Plant and Cell Physiology Advance Access published July 29, 2014
(Plant Cell Physiology)
Transient freezing behavior in photophobic responses of Euglena gracilis investigated in
a microfluidic device
Running Title: Transient freeze of Euglena photophobic responses
Kazunari OZASA
Bioengineering Lab., RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
e-mail : [email protected]
phone : +81-48-462-1111 ext.8409
fax
: +81-48-462-4658
Subject Areas: environmental and stress responses, new methodology
Number of b/w figures, color figures, tables: b/w = none, color = 7
type and number of supplementary material: none
© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant
Physiologists. All rights reserved. For Permissions, please e-mail: [email protected]
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Corresponding author
Transient freezing behavior in photophobic responses of Euglena gracilis investigated in
a microfluidic device
Running Title: Transient freeze of Euglena photophobic responses
Kazunari Ozasa1*, Jeesoo Lee2, Simon Song2, Mizuo Maeda1
RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2) Department of Mechanical Convergence Engineering, Hanyang University, 17
Haendang-dong, Seongdong-gu, Seoul, 133-791, Korea
Abbreviations: trace momentum (TM),
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1)
Abstract
We found that the transient freezing behavior in photophobic responses of Euglena gracilis is
a good indicator for the metabolic status of the cells. The transient blue-light photophobic
responses of Euglena gracilis cells were investigated on-chip using a new measurement,
‘trace momentum’ (TM), to quantitatively evaluate their swimming activity in real time.
When blue light of intensity >30 mW/cm2 was repeatedly switched on and off, a large
trace analysis at a blue light intensity of 40 mW/cm2 showed that 48% (on average, N = 15) of
tumbling Euglena cells ceased activity (‘freezing’) for 2–30s at the onset of blue-light-off
before commencing straightforward swimming,
while 45% smoothly commenced
straightforward swimming without delay. The proportion of freezing Euglena cells depended
on the blue light intensity (only 20% at 20 mW/cm2). When the cells were stimulated by four
blue light pulses at the higher intensity, without pre-exposure, the transient freezing behavior
was more prominent but, on repeating the stimuli after an 80 min interval in red light, the
same cells did not freeze. This shows that the metabolism of the cells had changed to
anti-freezing during the interval. The relation between the interval time with/without light
irradiation and the blue-light adaptation was elucidated experimentally. The origin of the
freezing behavior is considered to be a shortage of a metabolic substance that promotes
smooth switching of flagellum movement from on-site rotation mode to straightforward
swimming mode.
keywords: photophobic response, Euglena gracilis, blue light, transient responses,
adaptation, microfluidic aquarium
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negative spike in the TM was observed at the onset of the ‘blue-light-off’ phase. Single-cell
Introduction
Photosynthetic microorganisms have attracted much attention of researchers due to
their ability to produce oil and nutrition with environment-friendly manner [Chisti, 2007;
Schenk et al., 2008; Lardon et al., 2009; Brennan et al., 2010; Mahapatra et al., 2013]. Some
of them, such as Chlamydomonas reinhardtii [Stavis and Hirschberg, 1973; Rochaix, 1995;
Hegemann and Berthold 2009; Wakabayashi et al., 2011] and Euglena gracilis [Häder et al.,
photomovements [Nultsch and Häder, 1979; Sgarbossa et al., 2002; Braatsch and Klug, 2004],
in order to stay in a moderate light suitable for photosynthesis. They have developed
photo-kinesis [Chung et al., 2004], phototaxis [Sineshchekov et al., 2002], photophobic
responses [Diehn, 1969a; Bovee, 1982; Matsunaga et al., 1999], or photo-adaptation
[Greenblatt and Schiff, 1959]. These photoinduced kinetic responses have been investigated
by counting the number of cells that change their moving speed or directions according to
light irradiation [Lebert and Häder, 1997]. For instance, action spectra for the step-up/down
photophobic responses of Euglena gracilis have been measured by many researchers [Diehn,
1969b; Checcucci et al., 1976; Matsunaga et al., 1998] and have shown that blue light
(wavelength 350-500 nm) and UV light (250-300 nm) induce the photophobic responses
effectively [Diehn, 1973; Doughty, 1993; Matsunaga et al., 1998]. One of the recent
achievements for Euglena gracilis is the identification of the photoreceptor for their
photophobic response. Iseki et al. have found that photoactivated adenylyl cyclase (PAC) is
the photoreceptor [Iseki et al., 2002; Yoshikawa et al., 2005], which produces cAMP from
ATP by blue-light activation and mediates the photophobic response of Euglena. They also
showed that the PAC molecules are useful as photo-controllable signal-induction molecules
for optogenetics [Boyden et al., 2005; Tsunematsu et al., 2011], demonstrating
photo-activated neuron firing through genetically introduced PAC molecules [Nagahama et al.,
2007; Schröder-Lang et al., 2007]. Although the photoreceptor for the photophobic response
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1981; Ozasa et al., 2013a], are motile and show kinetic responses to light, so called
of Euglena has been identified, complicated metabolic processes involved in the photophobic
response of Euglena are still unclear. Micro-ethological approach will give an insight to the
metabolic processes of the photoresponses, as well as molecular level analysis of
proteins/enzymes related to the photoresponses.
