properties of blue light compared 244
green and red light. 245
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247
248
Discussion 249
A novel combination of multi-color light sheet microscopy with variable chlorophyll fluorescence 250
imaging enabled the mapping of light absorption and photosynthetic efficiencies in densely 251
pigmented tissues. By combining well tested methods of integrating sphere measurements and the 252
chlorophyll fluorescence profile technique (Takahashi et al., 1994; Vogelmann and Han, 2000; 253
Slattery et al., 2016) we propose a method for calculating profiles of photon absorption across plant 254
tissue sections, which can be combined with variable chlorophyll fluorescence imaging of 255
photosynthetic efficiency across tissue light gradients. 256
Conversion of PSII quantum yields measured by variable chlorophyll fluorescence to absolute rates 257
of photosynthetic electron transport activity requires precise measurements of i) mean effective 258
PAR, ii) the PSII absorption cross-section, and iii) knowledge about the partitioning between PSI 259
and PSII photochemistry (Klughammer and Schreiber, 2015). While such information can be 260
obtained in dilute suspensions of chloroplasts and microalgae (Klughammer and Schreiber, 2015), 261
to measure these parameters in dense algal solutions, plant tissue and algal biofilms is not trivial 262
(Szabó et al., 2014; Klughammer and Schreiber, 2015). In optically dense systems, light gradients 263
are affected by both multiple scattering and absorption and it is important to take diffuse light into 264
account when quantifying actinic light levels, i.e., by measuring the incident photon flux from all 265
directions with scalar irradiance microprobes (Kühl, 2005). While such sensors have tip diameters 266
down to 30 µm (Rickelt et al., 2016), it is difficult to perform scalar irradiance measurements in 267
thin, cohesive plant tissues, where measurements can be biased by tissue compression due to the 268
physical impact of the microprobe (Spilling et al., 2010; Lichtenberg and Kühl, 2015). The mean 269
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effective PAR can also be calculated from complex measurements of the angular radiance 270
distribution with field radiance microprobes (Vogelmann and Björn, 1984; Vogelmann et al., 1989; 271
Kühl and Jørgensen, 1994). Alternatively, information on the cell-size distribution, and the inherent 272
optical properties, i.e., the scattering phase function, and the scattering and absorption coefficients 273
allow calculations of PAR gradients, but these parameters are difficult to determine in optically 274
dense media (Privoznik et al., 1978; Berberoglu et al., 2009; Klughammer and Schreiber, 2015), 275
albeit recent experimental and theoretical advances in biomedical optics have allowed detailed 276
characterization of tissue optics using combinations of optical reflection spectroscopy, optical 277
coherence tomography and Monte Carlo simulations (Wang et al., 1995; Wangpraseurt et al., 278
2016a; Wangpraseurt et al., 2017). 279
An experimental solution to the above mentioned complications relies on measuring the chlorophyll 280
fluorescence profile, which represents the net outcome of photon absorption along the actinic light 281
gradient in the tissue (Takahashi et al., 1994; Vogelmann and Han, 2000). By correlating 282
fluorescence profiles to total absorption, we could measure the direct result of absorption and the 283
values obtained are thus only affected by the quantum yield of fluorescence and energy transfer 284
between antenna pigment molecules and PSII and PSI. However, due to the invasive nature of the 285
method, some actinic light will be lost from the cut surface causing some underestimation of the 286
tissue absorption in situ (Terashima et al., 2016). Furthermore, this method allows estimates of the 287
distribution of photon absorption, but does not enable a separation of possible changes in the 288
absorption cross-section or balance between PSI and PSII absorption in different tissue layers. 289
We found total absorption values from integrating sphere measurements that were similar to 290
terrestrial leaves (Gorton et al., 2010), although the absorption of green/yellow light was higher in 291
F. vesiculosus due to the presence of accessory brown algal pigments such as fucoxanthin that 292
displays a high efficiency of energy transfer to Chl a (~95% (Yukihira et al., 2017)). Due to the low 293
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reflection and transmission in the thallus, the absorption will be in the same order as the light 294
attenuation. Comparing, the calculated attenuation of light due to absorption with the light 295
attenuation calculated using scalar irradiance microprofile measurements from a previous study 296
(Lichtenberg and Kühl (2015); see Materials and Methods) we found a whole-thallus absorption 297
coefficient that was lower than cortex attenuation coefficients and higher than medulla attenuation 298
coefficients (Fig. 5). We predicted identical absorption coefficients in the cortex layers since these 299
layers are not anatomically different and in addition displayed similar levels of chlorophyll 300
fluorescence under uniform epi-illumination. Surprisingly, the absorption coefficient of the shaded 301
cortex was larger than the one found in the illuminated cortex (Fig. 5). We speculate that the light 302
field angularity could have caused this difference. Previously it has been shown that absorptance of 303
plant tissue can be different under collimated vs. diffuse light (Brodersen and Vogelmann, 2010; 304
Gorton et al., 2010), and here the incident light on the illuminated cortex was collimated while light 305
reaching the shaded cortex had a higher diffuse component due to internal scattering. Further, we 306
note that our calculations were based on profiles of blue light, which was almost completely 307
absorbed, making the calculations of absorption in the shaded cortex more prone to errors due to the 308
lower signal/noise ratio. Absorption profiles are further complicated due to the self-absorption of 309
red fluorescence by chlorophyll. Thus, red fluorescence profiles are likely to better represent 310
absorption profiles than profiles of far-red fluorescence (Vogelmann and Han, 2000). Here, we used 311
a long-pass filter (>670 nm) to detect fluorescence, and the resulting profiles therefore comprised 312
both red- and far-red chlorophyll fluorescence, and future studies should therefore divide the 313
