Transient Freezing Behavior in Photophobic Responses of
Euglena gracilis Investigated in a Microfluidic Device
Kazunari Ozasa1,*, Jeesoo Lee2, Simon Song2 and Mizuo Maeda1
1
*Corresponding author: E-mail, [email protected]; Fax, +81-48-462-4658.
(Received April 24, 2014; Accepted July 20, 2014)
We found that the transient freezing behavior in photophobic responses of Euglena gracilis is a good indicator of the
metabolic status of the cells. The transient blue light photophobic responses of E. gracilis cells were investigated on-chip
using a new measurement, ‘trace momentum’ (TM), to
evaluate their swimming activity quantitatively in real
time. When blue light of intensity >30 mW cm 2 was repeatedly switched on and off, a large negative spike in the
TM was observed at the onset of the ‘blue-light-off’ phase.
Single-cell trace analysis at a blue light intensity of
40 mW cm 2 showed that 48% (on average, n = 15) of tumbling Euglena cells ceased activity (‘freezing’) for 2–30 s at
the onset of blue-light-off before commencing forward
motion in a straight line (termed ‘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 cm 2). When the cells were stimulated by
four blue light pulses at the higher intensity, without preexposure, 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 relationship 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 in situ rotation mode to a straightforward
swimming mode.
Keywords: Adaptation Blue light Euglena gracilis Microfluidic aquarium Photophobic response Transient
responses.
Abbreviations: LC, liquid crystal; PAC,
adenylyl cyclase; TM, trace momentum.
photoactivated
Introduction
Photosynthetic microorganisms have attracted much attention
of researchers due to their ability to produce oil and nutrition
in an environmently friendly manner (Chisti 2007, Schenk
et al. 2008, Lardon et al. 2009, Brennan and Owende. 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. 1981, Ozasa et al. 2013a), are motile and
show kinetic responses to light, so-called 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 photokinesis (Chung
et al. 2004), phototaxis (Sineshchekov et al. 2002), photophobic
responses (Diehn 1969a, Bovee 1982, Matsunaga et al. 1999) or
photoadaptation (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 E. gracilis have been measured by many researchers
(Diehn 1969b, Checcucci et al. 1976, Matsunaga et al. 1998) and
it has been 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 E. 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 photocontrollable signal induction molecules for optogenetics
(Boyden et al. 2005, Tsunematsu et al. 2011), demonstrating
photoactivated neuron firing through genetically introduced
PAC molecules (Nagahama et al. 2007, Schröder-Lang et al.
2007). Although the photoreceptor for the photophobic response of Euglena has been identified, complicated metabolic
processes involved in the photophobic response of Euglena are
still unclear. A micro-ethological approach will provide an insight into 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 microorganisms
and to understand the mechanisms of metabolic adaptation
Plant Cell Physiol. 55(10): 1704–1712 (2014) doi:10.1093/pcp/pcu101, Advance Access publication on 29 July 2014,
available online at www.pcp.oxfordjournals.org
! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
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Regular Paper
Bioengineering Lab., RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan
Department of Mechanical Convergence Engineering, Hanyang University, 17 Haendang-dong, Seongdong-gu, Seoul, 133-791, Korea
2
Plant Cell Physiol. 55(10): 1704–1712 (2014) doi:10.1093/pcp/pcu101
Downloaded from http://pcp.oxfordjournals.org/ at Pennsylvania State University on September 13, 2016
to photointensity changes. For this purpose, single-cell level
studies on the photoresponses of the microorganisms with
millisecond 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. 2014). 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 light intensities and to trace the
photophobic movements of single cells.
Here we report an investigation of the photophobic
responses of E. 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 while increasing
its intensity. The detailed movements of single cells in the same
batch were subsequently traced through video analysis. Blue
light adaptation was also investigated by measuring the transient freezing behaviors after the interval of blue light pulse
exposures while changing the interval time and light irradiation
condition.
Results
Dependence on blue light intensity
Fig. 1b and c shows temporal changes of ‘trace momentum’
(TM; see the Materials and Methods) observed in response to
blue light pulses of constant duration and progressively increasing intensity. The blue light, whose spectrum is given in Fig. 1a,
was switched on and off at 75 s intervals. There were approximately 100 swimming cells in the micro-aquarium. The upper
limit of the TM envelope obtained during the blue-light-off
period slowly increased between 10 and 30 min 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
2 mW cm 2, 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
blue-light-off periods increased as the blue light intensity was
increased, up to approximately 30 mW cm 2. Above
30 mW cm 2, 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 and 40 mW cm 2 (Fig. 1c). The negative spike revealed
that the change in the locomotion of Euglena cells from rotational tumbling to forward motion in a straight line (termed
‘straightforward swimming’) in response to blue-light-off
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 (c). (c) Time-expanded
chart for blue light of intensity 20 and 40 mW cm 2. The time scale
has been shifted and overlaid for easy 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 cm 2.
was not instantaneous or smooth for blue light intensities
>30 mW cm 2. The TM recovery in Fig. 1c was fitted to an
exponential curve with a time constant of 6 s at 20 mW cm 2
and 17 s at 40 mW cm 2. A small delay of 1.5 s was inferred in
the exponential recovery at 40 mW cm 2, corresponding to the
downward spike of TM.