The detailed ethological study on the photoresponses of photosynthetic
microorganisms is also important to achieve the well-controlled growth/culture of the
changes. For this purpose, single-cell level studies on the photoresponses of the
microorganisms with milli-second resolution are required. Recently, we have developed an
optical feedback system, in which optical monitoring/stimulation can be performed
dynamically with arbitrary intensity of light and a high spatial resolution [Ozasa et al., 2011;
Ozasa et al., 2013b; Ozasa et al., in press]. We demonstrated that the spatial distribution of
Euglena cells confined in a microfluidic device can be controlled via optical feedback through
the photophobic response of Euglena. The system also enables us to measure quantitatively
the photophobic response of Euglena cells in-situ under various intensity of light and to trace
the photophobic movements of single cells.
In this paper, we report an investigation of the photophobic responses of
Euglena gracilis, including transient freezing behaviors, variations of photoresponses, and
blue-light adaptation. By confining a batch of Euglena cells in a microfluidic device, their
swimming activities were measured quantitatively in real time under blue light repeatedly
switched on and off with increasing its intensity. The detailed movements of single cells in the
same batch were traced subsequently through video analysis. Blue-light adaptation was also
investigated by measuring the transient freezing behaviors after the interval of blue light pulse
exposures with changing the interval time and light irradiation condition.
Results
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microorganisms and to understand the mechanisms of metabolic adaptation to photo intensity
Dependence on blue light intensity
Figures 1b and c show temporal changes of ‘trace momentum’ (TM, see Materials
and Methods section) observed in response to blue light pulses of constant duration and
progressively increasing intensity. The blue light was switched on and off at 75 s intervals.
There were approximately 100 swimming cells in the micro-aquarium. The upper limit of the
and then gradually decayed between 30 and 60 min. This increase was attributed to an
increase in swimming speed evoked by the blue light [Melkonian et al., 1986], whereas the
rate of the decay varied from experiment to experiment, for unidentified reasons. As the blue
light intensity increased above approximately 5 mW/cm2, the photophobic response to blue
light was observed as a decrease in TM during the blue light on period. The difference in the
TM value between the blue light on and off periods increased as blue light intensity was
increased, up to approximately 30 mW/cm2. Above 30 mW/cm2, the recovery of TM during
the blue-light-off period (step-down response) became slower and a downward spike was
observed at the onset of each blue light off period. The slower recovery and negative spike are
clearly observed in a comparison between expanded sections of the record obtained at 20
mW/cm2 and 40 mW/cm2 (Fig. 1c). The negative spike revealed that the change in the
locomotion of Euglena cells from rotational tumbling to straightforward swimming in
response to blue light off was not instantaneous or smooth for blue light intensities above 30
mW/cm2. The TM recovery in Fig. 1c was fitted to an exponential curve with a time constant
of 6 s at 20 mW/cm2 and 17 s at 40 mW/cm2. A small delay of 1.5 s was inferred in the
exponential recovery at 40 mW/cm2, corresponding to the downward spike of TM.
The downward spike observed at the onset of each blue light off period was to be
categorized as step-down photophobic response, according to the terminology recommended
by Diehn et al. [Diehn et al., 1977]. However, the downward spike is a part of the recovery
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TM envelope obtained during blue light off period slowly increased between 10 and 30 min
process from step-up photophobic response for relatively higher light intensities, and differs
from the conventional step-down photophobic response that Euglena cells in a moderately
illuminated area turn their swimming direction at the boundary between moderate-light and
dark area to avoid going into the unfavorably dark area. In order to avoid confusion, we
described the response we observed in this study just as photophobic response. The
illumination intensity of 40 mW/cm2 with a blue light spectrum centered at 465 nm (Fig. 1a)
conventional fluence rate (typically, 0.1–200 µmol/(m2 • s)) investigated for step-up
photophobic response of Euglena cells [Matsunaga et al., 1999]. The onset of step-up
photophobic response observed in Fig. 1b was approximately 2 mW/cm2 (90 µmol/(m2•s)).