detected fluorescence signals into red- and far-red fluorescence. We also note that our laser sheet 314
had a Gaussian beam profile, which makes positioning close to the edge of the cut thallus surface 315
difficult and may create a potential spill-over of photons onto the cut side. This limitation could be 316
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resolved by shaping the beam e.g. by the generalized-phase-contrast method (Bañas et al., 2014) to 317
transform the Gaussian beam profile to a sharp rectangular shape and such work is now underway. 318
The shape of the white fluorescence profile across the thallus was slightly different in appearance 319
than the profiles for blue, green and red light. Previously, it was shown that profiles of carbon 320
assimilation and chlorophyll fluorescence profiles followed each other closely depending on the 321
spectral quality (Sun et al., 1996; Vogelmann and Han, 2000), and it has been proposed that profiles 322
of carbon fixation under white light can be described as the mean when using blue, green and red 323
light (Sun et al., 1998; Vogelmann and Han, 2000). Here we show that for plant tissue harboring a 324
range of accessory pigments such as fucoxanthin, the absorptive properties are more complex 325
resulting in a different response to white light than what can be expected from the combination of 326
measurements made under monochromatic light. Furthermore, it was shown that green light drives 327
photosynthesis more efficiently deeper in terrestrial plant tissue than blue and red light due to a 328
larger penetration depth of green light in leaf tissues (Sun et al., 1998; Terashima et al., 2009). In 329
contrast, the presence of fucoxanthin and carotenes in Fucus caused the green light (525-575 nm) to 330
be absorbed equally effective as red light. However, not all wavelengths were absorbed equally 331
effective, and e.g. the spectral region around 570-605 nm displayed reduced absorption as compared 332
to the other spectral regions of photosynthetically active radiation. Since only the white light 333
treatment covered this part of the spectrum, it is possible that illumination with broadband white 334
light caused the differently shaped absorption profile. This might be confirmed by measuring 335
additional chlorophyll fluorescence profiles using yellow light (e.g. 570-605 nm) to validate if this 336
would result in fluorescence profiles with Fmax located deeper in the thallus, similar to fluorescence 337
profiles in terrestrial leaves illuminated with green light (Vogelmann and Han, 2000). 338
339
Photosynthesis 340
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We demonstrate that PSII quantum yields and derived apparent ETR across the thallus cross-341
sections strongly differed between homogeneous actinic light illumination of the cross-section and 342
unidirectional actinic light illumination on one side of the thallus with a laser light sheet. Under low 343
incident irradiance, the yields were very similar in all layers and between measurements. However, 344
as incident light directly on the cut side increased, the yields decreased across the tissue, with the 345
highest decreases found in the medulla (Fig. 6). This was not the case under laser light sheet 346
illumination perpendicular to one side of the thallus surface, where we found strongly reduced 347
yields in the illuminated cortex, while yields in the shaded cortex were unaffected (Fig. 6), even at 348
the highest incident photon irradiance (1108 µmol photons m-2
s-1
). Thus, when applying actinic 349
light directly on a cross-section our data show that it is possible to both underestimate and 350
overestimate PSII quantum yields as compared to yields found under natural light gradients. Using 351
a multilayer leaf model, Evans (2009) found that as irradiance increased on the adaxial side, the 352
quantum yields were progressively reduced deeper into the mesophyll. However, quantum yields at 353
the abaxial side were unaltered even under high blue irradiance, which is in good agreement with 354
the findings of this paper. Evans (2009) showed that surface based measurements of ϕPSII to 355
estimate ETR was only valid in some cases but overestimated ETR when the leaf was inverted. 356
Similarly, mesophyll conductance measurements could be influenced when estimated using surface 357
based ϕPSII values (Evans, 2009). With our new method, ϕPSII values can now be measured under 358
natural tissue light gradients and can be corrected for photon absorption thus making it possible to 359
get more detailed insights into mesophyll conductance heterogeneities e.g. by using depth resolved 360
ϕPSII data in combination with gas exchange measurements (Evans, 2009; Pons et al., 2009). 361
The differences in PSII quantum yield measured under illumination directly on the cross-section or 362
perpendicular to the side of the thallus surface indicate the difference between the photosynthetic 363
potential under equal illumination and the realized photosynthesis under tissue light gradients. We 364
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18
show here that even under high incident irradiance, photosynthetic electron transport in the lower 365
cortex was not saturated, and an even illumination of tissue cross-sections will therefore 366
underestimate the PSII quantum yield as compared to shaded parts during high unidirectional 367
illumination. Apparent ETR rates in the cortex layers were very similar when corrected for 368
absorption, while the medulla layer displayed saturation and lower ETR rates at increasing 369
irradiance indicating a lower photosynthetic capacity, probably due to lower pigment content (Fig. 3 370
and Fig. 7). The slope of the initial part of the ETR curves, which is a measure of the light 371
utilization efficiency at subsaturating photon flux, were similar in all thallus layers albeit 372
consistently slightly lower in the medulla (Table 1). In a recent study, absolute ETR rates of thin-373
tissued corals were calculated (Szabó et al., 2014) and the rates found in this study were of similar 374
magnitude. Szabó et al. (2014) also found initial slopes of the sub-saturated part of the ETR vs. 375
irradiance curves that were slightly higher, probably reflecting differences in photochemical 376
acclimatization that have been shown to be tightly linked to the optical properties of coral tissues 377
