Environ. Sci. Technol. 2004, 38, 4165-4168

Chlorophyll Fluorescence Imaging ofIndividual Algal Cells: Effects of
Herbicide on Spirogyra distenta
at Different Growth Stages
RYOSUKE ENDO AND KENJI OMASA*
Graduate School of Agricultural and Life Science,
The University of Tokyo, 1-1-1 Yayoi Bunkyo,
Tokyo 113-8657, Japan

Serious environmental degradation of aquatic ecosystems
has been caused by eutrophication and by pollutants
such as herbicides. Therefore, measurement of in situ algal
photosynthetic activity is important for environmental
monitoring. With ordinary nonimaging fluorometers, algal
chlorophyll fluorescence can be measured easily, butheterogeneity within samples cannot be detected. Effects
of a herbicide preparation containing 3-(3,4-dichlorophenyl)1,1-dimethylurea (DCMU) on photosynthetic activity at different
growth stages of Spirogyra distenta were investigatedby using a computer-aided microscopic imaging systemfor chlorophyll a fluorescence. Photosystem II photochemicalyield (φPSII) images were used to diagnose photosynthetic
activity of spiral filate chloroplasts in algal cells. The
herbicide treatment caused a stronger decline in φPSII values
in younger than in mature algae cells. This result indicatedthat heterogeneity within algal samples should beconsidered when algae are used for environmental
monitoring. Thus, measurement of chlorophyll fluorescencefrom young and mature chloroplasts with a microscopic
imaging system makes it possible to improve the sensitivity
for monitoring the environmental degradation of aquatic
ecosystems.

Introduction
In recent years, serious environmental degradation of aquatic
ecosystems in lakes, ponds, and rivers has been caused byhuman activities (1). Such ecosystems are greatly eutrophiedby high rates of nitrogen and phosphorus release fromagricultural fields (2). Furthermore, aquatic pollution arisingfrom applications of agrochemicals to fields also severely
affects aquatic ecosystems (3). Because green algae act as
producers in aquatic ecosystems, the ecological balance iseasily lost if algal photosynthesis is affected by environmental
degradation. Hence, measurement of in situ algal photosynthetic activity is important for monitoring environmental
degradation of aquatic ecosystems (4).
For nondestructive assessment of photosynthetic activity,
chlorophyll a fluorescence measurement is very effective(5-8). Various instrumentations have been developed foranalyzing chlorophyll fluorescence of aquatic plants and algae(9-11). Recently, for highly sensitive water toxicity biotests,
a new type of dual-channel pulse-amplitude modulation(PAM) chlorophyll fluorometer has become available (10).
* Corresponding author phone: +81-3-5841-5340; fax: +81-35841-8175; e-mail: aomasa@mail.ecc.u-tokyo.ac.jp.
10.1021/es035375+ CCC: $27.50
Published on Web 06/24/2004

 2004 American Chemical Society

Many results pertaining to aquatic degradation caused byeutrophication and herbicides have been obtained with thistype of instrument (1, 4, 12, 13). Haynes et al. (1) assessed
the impact of diuron pollution on photosynthesis of three
species of tropical seagrasses and pointed out that diuron isa potential risk to photosynthetic activity by seagrass innearshore sediments. Frickelf and Senger (14) reported aboutchanges in diuron sensibility during the life cycle of trichome.
Schreiber et al. (12) showed that measurement of chlorophyll
fluorescence in outdoor freshwater mesocosms was a reliable
and effective method for monitoring the effects of atrazine
pollution.
Nevertheless, the above-mentioned results were allachieved with nonimaging instruments; consequently, heterogeneity within samples was not taken into account. The
application of imaging techniques makes the spatial analysisof chlorophyll fluorescence possible (15-20). Recently,
microscopic fluorescence imaging systems have been developed for cellular-level, and even chloroplast-level, analyses(21-24). Oxborough and Baker (21) observed chlorophyll
fluorescence images of individual stomata of intact leaves
and calculated the photosystem II (PSII) photochemical yield,
φPSII, which is a measure of linear electron transport activity
through PSII. Additionally, Oxborough et al. (24) usedmicroscopic fluorescence and φPSII images to examine
individual and interspecific differences in photosynthetic
activity of benthic phytoplankton, caused by an increase oflight intensity. Confocal microscopic imaging of chlorophyll
fluorescence has also been used to analyze the phylogeny ofgreen algae and the origin of grana (25, 26).
Endo and Omasa (23) recently applied a microscopic
fluorescence imaging technique to observe the effects of theherbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)
on photosynthetic activity of mature algae. However, to dateno one has investigated the effect of any herbicide on
photosynthetic activities of algae at different growth stages.
We report the effect of the herbicide containing DCMU as
its main component on photosynthetic activity in individual
algae at different growth stages. Microscopic imaging dataof chlorophyll fluorescence were measured with our computer-aided microscopic imaging system, which is capableof resolving cell organelles. Inhibition of photosynthetic
activities by the herbicide was compared in young and mature
algae by calculating their φPSII image values.