The movements of Euglena cells at four selected times (A–D in Fig. 1c) at 40
mW/cm2 are compared in the trace images shown in Fig. 2. At the end of the blue light on
period (A), 91 traces of Euglena cells were counted. Most were tumbling rotationally but
about 10 were still swimming. At the onset of blue light off (B), 64 traces were counted, and
18 cells were swimming straightforward. Because the blue light was turned off, the tumbling
cells were recovering straightforward swimming as indicated by the increased number of
swimming cells. In contrast, the decrease in the total number of traces revealed that at least 27
cells that were moving at time A had stopped moving at time B. The negative spike observed
in Fig. 1c was caused by this decrease in the number of moving cells. As TM recovery
proceeded, the number of traces and the number of straightforward swimming cells were
increased to 68 and 48, respectively at time C, and to 89 and 76, respectively at time D. From
these observations, we infer that the recovery process at 40 mW/cm2 (Figs. 1c and 2) was
composed of (i) transition from rotational tumbling to straightforward swimming, and (ii)
freezing of locomotor activity and awakening from the freezing. The freezing observed here
may correspond to ‘delayed agitation’ mentioned but not fully described in previous reports
[Diehn, 1969a; Diehn, 1973].
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corresponds to a photon fluence rate of 1760 µmol/(m2 •s), relatively higher than the
Single-cell analysis
To elucidate the photoresponses of the cells, nine representative traces of individual
cells were tracked in a series of trace images and the TMs of these cells were plotted (Fig. 3a).
All nine cells showed irregular TM changes during the blue light on periods, corresponding to
continuous or intermittent rotational tumbling movements. At the onset of the blue light off
responses. Type-A cells continuously and smoothly recovered their original TMs, type-B cells
froze activity for 2–30s before recovering their original TMs, and type-C cells ceased moving
during blue-light-off period. The transient freezing behavior of type-B cells constituted a
complete arrest of motion, which resulted in the disappearance of their traces and hence the
decrease in cell number observed in Fig. 2 at time B. Typical swimming behavior of three
types of cells are plotted in Fig. 3b in blue light of intensity 40 mW/cm2. Swimming speeds of
type-A cells were relatively high compared with those of type-B and type-C cells, as shown in
Figs. 3a and 3b. Apart from their different swimming speeds, the traces of type-A and type-B
cells were similar, i.e. straightforward swimming during the blue-light-off phase and
rotational tumbling and intermittent swimming during the on phase. Of course, the transient
freezing of type-B is unobservable in this trace. Type-C cells drifted with rotational tumbling
during blue-light-on phase and became motionless during the off phase. We suggest that the
type-C cells may have a deficiency that prevents straightforward swimming.
We attributed the gradual recovery of TM shown in Fig. 1c at 40 mW/cm2 to the
frequency distribution of the freezing durations of type-B cells. The TM recovery in Fig. 1c
was a good fit to a single exponential curve, from which we deduced that the frequency of
freezing decayed exponentially with a time constant of 17 s and a delay of 1.5 s. As seen in
Fig. 3a, the freezing duration was not constant for individual cells. This implies that release
from transient freezing occurs stochastically with an equal probability for unit time.
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period, the cells could be separated into three distinct types based on their step-down
The numbers of cells of types A, B, and C can be counted by checking the behavior
of each cell before and after the onset of the blue light off period. We have counted the
number of cells of each type for 15 individual experiments, as shown in Fig. 4. For blue light
of intensity 40 mW/cm2, there were 45% type-A cells on average, 48% type-B, and 4%
type-C, with 3% indeterminate. Fewer type-B cells were observed for blue light at 20
mW/cm2, i.e. type-A cells 76%, type-B 20%, and type-C 2%, with 2% indeterminate. The
response of Euglena gracilis changes from type-A to type-B when blue light intensity
increases. The gradual increase of negative spikes in TM with blue light intensity suggests
that the threshold level of the transition from type-A to type-B varies from cell to cell.