(Lichtenberg et al., 2016; Wangpraseurt et al., 2016a; Wangpraseurt et al., 2016b). Fucus is often 378
found in the intertidal zone and thus, on a daily basis, experiences a variable light environment as a 379
function of water depth and concentration of organic and inorganic particles as well as dissolved 380
organic matter attenuating solar irradiance. Air exposed plant parts will therefore be subjected to the 381
full solar spectrum while the incident light field on algae situated in deeper oligotrophic waters will 382
be blue-shifted due to the absorption of red light by water. In contrast, the incident light field on 383
algae in shallow eutrophic waters will be green shifted due to absorption of blue and red 384
wavelengths by suspended phytoplankton. On a tissue scale, the medulla layer of Fucus will on 385
average experience the lowest photon irradiance compared to the cortex layers (Lichtenberg et al. 386
2015) and could therefore be thought of as a shade adapted compartment in the algal thallus. Shade-387
acclimatized phytoelements will normally display higher light use efficiencies (i.e. steeper initial 388
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19
slope on the photosynthesis-irradiance curve) but lower photosynthetic capacity (Pmax) due to 389
increased pigment content and biochemical regulations in the photosynthetic machinery and/or 390
ultrastructural changes in the chloroplasts (Lichtenthaler et al., 1981; Lichtenthaler and Babani, 391
2004; Lichtenthaler et al., 2007; Sarijeva et al., 2007). However, as the initial slopes of the ETR vs. 392
absorbed light curves in the medulla were both lower and displayed saturation at lower irradiances, 393
medulla layers cannot be described as photosynthetically shade-adapted in conventional terms. 394
Conversely, it appears that the structural organization of the thallus layers could be adapted to 395
maximize photon absorption in the outer cortex layers, while having a relatively translucent central 396
medulla with low absorptive properties. This is in contrast to terrestrial leaves, where even 397
illumination of tissue layers is achieved by increased internal scattering due to intercellular 398
airspaces with the concomitant absorption profiles following an exponential attenuation with depth 399
(Vogelmann and Han, 2000). This fundamental difference is in good agreement when considering 400
their respective position in terrestrial and aquatic habitats, as terrestrial leaves can organize their 401
position according to the angle of solar radiation, whereas aquatic macrophytes are limited in their 402
structural organization by strong drag and shearing forces imposed by waves and currents, 403
randomly exposing both sides of the thallus to direct light. By maximizing absorption in the outer 404
layers and having a translucent central layer, F. vesiculosus can thus maximize light harvesting by 405
allowing photons not absorbed in the illuminated thallus to propagate to tissue layers with unused 406
photosynthetic potential thereby ensuring a more efficient resource distribution. 407
The ETR vs. absorbed light curves measured in different tissue layers under laser light sheet 408
illumination were similar to what was previously found in Fucus (Lichtenberg and Kühl, 2015) 409
although these microfiber PAM-based measurements were associated with high standard deviations 410
due to the small measurement volume of the fiber-optic microprobe, which makes such 411
measurements prone to microscale tissue heterogeneity effects. With the experimental approach 412
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20
presented in this study, it is now possible to integrate photosynthetic responses from specific tissue 413
layers much more precisely, and in addition to correct them for the amount of photons absorbed by 414
that given tissue layer. Here we used a 10x microscope objective, but in principle such 415
measurements could be performed at even higher magnification e.g. to investigate single cell 416
gradients of light and photosynthetic efficiencies e.g. in large algal cells. The combination of multi-417
color light sheet microscopy with variable chlorophyll fluorescence imaging on plant tissue cross-418
sections provides an alternative to more destructive methods such as constructing profiles of CO2 419
fixation from paradermal sectioning (Evans and Vogelmann, 2003) or nanoscale secondary-ion-420
mass-spectroscopy (Kilburn et al., 2010; Wangpraseurt et al., 2016b) and allows sequential 421
measurements on the same sample e.g. under different levels of actinic irradiance or comparisons of 422
diffuse vs. collimated light fields (Brodersen et al., 2008). Here we demonstrated the application on 423
aquatic macrophyte tissue but the technique is readily applicable to many other types of plant 424
tissues including terrestrial leaves as well as photosynthetic biofilms and symbioses. 425
426
Conclusion 427
The combination of multicolor light sheet microscopy and variable chlorophyll fluorescence 428
imaging is a powerful technique that enables fine scale characterization of light absorption and PSII 429
quantum yields across plant tissue sections. Furthermore, quantification of photon absorption from 430
light sheet induced cross-tissue fluorescence profiles can be used in concert with variable 431
chlorophyll fluorescence imaging enabling calculations of ETR rates that otherwise require 432
knowledge of the absorption cross-section, and the mean effective PAR. 433
The spectral flexibility of a white super continuum laser source allows this method to be used in 434
other photosynthetic systems with different anatomical structures and pigmentation. In this manner, 435
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21
the role of specific accessory pigments in light propagation and photosynthesis can be further 436
investigated. 437
438
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22
Materials and Methods 439
Sample collection and preparation 440
Stands of the brown macroalga Fucus vesiculosus were collected in the littoral zone at various 441
locations around Helsingør, Denmark during late summer and were maintained in 10 liter buckets 442
continuously flushed with 0.2 µm filtered aerated seawater (temperature=16°C; salinity=32) for up 443
to one week prior to experiments. Samples were kept under a 12:12 h light:dark cycle under a 444
photon irradiance of ~50 µmol photons m-2
s-1
(PAR, 400-700 nm) as provided by a fluorescent 445
tube (Philips Master TL-D90, 18W; Philips, Amsterdam, the Netherlands). 446