Experimental Section
Culture Conditions and Herbicide Treatment. Spirogyra
distenta (green microalga) was cultured at the agricultural
field of the University of Tokyo. Algae were incubated in atest tube at 24 °C/22 °C (day/night) air temperature in acontrolled environment incubator (Eyela, Eyelatron FLI301N) under fluorescent lights with a photosynthetic photon
flux (PPF) of 180 µmol of photons m-2 s-1. The photoperiod
was 12 h/12 h (day/night).
The herbicide preparation Nekosogi-ace (Rainbow Yakuhin, Japan) was used in the experiment. Nekosogi-ace contains 6.0% DCMU, which inhibits photosynthetic electrontransport from PSII to photosystem I (PSI) at the thylakoid
membrane in the chloroplast.
Computer-Aided Microscopic Imaging System for Chlorophyll a Fluorescence. The computer-aided microscopic
imaging system for analyzing chlorophyll a fluorescence(Figure 1) uses an optical microscope with a 20× objective
lens (Mitutoyo, BD Plan Apo 20), which has a long working
distance and a high numerical aperture. A halogen lamp
(Nikon, PSM-1520) with a 620-nm cutoff filter (Corning, 4-96)
VOL. 38, NO. 15, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4165

FIGURE 1. Computer-aided microscopic imaging system forchlorophyll a fluorescence: (A) actinic light source; (B) light source
for saturation pulse; (C) short-pass filter (λ g 620 nm); (D) culture
solution containing Spirogyra distenta; (E) objective lens (20×); (F)long-pass filter (λ g 640 nm) for imaging only chlorophyll
fluorescence; the reflection image is taken by removing F filter; (G)cooled charge-coupled device (CCD) camera; (H) monitor; (I) digital
video recorder; (J) computer.
and heat-absorbing filters provides 200 µmol of photons m-2
s-1 actinic light. A metal halide lamp (Sumita, LS-M180),
filtered as described above, provides a 2-s saturating lightpulse of 2000 µmol of photons m-2 s-1, which causes atransient saturation of PSII photochemistry. The lightpulse is controlled by an electric shutter (EMS, shutter
controller). Chlorophyll a fluorescence excited by theselights is imaged by a cooled charge-coupled device (CCD)
camera (Hamamatsu Photonics, C5985-02) through an optical filter (Corning 2-64, wavelength λ g 640 nm) in the lensbarrel of the microscope. Subsequently, fluorescence images
are recorded by a digital video recorder (Sony, DSR-V10) andconverted into 8-bit gray scale images with a resolution of
640 × 480 pixels. A fluorescent card that has a constantfluorescence yield at any light intensity was used for
confirming the relationship between fluorescence intensity
and the analog-to-digital conversion level of the obtained
images. The result showed good linearity.
Analysis of Effects of the Herbicide on Spirogyra distenta.
A drop of culture solution containing S. distenta was placedin the hollow of a depression slide. Then the preparation
was set on the microscope stage, where it was exposed to a
PPF of 200 µmol of photons m-2 s-1 actinic light. Air
temperature was kept at 24 °C. After 10 min of adaptation
time on the microscope stage, a fluorescence intensity image
F was measured under the actinic light. Just after that measurement, a fluorescence intensity image Fm′ was measured
during a 2-s saturation light pulse during steady-state
photosynthesis. Each intensity image (F and Fm′) was divided
by the average value of fluorescence emitted from afluorescent standard induced by the corresponding lightintensity to convert it into a relative yield image. Finally, theφPSII was calculated (27, 28):Fm ′F
RSL RAL
ΦPSII )Fm ′RSL
where RSL and RAL are values of fluorescence emitted by thefluorescent standard induced by the saturation light pulse
and the actinic light, respectively.
4166 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 15, 2004

FIGURE 2. Schematic diagram of the life cycle of Spirogyra distenta.
S. distenta has two reproductive modes: sexual, in which a diploidzygote divides by meiosis, and asexual, in which haploid cells
divide by mitosis. In the asexual mode, two small chloroplasts
located centrally extend to both ends of the cell, creating a double
spiral shape (stages 1-3). Strands grow in length by somatic cell
division and then break, thus forming more individuals.
Subsequently, the Nekosogi-ace solution was preparedand then a drop was added to the S. distenta solution on the
preparation. The DCMU concentration in the final S. distentasolution was approximately 3 × 10-6 M. After a 15-min
adaptation period, the measurement and analysis sequencedescribed above was repeated.