Anti-freezing adaptation
When blue light pulses of high intensity were applied without pre-exposure to
increasing intensity, much larger negative spikes were observed in TM. Figure 5a shows the
changes in TM produced by four pulses of blue light of 40 mW/cm2, applied without
pre-pulses. The TM at the onset of blue light off decreased to 50% of the original TM level
and the number of moving cells in the micro-aquarium was reduced from approximately 55 to
30. Combining the observations shown in Figs. 1c and 5a, we conclude that the threshold for
transient freezing was low at the beginning of the Fig. 1b experiment but gradually increased
as blue light pulses of increasing intensity were applied, resulting in the relatively small
negative spikes at around 40 mW/cm2.
An important observation in Fig. 5a is that the TM was increased by blue light pulses.
The TM initially decreased from 4000 to 2700 at the first pulse of blue light but it recovered
during the light pulse and increased further to 5000 after the blue light was turned off. It is
well-known that the swimming speed of Euglena gracilis is increased by blue light, a
behavior termed photokinesis [Wolken and Shin, 1958; Lebert, 2001; Melkonian et al., 1986].
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decrease (increase) in the number of type-A (type-B) cells indicates that the step-down
The TM increase could be partly attributed to photokinesis but also partly reflected an
increase in the number of moving cells (Fig. 5a) [Melkonian et al., 1986]. The number of
moving cells increased from 55 to approximately 60 during blue light pulses, revealing that
some cells had been inactive before the first blue pulse. After the last blue pulse, the TM
gradually decreased reaching the original level after about 7 min. This decrease paralleled a
decrease in the number of moving cells to approximately 50. We speculate that some resting
state when the blue light was terminated.
Figure 5b shows the changes of TM obtained during a second test with four
blue-light pulses performed on the same Euglena cells, commencing 80 min after the last
pulse of the first test (i.e., 80 min interval between the blue-light pulses). Although the
envelope of TM in Fig. 5b is almost identical to that in Fig. 5a, no large negative spikes are
apparent in Fig. 5b, indicating that transient freezing behavior was suppressed after the 80
min interval. This difference suggests that the Euglena cells had acquired a capability to
recover straightforward swimming smoothly without freezing after the interval. Type-B cells
occupied 49% in the 1st experiment of Fig. 5a, whereas only 8% in the 2nd experiment of Fig.
5b.
Adaptation versus interval time
The adaptation to strong blue light was examined with changing the interval time
between the first and second test. Figure 6 shows the dependence of spike ratio on interval
time in red light, measured in the similar experiments as that in Fig. 5 with various interval
times. Spike ratio was defined as the depth of the negative spike to the TM level before the
spike, for the 4th pulse in the second test. During the interval time, red light of 16.8 mW/cm2
was continuously irradiated to the micro-aquarium. The dependence shows that the adaptation,
i.e., the acquisition of a capability to recover straightforward swimming smoothly without
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cells were awakened by blue light pulses to escape from the light and returned to the resting
freezing, proceeds faster at the beginning of the interval, and gradually slows down with time.
When the red light illumination was cut during the interval time, large negative spikes were
observed even after 80-min interval, indicating that the cells do not acquire the blue-light
adaptation in the dark. This observation reveals that the blue-light adaptation sensitively
reflects the metabolic status of Euglena cells, which is changed by the photosynthesis in red
light.
Transition between type-A and type-B
We consider that the metabolism of type-B cells was changed to that of type-A
during photosynthesis under the red light (Fig. 6). The rather scattered data of spike ratio in
Fig. 6 suggests that the adaptation may also be influenced by the circadian rhythm of Euglena
gracilis [Hagiwara et al., 2002; Bolige et al., 2005], since the examination in this study was
carried out without culture synchronization [Osafune et al., 1975]. The adaptation to strong
blue light is presumably intimately related to the survival strategies of the organism. When the
same Euglena cells were kept in the dark for more than 24 hours, large negative spikes were
again observed, indicating that some of the type-A cells were transformed back to type-B.
Interestingly, the freezing of the flagellum of Euglena gracilis was also found when the cells
encounter sudden gravity change from hyper to micro-gravity [Strauch et al., 2010], which
may be related to the survival strategies of the organism as well.