Prior to measurements, an apical thallus fragment was cut ~1 cm from the thallus tip with a razor 447
blade, and the cut side was rinsed in seawater with a transfer pipette to wash away pigments leaking 448
from cut chloroplasts. The sample holder (Fig. 1) consisted of a standard plastic cuvette, cut down 449
to a height of 11 mm to allow insertion under the microscope (internal size = 10 x 10 x 10 mm). To 450
fix the sample in the cuvette, ~300 µL of 20 g L-1
seawater agar (Sigma-Aldrich) was transferred to 451
the cuvette and allowed to cool to 20°C, after which a slit was cut parallel to the cuvette window to 452
allow insertion of the thallus fragment. The thallus was inserted flush with the edge of the cuvette, 453
filled with seawater (16°C; salinity=32) and closed with a coverslip. 454
455
Chlorophyll fluorescence profiles
456
Profiles of absorbed light as estimated from chlorophyll fluorescence profiles across tissue sections 457
were imaged with a customized microscope setup (Fig. 1). The sample cuvette (see above) was 458
mounted on an inverted microscope (IX81; Olympus, Japan) with a 10x objective (UPlanSApo 459
10x/0.40; Olympus, Japan). The sample was illuminated perpendicular to one side of the thallus 460
surface by a supercontinuum laser (SuperK Extreme, EX-B, NKT Photonics, Denmark). The laser 461
was connected to a tunable single line filter module (SuperK Varia, NKT Photonics, Denmark) via 462
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23
a single mode fiber with a collimated output. The tunable single line filter could be tuned from 400-463
840 nm to produce bandwidths from 1-400nm. Light from the single line filter module was 464
delivered, via an alignment tool (SuperK Connect, NKT Photonics, Denmark), to an endlessly-465
single-mode-large-mode-area photonic crystal fiber (FD7, NKT Photonics, Denmark) with a 466
collimated output. The collimated output was connected to a cylindrical laser sheet generator (NKT 467
Photonics, Denmark) with a 14° “light-sheet half angle” yielding a 5 cm longitudinal line at 10 cm 468
distance. The generated laser sheet had a Gaussian beam profile of ~1 mm on the latitudinal axis. 469
The output laser optics was mounted on a manual micromanipulator (MM33; Märzhäuser, Wetzlar, 470
Germany) that allowed easy positioning of the laser sheet in the focal plane of the microscope. 471
The sample was positioned with the cut side facing the microscope objective and the laser sheet was 472
adjusted to hit as close to the edge of the cut as possible without illumination spill-over to the cut 473
side. After positioning, the sample was allowed to dark adapt for 15 min. 474
Images of chlorophyll fluorescence from the cross-section were taken with a sensitive charge-475
coupled device (CCD) camera (iXon, Andor, UK) using a fixed exposure time of 70 ms. 476
Chlorophyll fluorescence was detected by placing a ultra-steep longpass edge filter (BLP01-664R, 477
Semrock, USA) in the light path between objective and the camera with a transmission close to 478
100% at wavelengths >670nm and close to 0% at wavelengths <670nm. Illumination in different 479
spectral bands was achieved by control of the laser and the spectral filtering module with the 480
manufacturers software (NKTP Control, NKT Photonics, Denmark). Four different spectral 481
illumination bands were composed (Fig. 1) and adjusted to the same photon irradiance (1190.3 ± 482
1.9 µmol photons m-2
s-1
) as measured with a micro quantum sensor (MC-MQS, Walz GmbH, 483
Germany) connected to a calibrated quantum irradiance meter (ULM-500, Walz GmbH, Germany). 484
We used broadband illumination (≥50nm) to ensure excitation of both PSII and PSI, thus avoiding 485
eventual red-drop effects. 486
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24
Illumination of the sample during individual image acquisitions was limited to <2 s per image, and 487
the spectral sequence was randomized between replicates. The effect of irradiance on the emitted 488
fluorescence was tested by treatment with DCMU (3-(3,4-dichlorophenyl)-1, 1-dimethyl-urea) (see 489
Supplementary Information; Fig. S2). Images were analyzed in ImageJ (v. 1.50B), where grey 490
values were extracted either by the line profile tool or by extracting all grey values. The images had 491
a spatial resolution of 0.8x0.8 µm pixel-1
. Maximum chlorophyll fluorescence was normalized to 1 492
in all images to allow comparison between images. Plots were made in OriginPro (v. 9.3, Origin 493
Lab, MA, USA). The thickness of the thallus varied, both between samples and depending on 494
location in the cross-section (Fig. 2). Therefore, profiles of fluorescence were taken at thallus 495
thicknesses of ~450 µm to allow comparison of thallus light gradients over the same tissue 496
thickness. 497
498
Integrating sphere measurements of reflectance and transmittance 499
Thallus reflectance and transmittance were measured using an integrating sphere (diameter = 10 500
cm; port diameters = 2.5 cm, Labsphere Instruments Inc., USA). The sphere had 3 port openings; 501
two located opposite each other and one orthogonal to the two other openings (Fig. S1). The 502
incident light from the supercontinuum laser was tuned to different spectral ranges each with the 503
same photon irradiance (see text above and Fig. 1). Light was measured with a calibrated spectral 504
irradiance meter (MSC15, Gigahertz Optik GmbH, Germany) connected to the orthogonally located 505
port on the integrating sphere. For transmittance measurements, a thallus fragment was mounted in 506
front of the entrance port between the light source and the integrating sphere and the port opposite 507
to the entrance was covered with a white reflecting plate. For reflectance measurements, a thallus 508
fragment was placed in the port opening opposite to the incident laser beam at an angle of 5-10° to 509
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25
capture both the specular and diffuse reflection. Total absorptance (A) by the thallus was estimated 510
as: 511
512
𝐴 = ∫(𝐼−𝑅−𝑇)
𝐼
𝜆𝑛
𝜆𝑖 (1) 513
514
where I is the incident photon irradiance, R is the reflectance and T is the transmittance, all 515
integrated over the spectral region of interest [λi - λn]. 516
517
Modelling of light attenuation 518
Light attenuation profiles were modelled using attenuation coefficients, α, of blue scalar irradiance 519
(420-520 nm) in the cortex and medulla layers measured in a previous study (Lichtenberg and Kühl, 520