Results and Discussion
A schematic diagram of the life cycle of an individual cell of
Spirogyra distenta is shown in Figure 2. S. distenta has two
reproductive modes: sexual, in which a diploid zygote divides
by meiosis, and asexual, in which haploid cells divide bymitosis. Each haploid S. distenta cell has one centrally locatednucleus and one to three spiral filate chloroplasts, and eachcell is part of a continuous line of numerous cells. The spiralchloroplasts increase in length and density as they grow(Figure 2, stages 1-3). Reproduction in this species is usually
asexual. Therefore, the effects of DCMU on S. distenta were
examined at different growth stages of haploid cells.
Figure 3A shows a reflection image of two S. distenta
filaments observed at a resolution of 0.5 µm under actinic
light at 200 µmol m-2 s-1 PPF. The spiral ribbonlike organelles
are algal chloroplasts at two different haploid growth stages(see Figure 2). Other kinds of cell organelles without
pigmentation could not be observed. The algae on the rightside of Figure 3 are mature (stage 3 in Figure 2) and thoseon the left are younger (stage 1 in Figure 2), as determinedfrom the shapes of their chloroplasts.
The distribution of φPSII values in control algal chloroplasts,
before the herbicide treatment, was calculated from the Fm′and F images (Figure 3B). The microscope background, slide,
and aquatic solution did not fluoresce in the experiment.
The φPSII images contained heterogeneity caused by various
factors. Defocus and nonuniform absorption of light causedby the spiral and uneven shapes of the chloroplasts in theFm′ and F images might lead to inaccurate values of φPSII atsome sites. However, these sites were omitted by comparing
the Fm′ and F images with the reflection image shown in
Figure 3A. An example of a possible physiological factor was
an area with low φPSII values, indicated by the dotted arrow
in Figure 3B, where the reflection image showed no difference.
This phenomenon was temporary; it disappeared after lessthan a minute (data not shown).
Two areas (area a within a young chloroplast and area b
within a mature chloroplast; Figure 3B) showed in-focus anduniform reflection (see Figure 3A). The φPSII values in thoseareas ranged from 0.39 to 0.46 in the mature alga (site b in

FIGURE 4. Effects of DCMU (3 × 10-6 M) treatment on OPSII valuesof Spirogyra distenta at different growth stages (stages 1-3 inFigure 2; see Figure 3). The OPSII values were obtained at sites with
in-focus and uniform reflectance, such as a and b in Figure 3.
Experimental conditions were the same as those in Figure 3.