Origin of type difference and adaptation
The observation in this study revealed that the mechanism of freezing behavior
differs from that of conventional photophobic response of Euglena, since the photophobic
response continues even after the freezing behavior disappeared after the red light
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Discussion
illumination of 80 min. We consider that the metabolic status of Euglena cells evolves by red
light illumination of a short period of some ten minutes, and then the cells are able to avoid to
fall in freezing after the light (type-A). The difference between type-A and type-B is distinct,
and no intermediate response was observed. This indicates that freezing behavior is an
independent mode of flagellum motion, differentiated from straightforward swimming and
on-site rotation (tumbling).
substance produced by light is required to switch the mode of flagellum movement from
on-site rotation to straightforward swimming immediately. The shortage of the substance
(type-B) results in delayed ignition of the straightforward swimming; the cells stop on-site
tumbling when the blue light is terminated but cannot start swimming, i.e., freezing for 2-30 s.
The photoproduced substance will be decomposed gradually during the nighttime (dark
situation), resulting in the transformation from type-A back to type-B.
The correlation of the freezing behavior to a metabolic substance is still speculative
and not conclusive at the moment. Specific experiments are required to determine the effect of
blue light on the metabolic activity of Euglena gracillis, such as co-immunoprecipation
studies to look at what binds to PAC following activation in the presence of blue light and in
it's absence, gene expression profiling of common metabolic proteins and mitochondrial
proteins, or quantification studies on glucose utilization induced by blue light. Unfortunately,
these molecular-level studies are hard for non-sustainable phenomena including transient
freezing behaviors investigated in this study. Instead, we are trying to figure out the signal
path related to the transient freezing by employing enzyme knock-down technique, expecting
that the enzymes responsible for photophobic response and for the mode of flagellum
movements will be made clear.
The effect of photosynthesis on freezing behavior will be examined indirectly by
employing
chloroplast-free
Euglena
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cells,
or
by
using
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A reasonable mechanism for the freezing behavior is that a certain metabolic
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to suppress photosynthesis. Further,
oxygen concentration in culture medium or in the cells, or other photoreceptors such as
phytochrome for red light [Bolige and Goto, 2007] may play an important role to
induce/suppress the freezing behavior. We will perform the further experiments on the
freezing behavior (i) for by chloroplast-free Euglena cells, (ii) with DCMU, (iii) under
reduced red light to test the contribution of phytochrome, and (iv) with excess oxygen in the
reported as separated paper in the near future.
In conclusion, transient behaviors in the blue-light photophobic response of Euglena
gracilis were investigated with the introduction of the TM measurement to evaluate the
swimming activity of Euglena cells. Three types of transient behaviors at the time of blue
light turn-off were observed by single-cell trace-analysis: smooth recovery from tumbling to
straight-line swimming (type-A); transient freezing for 2–30 s before starting swimming
(type-B); and slow tumbling only under the blue light (type-C). The same Euglena cells that
experienced transient freezing for blue light pulses did not show the transient freezing
behavior after an 80 min interval in red light, indicating that they acquired anti-freezing
metabolism during the interval. From the analysis of the dependence of blue-light adaptation
on interval time and red light, we concluded that the adaptation is determined by metabolic
change evoked by photosynthesis. The adaptation suggests that a metabolic substance, not
identified yet, is produced by light illumination and promotes the smooth switching of the
mode of flagellum movement from on-site rotation mode to straightforward swimming mode.
The analysis of transient freezing is significant to figure out the signal path responsible for the
photoinduced responses of green algae, and gives wide application for photosynthetic
production of biomass.
Materials and Methods
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culture medium by adding hydrogen peroxide. The results of these experiments will be
Euglena gracilis Z strain were supplied by Euglena Co., Ltd. as a suspension
cultured in Cramer–Myers’ (CM) medium (without sodium citrate) [Cramer and Myers, 1952].
The suspension was stored in a conical microtube (1.0 mL) before experiments, which was
kept at room temperature without agitation under white light illumination of approximately
0.15 mW/cm2 (daytime) and 60 nW/cm2 (nighttime). The cells were in the stationary phase
with possibly less oxygen due to complete confinement in a plastic microtube. For each
placed on a poly dimethyl siloxane (PDMS) circular micro-aquarium 2.49 mm in diameter
and 140 µm deep (0.7 µL). The micro-aquarium was then sealed by a cover glass, and the
excess volume of 9.3 uL overflowed away from the microchip. The micro-aquarium was then
sandwiched between two cover glasses and the space between the two glasses filled with pure
water to prevent evaporation (Fig. 7a). Approximately 50–200 cells were confined in the
micro-aquarium. Oxygen supply was recovered when the cells were transferred to the
microchip, since the microchip permeates oxygen well. The micro-aquarium was then placed
in a 30 mm diameter dish with a glass top and bottom and sealed with plastic tape to maintain
internal humidity. The cells are able to survive for more than two weeks in these
evaporation-resistant surroundings. The photophobic response in this study was also observed
even two weeks after transferring the cells from the microtube into the microchip. The growth
rate of the cell number can be ignored for time durations used in this study.