2015). The model assumed monoexponential attenuation of incident irradiance, I0, over tissue depth 521
intervals Δz and the light availability in different tissue depths was then calculated as: 522
523
𝐼𝑧 = 𝐼0 ∙ 𝑒−𝛼∙∆𝑧 (2) 524
525
These data were compared to the estimated attenuation, due to absorption quantified as induced 526
fluorescence and corrected for total absorption (Fig. S3) across the thallus under blue irradiance 527
(425-475 nm) in this study. The attenuation due to absorption was calculated by subtracting the 528
cumulative absorption (Fig. 4), integrated in 50 µm increments, from the incident irradiance. 529
530
Variable chlorophyll fluorescence imaging 531
Pulse-amplitude-modulated variable chlorophyll fluorescence imaging (Imaging-PAM) with the 532
saturation pulse method (Schreiber et al., 1995; Schreiber, 2004; Kühl and Polerecky, 2008) was 533
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26
used to assess the photosynthetic performance over cross-sections of the algal thallus mounted in 534
cuvettes as described above. Measurements were performed with a microscope Imaging-PAM 535
system (Fig. 1) fitted with a Red-Green-Blue (RGB) LED excitation lamp (IMAG-RGB; Heinz 536
Walz GmbH, Effeltrich Germany) as described in detail elsewhere (Trampe et al., 2011). The 537
microscope was fitted with a high numerical aperture objective (10x/NA0.8, Plan-Apochromate, 538
Carl Zeiss GmbH, Germany). Fast measurements of the effective PSII quantum yield under 539
increasing photon irradiance (each irradiance step was applied for 20 s) were used to measure so-540
called rapid light curves (RLC) (White and Critchley, 1999) of relative PSII electron transport vs. 541
irradiance curves. Two different approaches were applied: i) Using increasing actinic irradiances of 542
red light (590-650nm) as provided by the system-default internal RGB-LED lamp for equal 543
excitation of the exposed cross-section of the thallus from above, in combination with the 544
customizable automated light curve function of the software provided by the system software 545
(ImagingWin, Heinz Walz GmbH, Germany); ii) Using the supercontinuum laser setup as described 546
above as an external actinic light source, illuminating the thallus with a light sheet perpendicular to 547
the thallus surface. The actinic light sheet was controlled manually in stepwise increments in sync 548
with the custom defined automated light curve function of the ImagingWin software facilitating a 549
semi-automated acquisition of RLC’s, while the system-default internal actinic RGB-LED light 550
source was disconnected. RLC’s with the laser light sheet were obtained with increasing actinic 551
irradiance of blue (425-475 nm), green (525-575 nm), red (615-665 nm) or white (400-700 nm) 552
light, respectively. Non-actinic modulated blue measuring light was provided by the build-in LEDs 553
of the ImagingPAM system during both approaches. The two setups were calibrated using a photon 554
irradiance meter connected to a cosine corrected mini quantum PAR irradiance sensor, (ULM-500, 555
MQS-B, Walz GmbH, Germany). All samples were allowed to dark adapt for 15 min before the 556
measurements started. When in the dark-acclimated state, all reactions centers of PSII were open, 557
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27
enabling imaging of the minimal fluorescence yield (F0). Upon exposure to a high-intensity 558
saturation pulse, all PSII reaction centers closed permitting imaging of the maximal fluorescence 559
yield (Fm) over the thallus cross-section. From these images, the maximum PSII quantum yield 560
could be calculated as in Schreiber (2004): 561
562
𝐹𝑉/𝐹𝑚 = (𝐹𝑚 − 𝐹0)/𝐹𝑚 (3) 563
564
From imaging of the fluorescence yield, F, while the sample was illuminated with a predefined 565
level of actinic light (PAR, in μmol photons m-2
s-1
), and the maximum fluorescence yield during a 566
saturation pulse, 𝐹𝑚′, images of the effective PSII quantum yield could be calculated as: 567
568
ϕ𝑃𝑆𝐼𝐼 = (𝐹𝑚′ − 𝐹)/𝐹𝑚′ (4) 569
570
From these values, the relative photosynthetic electron transport rate (rETR), is usually derived 571
using the equation: 572
573
𝑟𝐸𝑇𝑅 = ϕ𝑃𝑆𝐼𝐼 × PAR × AF (5) 574
575
where PAR is the incident photon irradiance, and AF (the absorption factor) is a constant set to 0.42 576
assuming that 84% of the incident light is absorbed (Demmig and Björkman, 1987) and an even 577
distribution of absorbed photons between photosystem II and I (Schreiber et al., 2012). In our study, 578
we estimated internal ETR rates (in units of µmol electrons m-2
s-1
) in different regions of the 579
thallus (cortex/medulla) by multiplying average ϕ𝑃𝑆𝐼𝐼 values (in units of µmol electrons m-2
s-1
580
(µmol photons m-2
s-1
)-1
) for a given area with the total amount of absorbed photons (in units of 581
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28
µmol photons m-2
s-1
) in that area. The wavelength-dependent absorption was calculated by 582
normalizing the chlorophyll fluorescence profiles to the total absorption (Fig. S3). This essentially 583
gives an estimate of the wavelength-dependent photon absorption at any given depth in the thallus, 584
assuming that one unit of fluorescence was the direct result of one unit of absorption. In reality 585
however, this is affected by factors such as the quantum yield of fluorescence and energy transfer 586
between antenna pigment molecules and PSII/PSI. The true photon absorption will therefore vary 587
slightly, and the calculated photon absorptions are thus only estimates. 588
The light saturation coefficient, i.e., the photon scalar irradiance at onset of light saturation of 589
photosynthesis, Ek was calculated as 𝐸𝑘 = 𝐸𝑇𝑅𝑚𝑎𝑥/𝛼, where 𝐸𝑇𝑅𝑚𝑎𝑥 is the maximum activity, 590
and α is the initial slope of the ETR vs. photon absorption curve; both parameters were obtained by 591
curve fitting of ETR measurements using an exponential function (Webb et al., 1974) by means of a 592
non-linear Levenberg-Marquardt fitting algorithm (OriginPro 2015, OriginLab Corporation, 593
Northampton, MA, USA). 594
595
Statistics 596
One-way ANOVAs were applied to test differences in the slopes of the ETR curves between 597
different light treatments. Data was first tested for normality (Shapiro-Wilk) and for equal variance. 598
Statistical analysis was performed in SigmaPlot (SigmaPlot v. 12.5) and significance level was set 599
to p<0.01. 600
601
Supplemental information 602
Figure S1. Schematic drawing of integrating sphere measurements of transmittance and reflectance. 603