FIGURE 3. Microscopic reflection image (A) and OPSII images justbefore DCMU (3 × 10-6 M) treatment (B) and 15 min after treatment(C) of Spirogyra distenta at different growth stages. Haploid stages
correspond to stages 1 and 3 in Figure 2. (a, b) Areas with in-focus
and uniform reflectance. Images were taken at PPFs of 200 µmol
m-2 s-1 actinic light and during a saturating light pulse of 2000
µmol m-2 s-1. Scale bar ) 50 µm.
Figure 3B) and from 0.35 to 0.44 in the younger alga (site ain Figure 3B). The difference in φPSII values between chloroplasts of site a and those of site b was small. This result
indicates that in the younger chloroplast the electrontransport activity from PSII to PSI was already similar to thatin the mature chloroplast under ordinary conditions. Moreover, that activity was nearly uniform within the algal
chloroplast.
After a 15-min herbicide treatment of these algae (Figure3C), the φPSII values decreased. A different response wasobserved, however, between mature and younger chloroplasts. The φPSII values decreased to 0.28-0.35 at sites in areab, in the mature alga, and to 0.13-0.23 at sites in area a, inthe younger alga. Thus, after the treatment, the φPSII valuesof the younger chloroplast decreased more than those of themature chloroplast. This result shows the younger chloroplast
was more sensitive to the herbicide than the mature
chloroplast. Incidentally, no treatment effect was observedin the reflection image.
Figure 4 shows the effects of the herbicide treatment on
the φPSII values of chloroplasts in S. distenta cells at different
growth stages (stages 1-3 in Figure 2). The φPSII values wereobtained at sites with in-focus and uniform reflectance. Before
the treatment, no difference in φPSII values was found among
the three stages (control in Figure 4). However, differencesin the amount of decrease of φPSII values were found between
these stages after the herbicide treatment; in younger algae,
the decrease was higher than in mature algae. This result
indicates that the photosynthetic response of the algae tothis herbicide depends on their growth stage. The differencein the effect may indicate differences in permeability of thecell wall or the cell membrane to the herbicide solution. It
would be expected that younger chloroplasts would be more
permeable and therefore would take up more DCMU. This
scenario would result in a higher concentration of DCMU in
younger algae cells than the mature algae and would causea larger difference in φPSII values in the younger algae cells.
Because the surface area and volume of chloroplasts differ
at each stage, uptake of the herbicide into the chloroplast
may also differ, leading to varying concentrations within thechloroplast. However, to determine the effect of the herbicide
on photosynthetic PSII activity per unit chloroplast atdifferent growth stages, an in-depth physiological analysis
would be required.
Algal chlorophyll fluorescence can be measured easily
with ordinary nonimaging fluorometers. However, photosynthetic heterogeneity within samples is not detectable bythat technique. Our results indicated that young chloroplasts
are more sensitive to a herbicide solution than mature
chloroplasts, indicating that heterogeneity within algal
samples should be taken into account when the algae areused for environmental monitoring. Thus, measurement ofchlorophyll fluorescence from young chloroplasts with amicroscopic imaging system makes it possible to improvesensitivity when monitoring environmental degradation of
aquatic ecosystems.

Acknowledgments
We thank Takateru M. Uenishi and Kotaro Takayama for
helpful discussion and comments on the manuscript. We
are grateful to the Japan Society for the Promotion of Sciencefor funding this study.

Literature Cited(1) Haynes, D.; Ralph, P.; Prange, J.; Dennison B. The impact of theherbicide diuron on photosynthesis in three species of tropicalseagrass. Mar. Pollut. Bull. 2000, 41, 288-293.
(2) Carpenter, S. R.; Caraco, N. F.; Correll, D. L.; Howarth, R. W.;
Sharpley, A. N.; Smith, V. H. Nonpoint pollution of surface waters
with phosphorus and nitrogen. Ecol. Appl. 1998, 8, 559-568.
(3) Christopher, S. V.; Bird, K. T. J. The effects of herbicides ondevelopment of Myriophyllum spicatum L. cultured in vitro.
Environ. Qual. 1992, 21, 203-207.
VOL. 38, NO. 15, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4167