Transient behavioral responses of Euglena gracilis to blue light stimulation were
examined with the 2D optical feedback system that we used previously for the demonstration
of the spatial and dynamic control of the density of Euglena cells in a micro-aquarium [Ozasa
et al., 2011]. The system was the combination of an upright optical microscope (BX51,
Olympus), a video camera (IUC-200CK2, Trinity), a data processing computer (PC,
MG/D70N, Fujitsu), a liquid crystal (LC) projector (LP-XU84, Sanyo), and optics, as shown
in Fig. 7c. The micro-aquarium was placed on the microscope stage and illuminated from
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experiment, an approximately 10 µL droplet of CM medium containing Euglena cells was
below by the LC projector through reduction lenses. The illuminated area was 5.1 mm × 3.8
mm. During experiments, the whole area of the micro-aquarium was illuminated constantly
by red light (570–700, 16.8 mW/cm2) to observe the movements of the cells. The stimulating
blue light (430–520 nm) was also provided from the LC projector whose intensity and
duration were controlled by the data processing computer. The spectra of the red and blue
light provided by the LC projector are given in Fig. 1a. Because the micro-aquarium was only
direction of the stimulating light.
Real-time raw images of Euglena cells swimming in the micro-aquarium were taken
by the video camera through a 5x objective lens (MPLFLN5X, Olympus) at intervals of
approximately 0.1 s at a resolution of 200 pixels/mm and an image size of 800 × 600 pixels.
To quantify the swimming activity of Euglena cells, the raw images were processed according
to the flow chart shown in Fig. 7d. We produced a differential image from two consecutive
raw images and converted the differential image into a binary image by thresholding. We
superimposed N (N = 3 to 10) binary images to generate a ‘trace image’, which showed the
swimming traces of Euglena cells as footprints during N time intervals. Finally, a
measurement, termed the ‘trace momentum’ (TM), was calculated by counting the number of
pixels covered by Euglena traces in each trace image. Each TM value approximates the scalar
summation of the swimming speeds of Euglena cells in the micro-aquarium. Typically, one
swimming Euglena cell produced a TM value of 60–70, whereas one rotating and tumbling
cell produced a TM of 40–50. In contrast to particle tracking techniques [Wu et al., 2006],
which require extensive calculation and are weak in trace identification, the TM calculation
can be performed in real time and reliably allows for crossing and disappearance of
swimming cells in the trace image. The forward motion of Euglena gracilis in a straight line
(termed ‘straightforward swimming’) under red light and the ‘rotational tumbling’ motion
under blue light are presented in Fig. 7b.
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140 µm deep, the swimming directions of all Euglena cells were perpendicular to the
The results described in this paper have been reproduced for three independent cell
samples of Euglena Z strain supplied from three different organizations.
Funding
This work was supported by the Ministry of Education, Science, Sports and Culture,
Grant-in-Aid for Scientific Research (B) [grant number 21360192 (2009-2012), 25280092
funded by the Ministry of Education, Science and Technology [2013R1A2A2A01014234,
2012R1A6A1029029].
Disclosures
No conflict of interest declared.
Acknowledgements
The authors would like to thank Mr. Kengo Suzuki, Ms. Sharbanee Mitra, and Ms.
Ayaka Nakashima at Euglena Co. Ltd. (http://euglena.jp/english) for supplying the Euglena
cells and culture medium, together with information on cell culture. They also thank Dr. Ken
Goto at Obihiro University of Agriculture and Veterinary Medicine for information on the
culture conditions and the blue light responses of Euglena gracilis. The authors greatly
appreciate useful suggestions from Dr. Masakatsu Watanabe at The Graduate School for the
Creation of New Photonics Industries and Dr. Shigeru Matsunaga at Hamamatsu Photonics
K.K.