Figure S2. Chlorophyll fluorescence profiles over cross-sections of apical thallus fragments of F. 604
vesiculosus. 605
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29
Figure S3. Concept of the calculations of photon absorption profiles across plant tissue cross-606
sections. 607
Figure S4. PSII quantum yield (Y(II)) and non-photochemical quenching (NPQ) as a function of 608
absorbed photons in the upper cortex, medulla and lower cortex of Fucus vesiculosus. 609
610
Acknowledgements 611
This study was supported by a Sapere-Aude Advanced grant from the Danish Council for 612
Independent Research ǀ Natural Sciences (MK), and instrument grants from the Carlsberg 613
Foundation (MK). We thank NKT Photonics, Denmark, for generous loan of the Supercontinuum 614
Laser and for excellent technical support. 615
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30
Table 1: Photosynthetic electron transport from PSII (ETR) vs. photon absorption curve parameters 616
and the fractional photon absorption (in % of total absorption) calculated for the upper cortex, 617
medulla and lower cortex under blue (425-475 nm), green (525-575 nm), red (615-665 nm) and 618
white (400-700 nm) irradiance applied perpendicular to one side of the thallus surface of F. 619
vesiculosus. Slopes on the sub-saturated part of the ETR vs. light curve, maximum ETR values and 620
the light acclimation index Ek were estimated from curve fitting of the ETR vs. photon absorption 621
curves with an exponential function (Webb et al., 1974) using a non-linear Levenberg-Marquardt 622
fitting algorithm. The RGB values were calculated as average curves of blue, green and red. 623
Pairwise superscript letters indicate statistically significant differences (One-way ANOVA; p<0.01; 624
n=5 for white; n=4 for RGB). 625
Upper 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 30.0
Green 0.47 225.31 482.46 44.0 0.45 81.33 180.71 26.0 0.47 228.90 489.60 30.0
Blue 0.54 878.52 1620.05 57.0 0.54 254.76 469.67 21.0 0.55 599.82 1082.76 22.0
White 0.60a 378.40 629.79 45.0 0.58b 125.48 216.22 26.0 0.60c 349.31 586.39 29.0
RGB 0.54a - - - 0.53b - - - 0.54c - - -
626
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31
Figure legends 627
Figure 1. Experimental setup for light sheet microscopy in combination with variable chlorophyll 628
fluorescence imaging. A) The sample holder consisted of a cuvette cut down to 11 mm height 629
(internal dimensions of 10 x 10 x 10 mm). The sample was mounted in agar in the botttom of the 630
sample holder, which was filled with seawater and then closed with a coverslip. B) Spectral 631
composition of the laser light used for measurements of chlorophyll fluorescence profiles and the 632
actinic light in measurement of variable chlorophyll fluorescence. The laser was adjusted to have 633
the same absolute photon irradiance independent of spectral composition. C) Schematic drawing of 634
the experimental setup for measuring chlorophyll fluorescence profiles. (a) The algal thallus sample 635
positioned in the cuvette, (b) microscope objective, (c) filter cube with longpass filter, (d) CCD 636
camera. Illumination of the sample was done with a PC-controlled super continuum laser connected 637
to a spectral line filter unit and a laser sheet generator. D) Schematic drawing of the experimental 638
setup for variable chlorophyll fluorescence microscopy. (a) sample fixed in the cuvette, (b) 639
microscope objective, (c) emission filter, (d) dichroic beam splitter cube, (e) dichroic filter, (f) 640
mirror (to occular), (g) CCD camera. Weak non-actinic modulated measuring light was provided by 641
a software-controlled RGB LED unit. Actinic light was provided perpendicular to one side of the 642
thallus surface by a PC-controlled super continuum laser connected to a spectral line filter unit and 643
a laser sheet generator. 644
645
Figure 2. Color-coded maps showing the distribution of Chl a fluorescence (normalized to max 646
fluorescence) of A) a Fucus vesiculosus thallus cross-section illuminated evenly over the cut side 647
with 430 nm light from a Xe-lamp (the plot is composed of multiple images taken through a 10x 648
objective, and stitched together using Adobe Photoshop), and B-E) Thallus cross-sections 649
illuminated perpendicular to one side of the thallus surface with a super continuum laser sheet of 650
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32
different spectral compositions of B) blue (425-475 nm), C) green (525-575 nm), D) red (615-665 651
nm) and E) white (400-700 nm) light. 652
653
Figure 3. A) Epifluorescence microscopy image (false colors) of a F. vesiculosus thallus cross-654
section illuminated evenly with blue light (430 nm) from a Xe-lamp, and B) the associated 655
fluorescence profile (normalized to max fluorescence). C) Example of a fluorescence image (false 656
colors) of a thallus fragment irradiated perpendicularly to one side of the thallus surface (arrow) 657
with a laser sheet of red light (615-665 nm) from a super continuum laser, and D) chlorophyll 658
fluorescence profiles of cross-sections of apical thallus fragments of F. vesiculosus irradiated 659
perpendicular to the thallus surface with different spectral bands of blue (425-475nm), green (525-660
575 nm), red (615-665) and white (400-700nm) light. Data was normalized to max fluorescence and 661
actual data points are spaced 0.8 µm apart. (n=5. Error bars are not shown for clarity but mean 662
relative S.D. was ±7.7%). 663
664
Figure 4. A) Spectral measurements of reflectance, transmittance and absorptance of a F. 665
vesiculosus thallus fragment using an integrating sphere (see Fig. S1). Data were recorded using 666
either incident blue (425-475 nm; blue lines), green (525-575 nm; green lines), red (615-665 nm; 667
red lines) or white (400-700 nm; black lines) laser light. Dashed lines indicate ± 1 S.D.; n=3. B) 668
Calculated absorption profiles of cross-sections of apical thallus fragments of F. vesiculosus 669
irradiated perpendicular to one side of the thallus surface with different spectral bands of blue (425-670
475 nm), green (525-575 nm), red (615-665 nm) and white (400-700 nm) laser light. Absorption 671
was calculated from the measured chlorophyll fluorescence profiles (Fig. 3D) and was normalized 672
to the bulk absorption measured with an integrating sphere (A). Dashed lines indicate the borders of 673
the cortex and medulla tissue layers. Data points are spaced 0.8 µm apart (n=5). 674
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33
675