The downward spike observed at the onset of each bluelight-off period was to be categorized as a step-down photophobic response, according to the terminology recommended
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K. Ozasa et al. | Transient freeze of Euglena photophobic responses
by Diehn et al. (1977). However, the downward spike is a part of
the recovery process from the step-up photophobic response
for relatively higher light intensities, and differs from the conventional step-down photophobic response in which Euglena
cells in a moderately illuminated area turn their swimming
direction at the boundary between moderate light and a 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 the photophobic response. The illumination intensity of 40 mW cm 2 with a blue light spectrum centered at
465 nm (Fig. 1a) corresponds to a photon fluence rate of
1,760 mmol (m2 s) 1, relatively higher than the conventional
fluence rate [typically, 0.1–200 mmol (m2 s) 1] investigated for
the step-up photophobic response of Euglena cells (Matsunaga
et al. 1999). The onset of the step-up photophobic response
observed in Fig. 1b was approximately 2 mW cm 2 [90 mmol
(m2 s) 1].
The movements of Euglena cells at four selected times (A–D
in Fig. 1c) at 40 mW cm 2 are compared in the trace images
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shown in Fig. 2. At the end of the blue-light-on period (Fig. 2A),
91 traces of Euglena cells were counted. Most were tumbling
rotationally, but about 10 were still swimming. At the onset of
blue-light-off (Fig. 2B), 64 traces were counted, and 18 cells
were straightforward swimming. Because the blue light was
turned off, the tumbling cells were recovering to 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 cm 2
(Figs. 1c, 2) was comprised of (i) transition from rotational
tumbling to straightforward swimming and (ii) freezing of
locomotor activity and awakening from the freezing. The
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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 time order blue, yellow, red and white. Scale bar = 500 mm. 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.
Plant Cell Physiol. 55(10): 1704–1712 (2014) doi:10.1093/pcp/pcu101
freezing observed here may correspond to ‘delayed agitation’
mentioned but not fully described in previous reports (Diehn
1969a, Diehn 1973).
Single-cell analysis
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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
period, the cells could be separated into three distinct types
based on their photophobic (step-down) responses. Type-A
cells continuously and smoothly recovered their original TMs,
type-B cells froze activity for 2–30 s before recovering their
original TMs, and type-C cells ceased moving during the bluelight-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. The typical swimming behavior of three types of cells are plotted in Fig. 3b
in blue light of intensity 40 mW cm 2. Swimming speeds of
type-A cells were relatively high compared with those of
type-B and type-C cells, as shown in Fig. 3a and b. 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 the traces
in Fig. 3b. Type-C cells drifted with rotational tumbling
during the blue-light-on phase and became motionless during
the off phase. We suggest that the type-C cells may be in
diapause, or have a deficiency that prevents straightforward
swimming.
We attributed the gradual recovery of TM shown in Fig. 1c
at 40 mW cm 2 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.
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 cm 2, 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 cm 2, i.e. 76% type-A cells, 20% type-B and 2% typeC, with 2% indeterminate. The decrease (increase) in the
number of type-A (type-B) cells indicates that the photophobic
(step-down) response of E. gracilis changes from type-A to typeB when the 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. Fig. 5a shows the changes in
TM produced by four pulses of blue light of 40 mW cm 2,
applied without pre-pulses. The TM at the onset of bluelight-off decreased to 50% of the original TM level and the
number of moving cells in the micro-aquarium was reduced
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 three
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 (a), observed for the same period. Circles, type-A; triangle, type-B; squares, type-C; open symbols, blue-light-on; filled
symbols, blue-light-off. Scale bar = 500 mm. 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 tumbling during the blue-light-on phase and became
motionless during the off phase.
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K. Ozasa et al. | Transient freeze of Euglena photophobic responses
Fig. 4 Population of cell types A–C observed at 20 and 40 mW cm 2
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 cm 2 than that at 20 mW cm 2, revealing that
the photophobic (step-down) response of Euglena gracilis changes
from type-A to type-B when blue light intensity increases. The average
population of type-A and type-B cells was 76% and 20% at
20 mW cm 2, respectively, and 45% and 48% at 40 mW cm 2, 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.