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(2013-2016)], and also partially by a National Research Foundation of Korea (NRF) grant
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Figure Legends
Fig. 1
(a) Spectra of the blue light used for stimulation and red light used for observation, as
measured on the microscope stage. (b) Temporal change of TM observed in response to
switching blue light on and off periodically with progressively increasing intensity. Square
brackets indicate regions expanded in Fig. 1c. (c) Time-expanded chart for blue light of
comparison. Trace images for the marked points are presented in Fig. 2. The negative spike
indicates non-instantaneous and non-smooth change in the locomotion of Euglena cells from
rotational tumbling to straightforward swimming at 40 mW/cm2.
Fig. 2
Trace images at the four points (A-D) indicated in Fig. 1c. Traces were superimposed
for a period of 4.5 s to show swimming movements. To indicate the swimming direction,
traces are colored in the order blue, yellow, red, and white. Scale bar indicates 500 µm. The
decrease in the total number of traces from time A to B revealed that at least 27 cells that were
moving at time A had stopped moving at time B.
Fig. 3
(a) Temporal changes in the TMs of nine individual Euglena cells corresponding to
the TM change shown in Fig. 1b. The plots have been shifted vertically to display them in a
single graph, while preserving the relative intensities of the plots. These nine cells were
grouped into 3 types, A, B, and C, according to their behaviors during the blue-light-off
period, as described in the text. (b) Typical traces of the three types of Euglena cells shown in
Fig. 3a, observed for the same period. Circles, type-A; triangle, type-B; squares, type-C; open
symbols, blue-light-on; closed symbols, blue-light-off. Scale bar indicates 500 µm. Type-A
and type-B cells were swimming during the blue-light-off phase and tumbling with
intermittent swimming during the on phase, whereas type-C cells drifted with rotational
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intensity 20 and 40 mW/cm2. The time scale has been shifted and overlaid for easy
tumbling during blue-light-on phase and became motionless during the off phase.
Fig. 4
Population of cell types A-C observed at 20 mW/cm2 and 40 mW/cm2 for 15
independent trials with the same blue-light process shown in Fig. 1b. The population of
type-B (type-A) cells became larger (smaller) at 40 mW/cm2 than that at 20 mW/cm2,
revealing that the step-down response of Euglena gracilis changes from type-A to type-B
76% and 20% at 20 mW/cm2, respectively, whereas 45% and 48% at 40 mW/cm2,
respectively. The trial-by-trial variation of the population suggests that the ratio of type-B
cells to type-A cells depends on culture environments and culture history.
Fig. 5
Temporal change of summed-up TM and the numbers of moving Euglena cells
observed during four 75 s pulses of blue light of 40 mW/cm2. The TMs of all the cells were
summed up and plotted. The first test (a) and the second test (b) used the same cells. The
interval between the last blue light pulse in (a) and the first blue light pulse in (b) was 80 min.
The absence of large negative spikes in (b) suggests that the Euglena cells had acquired a
capability to recover straightforward swimming smoothly without freezing after the interval.
Fig. 6
Dependence of spike ratio on interval time in red light, measured in the similar
experiments as that in Fig. 5 with various interval times. Spike ratio was defined as the depth
of the negative spike to the TM level before the spike, for the 4th pulse. Circles indicate the
average for each interval time, whereas squares represent the result of each independent
experiment. The dependence shows that the acquisition of a capability to recover
straightforward swimming smoothly without freezing proceeds faster at the initial stage of the
interval and slows down with time. When the red light illumination was cut during the
interval time, large negative spikes were observed even after 80-min interval, indicating that
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when blue light intensity increases. The average population of type-A and type-B cells was
the cells do not acquire the capability to recover straightforward swimming smoothly without
freezing in the dark.
Fig. 7 (a) Cross-sectional drawing of micro-aquarium and surroundings. The setup can sustain
live Euglena cells for more than two weeks. (b) Examples of Euglena motion. The three
traces at the left show straight-line forward swimming with slight curvature under red light,
movement duration was 9.4 s in the direction from green to yellow. Scale bar indicates 200
µm. (c) Schematic illustration of the experimental system, including Euglena micro-aquarium
(E), objective lens (OL), reduction lenses (RL1, RL2), filters (F1: infrared light cutoff, F2:
blue light cutoff), and a mirror (M). The system achieves real time measurement and optical
feedback to motile microorganisms. (d) Flow diagram of image processing to measure TM
from a series of raw images of swimming Euglena cells. The TM calculation can be
performed in real time and reliably allows for crossing and disappearance of swimming cells
in the trace image.
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and the three at the right show rotational tumbling under blue light. In each case, the
Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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Fig. 7
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