Figure 5. Left panel shows plots of attenuation profiles of blue light (420-520 nm) calculated using 676
attenuation coefficients from the cortex (blue triangles) and medulla (red circles) layers 677
(Lichtenberg and Kühl, 2015), and a profile of the attenuation due to absorption of blue light (425-678
475 nm) estimated from the observed fluorescence profile (black squares). Right panel shows the 679
natural logarithm transformed absorption profile with linear fits in the cortex and medulla layers, 680
respectively (R2>0.95 for all fits). 681
682
Figure 6. Isopleths (A, B) and images (C-H) of effective PSII quantum yield in apical thallus 683
fragments of F. vesiculosus illuminated evenly on a cross-section or perpendicular on one side of 684
the thallus surface. Images were acquired under red light using either direct light from the build-in 685
LEDs of the variable chlorophyll fluorescence imaging system (590-650 nm), or light perpendicular 686
to the thallus surface provided by a super continuum laser (615-665 nm) connected to a tunable 687
single line filter and delivered via a laser sheet generator. The isopleths (A, B) show the influence 688
of actinic irradiance on the effective PSII quantum yield (in µmol electrons m-2
s-1
(µmol photons 689
m-2
s-1
)-1
) as function of depth in the tissue when illuminated either directly on the cross-section (A) 690
or perpendicular to the surface of the thallus (B). Illumination in B was given from left towards 691
right of the panel. Line profiles (line width = 15 pixel) were taken on thallus parts with similar 692
thickness (~250 µm) with cortex layers also displaying similar thicknesses (~50-75 µm). Panels C-693
H show images of effective PSII quantum yield in darkness, moderate (567 ± 18 µmol photons m-2
694
s-1
) and, saturating irradiance (1087 ± 30 µmol photons m-2
s-1
) under direct even illumination of the 695
cross-section (C-E) and with laser light sheet illumination perpendicular to the thallus surface 696
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34
(arrows) (F-H). Illumination in F-H was given from the bottom towards the top of the panels. 697
Scale-bar = 0.2 mm. 698
Figure 7. Apparent electron transport rates through PSII corrected for absorbed photons (Fig. 4). 699
Measurements were performed with 20 s acclimation to each increasing actinic irradiance level of 700
A) blue (425-475 nm), B) green (525-575 nm), C) red (615-665 nm) and D) white (400-700 nm) 701
light as provided by a laser sheet illuminating a thallus fragment perpendicular to one side of the 702
thallus surface. Data point represents means ± S.E. (n=5, except panel A where n=4). 703
704
Figure S1. A) Schematic drawing of integrating sphere measurements of transmittance (left) and 705
reflectance (right). Spectral quantities were calculated over the wavelengths of interest as 𝐴 =706
∫(𝐼−𝑅−𝑇)
𝐼
𝜆𝑛
𝜆𝑖 where I is the incident photon irradiance, R is the reflectance and T is the 707
transmittance, all integrated over the spectral region of interest [λi - λn]. B) Attenuance spectrum 708
calculated as –log(transmittance). 709
710
Figure S2. Chlorophyll fluorescence profiles over cross-sections of apical thallus fragments of F. 711
vesiculosus treated with 10 µM DCMU for 1h and then irradiated perpendicular to one side of the 712
thallus surface with different spectral bands of blue (425-475 nm), green (525-575 nm), red (615-713
665 nm) and white (400-700 nm) light (n=4. SD not shown for clarity but mean relative SD was ± 714
4.3%). 715
716
Figure S3. Concept of the calculations of photon absorption profiles across plant tissue cross-717
sections here illustrated for blue (425-475 nm) laser light sheet measurements on the brown alga 718
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35
Fucus vesiculosus. A) shows the raw fluorescence profile across the tissue as detected by the CCD 719
camera expressed in pixel grey values. B) shows the relative fluorescence normalized to the 720
maximum grey value. C) shows the wavelength-dependent, whole-thallus absorption calculated 721
from integrating sphere measurements of transmission and reflection and expressed in % of incident 722
irradiance. D) The depth distribution of photon absorption was calculated by normalizing the 723
fluorescence profile to the whole-thallus photon absorption, such that the area under the curve is 724
equal to the whole-thallus absorption. 725
726
Figure S4. PSII quantum yield (Y(II)) and non-photochemical quenching (NPQ) as a function of 727
absorbed photons in the upper cortex, medulla and lower cortex of Fucus vesiculosus. The tissue 728
was irradiated perpendicular to one side of the thallus surface with blue (425-475 nm) green (525-729
575 nm), red (615-665 nm) or white (400-700 nm) light as provided by a supercontinuum laser 730
connected to a tunable single line filter unit. See main-text for measurement details. 731
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36
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Figure 1. Experimental setup for light sheet microscopy in combination with variable chlorophyll �uorescence imaging. A) The sample holder consisted of a cuvette cut down to 11 mm height (inter-nal dimensions of 10 x 10 x 10 mm). The sample was mounted in agar in the botttom of the sample holder, which was �lled with seawater and then closed with a coverslip. B) Spectral composition of the laser light used for measurements of chlorophyll �uorescence pro�les and the actinic light in measurement of variable chlorophyll �uorescence. The laser was adjusted to have the same absolute photon irradiance independent of spectral composition. C) Schematic drawing of the experimental setup for measuring chlorophyll �uorescence pro�les. (a) The algal thallus sample positioned in the cuvette, (b) microscope objective, (c) �lter cube with longpass �lter, (d) CCD camera. Illumination of the sample was done with a PC-controlled super continuum laser connected to a spectral line �lter unit and a laser sheet generator. D) Schematic drawing of the experimental setup for variable chloro-phyll �uorescence microscopy. (a) sample �xed in the cuvette, (b) microscope objective, (c) emission �lter, (d) dichroic beam splitter cube, (e) dichroic �lter, (f ) mirror (to occular), (g) CCD camera. Weak non-actinic modulated measuring light was provided by a software-controlled RGB LED unit. Actinic light was provided perpendicular to one side of the thallus surface by a PC-controlled super continu-um laser connected to a spectral line �lter unit and a laser sheet generator.