(4) Conrad, R.; Büchel, C.; Wilhelm, C.; Arsalane, W.; Berkaloff, C.;
Duval, J. C. J. Changes in yield in in-vivo fluorescence ofchlorophyll a as a tool for selective herbicide monitoring. Appl.
Phycol. 1993, 5, 505-516.
(5) Krause, G. H.; Weis, E. Chlorophyll fluorescence and photosynthesis - the basics. Annu. Rev. Plant Physiol. Mol. Biol. 1991,
42, 313-349.
(6) Govindjee. 63 years since Kautsky - Chlorophyll-a fluorescence.
Aust. J. Plant Physiol. 1995, 22, 131-160.
(7) Lichtenthaler, H. K. In vivo chlorophyll fluorescence as a tool
for stress detection in plants. In Applications of Chlorophyll
Fluorescence; Lichtenthaler H. K.; Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1988; pp 129-142.
(8) Lichtenthaler, H. K.; Rinderle, U. The role of chlorophyll
fluorescence in the detection of stress conditions in plants. CRC
Crit. Rev. Anal. Chem. 1988, 19, S29-S85.
(9) Häder, D. P.; Kumar, H. D.; Smith, R. C.; Worrest, R. C. Effects
on aquatic ecosystems. Annu. Rev. Plant Physiol. Mol. Biol. 1998,
46, 53-68.
(10) Lu, C. M.; Chau, C. W.; Zhang, J. H. Acute toxicity of excess
mercury on the photosynthetic performance of cyanobacterium,
S. platensis - assessment by chlorophyll fluorescence analysis.
Chemosphere 2000, 41, 191-196.
(11) Jochem, F. J. Probing the physiological state of phytoplankton
at the single-cell level. Sci. Mar. 2000, 64, 183-195.
(12) Schreiber, U.; Müller, J. F.; Haugg, A.; Gademann, R. New typeof dual-channel PAM chlorophyll fluorometer for highly sensitive
water toxicity biotests. Photosynth. Res. 2002, 74, 317-330.
(13) Seguin, F.; Le Bihan, F.; Leboulanger, C.; Berard, A. A risk
assessment of pollution: induction of atrazine tolerance inphytoplankton communities in freshwater outdoor mesocosms,
using chlorophyll fluorescence as an endpoint. Water Res. 2002,
36, 3227-3236.
(14) Frickelf, B.; Senger, H. Changes in DCMU-sensibility during
life-cycle of Scenedesmus obliquus. Ber. Deut. Bot. Ges. 1972,
85, 401-408.
(15) Omasa, K.; Shimazaki, K.; Aiga, I.; Larcher, W.; Onoe, M. Image
analysis of chlorophyll fluorescence transients for diagnosing
the photosynthetic system of attached leaves. Plant Physiol.
1987, 84, 748-752.
(16) Daley, P. F.; Raschke, K.; Ball, J. T.; Berry, J. A. Topography ofphotosynthetic activity of leaves obtained from video imagesof chlorophyll fluorescence. Plant Physiol. 1989, 90, 1233-1238.
(17) Lichtenthaler, H. K.; Miehe, J. A. Fluorescence imaging as adiagnostic tool for plant stress. Trends Plant Sci. 1997, 2, 316320.
(18) Govindjee; Nedbal, G. L. Seeing is believing. Photosynthetica
2000, 38, 481-482.

4168 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 15, 2004

(19) Kim, M. S.; McMurtrey, J. E.; Mulchi, C. L.; Daughtry, C. S. T.;
Deitzer, G.; Chappelle, E. W. Steady-state multispectral fluorescence imaging system for plant leaves. Appl. Opt. 2001, 40,
157-166.
(20) Omasa, K.; Takayama, K. Simultaneous measurement of stomatal conductance, nonphotochemical quenching, and photochemical yield of photosystem II in intact leaves by thermaland chlorophyll fluorescence imaging. Plant Cell Physiol. 2003,
44, 1290-1300.
(21) Oxborough, K.; Baker, N. R. An instrument capable of imaging
chlorophyll a fluorescence from intact leaves at very low
irradiance and at cellular and subcellular levels of organization.
Plant Cell Environ. 1997, 20, 1473-1483.
(22) Küpper, H.; Šetlı́k, I.; Trtı́lek, M.; Nedbal, L. A microscope fortwo-dimensional measurements of in vivo chlorophyll fluorescence kinetics using pulsed measuring radiation, continuous
actinic radiation, and saturating flashes. Photosynthetica 2000,
38, 553-570.
(23) Endo, R.; Omasa, K.; Kondo, J. Microscopic image instrumentation of chlorophyll a fluorescence from in situ microalgae. EcoEngineering 2002, 14, 17-22.
(24) Oxborough, K.; Hanlon, A. R. M.; Underwood, G. J. C.; Baker,
N. R. In vivo estimation of the photosystem II photochemical
efficiency of individual microphytobenthic cells using highresolution imaging of chlorophyll a fluorescence. Limnol.
Oceanogr. 2000, 45, 1420-1425.
(25) Gunning, B. E. S.; Schwartz, O. Confocal microscopy of thylakoid
autofluorescence in relation to origin of grana and phylogeny
in the green algae. Aust. J. Plant Physiol. 1999, 26, 695-708.
(26) Osmond, C. B.; Schwartz, O.; Gunning, B. E. S. Photoinhibitory
printing on leaves, visualised by chlorophyll fluorescenceimaging and confocal microscopy, is due to diminishedfluorescence from grana. Aust. J. Plant Physiol. 1999, 26, 717724.
(27) Genty, B.; Briantais, J. M.; Baker, N. R. The relationship between
the quantum yield of photosynthetic electron-transport andquenching of chlorophyll fluorescence. Biochim. Biophys. Acta
1989, 990, 87-92.
(28) van Kooten, O.; Snel, J. F. H. The use of chlorophyll fluorescence
nomenclature in plant stress physiology. Photosynth. Res. 1990,
25, 147-150.

Received for review December 10, 2003. Revised manuscript
received April 29, 2004. Accepted May 6, 2004.
ES035375+