Figure 1.
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Figure 2. Color-coded maps showing the distribution of Chl a �uorescence (normalized to max �uo-rescence) of A) a Fucus vesiculosus thallus cross-section illuminated evenly over the cut side with 430 nm light from a Xe-lamp (the plot is composed of multiple images taken through a 10x objec-tive, and stitched together using Adobe Photoshop), and B-E) Thallus cross-sections illuminated perpendicular to one side of the thallus surface with a super continuum laser sheet of di�erent spec-tral compositions of B) blue (425-475 nm), C) green (525-575 nm), D) red (615-665 nm) and E) white (400-700 nm) light.
Figure 2.
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Figure 3. A) Epi�uorescence microscopy image (false colors) of a F. vesiculosus thallus cross-section illuminated evenly with blue light (430 nm) from a Xe-lamp, and B) the associated �uorescence pro�le (normalized to max �uorescence). C) Example of a �uorescence image (false colors) of a thal-lus fragment irradiated perpendicularly to one side of the thallus surface (arrow) with a laser sheet of red light (615-665 nm) from a super continuum laser, and D) chlorophyll �uorescence pro�les of cross-sections of apical thallus fragments of F. vesiculosus irradiated perpendicular to the thallus surface with di�erent spectral bands of blue (425-475nm), green (525-575 nm), red (615-665) and white (400-700nm) light. Data was normalized to max �uorescence and actual data points are spaced 0.8 µm apart. (n=5. Error bars are not shown for clarity but mean relative S.D. was ±7.7%).
Figure 3.
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Figure 4. A) Spectral measurements of re�ectance, transmittance and absorptance of a F. vesiculo-sus thallus fragment using an integrating sphere (see Fig. S1). Data were recorded using either incident blue (425-475 nm; blue lines), green (525-575 nm; green lines), red (615-665 nm; red lines) or white (400-700 nm; black lines) laser light. Dashed lines indicate ± 1 S.D.; n=3. B) Calculated absorption pro�les of cross-sections of apical thallus fragments of F. vesiculosus irradiated perpen-dicular to one side of the thallus surface with di�erent spectral bands of blue (425-475 nm), green (525-575 nm), red (615-665 nm) and white (400-700 nm) laser light. Absorption was calculated from the measured chlorophyll �uorescence pro�les (Fig. 3D) and was normalized to the bulk absorption measured with an integrating sphere (A). Dashed lines indicate the borders of the cortex and medul-la tissue layers. Data points are spaced 0.8 µm apart (n=5).
Figure 4.
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Figure 5. Plots of attenuation profiles of blue light (420-520 nm) calculated using attenuation coefficients from the cortex (blue triangles) and medulla (red circles) layers (Lichtenberg and Kühl, 2015), and a profile of the attenuation due to absorption of blue light (425-475 nm) estimated from the observed fluorescence profile (black squares).
Figure 5.
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Figure 6. Isopleths (A, B) and images (C-H) of effective PSII quantum yield in apical thallus fragments of F. vesiculosus illuminated evenly on a cross-section or perpendicular on one side of the thallus surface. Images were acquired under red light using either direct light from the build-in LEDs of the variable chlorophyll fluorescence imaging system (590-650 nm), or light perpendicular to the thallus surface provided by a super continuum laser (615-665 nm) connected to a tunable single line filter and delivered via a laser sheet generator. The isopleths (A, B) show the influence of actinic irradiance on the effective PSII quantum yield (in µmol electrons m-2 s-1 (µmol photons m-2 s-1)-1) as function of depth in the tissue when illuminated either directly on the cross-section (A) or perpendicular to the surface of the thallus (B). Illumination in B was given from left towards right of the panel. Line profiles (line width = 15 pixel) were taken on thallus parts with similar thickness (~250 µm) with cortex layers also displaying similar thicknesses (~50-75 µm). Panels C-H show images of effective PSII quantum yield in darkness, moderate (567 ± 18 µmol photons m-2 s-1) and, saturating irradiance (1087 ± 30 µmol photons m-2 s-1) under direct even illumination of the cross-section (C-E) and with laser light sheet illumination perpendicular to the thallus surface (arrows) (F-H). Illumination in F-H was given from the bottom towards the top of the panels. Scale-bar = 0.2 mm.
Figure 6.
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Figure 7. Apparent electron transport rates through PSII corrected for absorbed photons (Fig. 4). Measurements were performed with 20 s acclimation to each increasing actinic irradiance level of A) blue (425-475 nm), B) green (525-575 nm), C) red (615-665 nm) and D) white (400-700 nm) light as provided by a laser sheet illuminating a thallus fragment perpendicular to one side of the thallus surface. Data point represents means ± S.E. (n=5, except panel A where n=4).
Figure 7.
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