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escape from the light and returned to the resting state when
the blue light was terminated.
Fig. 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.
an 80 min interval between the blue light pulses). Although the
up-down trend 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 without freezing (anti-freezing capability) after the interval. Type-B
cells comprised 49% of all cells in the first experiment of
Fig. 5a, but only 8% in the second experiment of Fig. 5b.
Adaptation vs. interval time
The adaptation to strong blue light was examined by changing
the interval time between the first and second test. Fig. 6 shows
the dependence of the spike ratio on the interval time in red
light, measured in similar experiments to that in Fig. 5 with
various interval times. The spike ratio was defined as the
depth of the negative spike to the TM level before the spike,
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 cm 2. The TMs of all the cells were summed up and plotted.
The first (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.
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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 cm 2.
An important observation in Fig. 5a is that the TM was
increased by blue light pulses. The TM initially decreased
from 4,000 to 2,700 at the first pulse of blue light but it recovered during the light pulse and increased further to 5,000
after the blue light was turned off. It is well known that the
swimming speed of E. gracilis is increased by blue light, a behavior termed photokinesis (Wolken and Shin 1958, Melkonian
et al. 1986, Lebert 2001). 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 cells were awakened by blue light pulses to
Plant Cell Physiol. 55(10): 1704–1712 (2014) doi:10.1093/pcp/pcu101
for the fourth pulse in the second test. During the interval time,
the micro-aquarium was continuously irradiated with red light
of 16.8 mW cm 2. The dependence shows that the adaptation,
i.e. the acquisition of the anti-freezing capability proceeds faster
at the beginning of the interval, and gradually slows down with
time. When the red light was not irradiated during the interval
time, large negative spikes were observed even after an 80 min
interval, indicating that the cells do not acquire the anti-freezing
capability 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 the spike ratio in Fig. 6
suggest that the adaptation may also be influenced by the circadian rhythm of E. 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 micro organism. When the same
Euglena cells were kept in the dark for >24 h, 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 E. gracilis was also found when the cells
encounter sudden gravity change from hyper- to micro-gravity
(Strauch et al. 2010), which may also be related to the survival
strategies of the micro organism.
Fig. 6 Dependence of the spike ratio on the interval time in red light,
measured in similar experiments to that in Fig. 5 with various interval
times. The spike ratio was defined as the depth of the negative spike to
the TM level before the spike, for the fourth 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 the anti-freezing capability proceeds faster at the initial
stage of the interval and slows down with time. When the red light
was not irradiated during the interval time, large negative spikes were
observed even after the 80 min interval, indicating that the cells do
not acquire the anti-freezing capability in the dark.
The observation in this study revealed that the mechanism of
freezing behavior differs from that of the conventional photophobic response of Euglena, since the photophobic response
continues even after the freezing behavior disappeared after the
red light illumination of 80 min. We consider that the metabolic
status of Euglena cells evolves by red light illumination for
a short period of some 10 min, and then the cells are able
to avoid 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 in situ rotation
(tumbling).
A reasonable mechanism for the freezing behavior is that a
certain metabolic substance produced by light is required to
switch the mode of flagellum movement from in situ rotation
to forward swimming immediately. The shortage of the substance (type-B) results in delayed ignition of the swimming; the
cells stop in situ tumbling when the blue light is terminated but
cannot start swimming, i.e. they freeze for 2–30 s. The photoproduced substance will be decomposed gradually during the
night-time (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 E. gracillis,
such as co-immunoprecipation studies to look at what binds to
PAC following activation in the presence of blue light and in its
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 determine the signal path
related to the transient freezing by employing an 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
cells, or by using DCMU to suppress photosynthesis. Further,
the oxygen concentration in the 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) with 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 culture medium by adding hydrogen peroxide. The results of these experiments will be reported as a
separate paper in the near future.
In conclusion, transient behaviors in the blue light photophobic response of E. gracilis were investigated with the
introduction of the TM measurement to evaluate the
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Discussion
Origin of type difference and adaptation
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K. Ozasa et al. | Transient freeze of Euglena photophobic responses
photoinduced responses of green algae, and provides wide application for photosynthetic production of biomass.
Materials and Methods
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; the tube was kept at room temperature without agitation
under white light illumination of approximately 0.15 mW cm 2 (daytime) and
60 nW cm 2 (night-time). The cells were in the stationary phase with
possibly less oxygen due to complete confinement in a plastic microtube. For
each experiment, an approximately 10 ml droplet of CM medium containing
Euglena cells was placed on a poly dimethyl siloxane (PDMS) circular microaquarium 2.49 mm in diameter and 140 mm deep (0.7 ml). The micro-aquarium
was then sealed by a cover glass, and the excess volume of 9.3 ml overflowed
away from the microchip. The micro-aquarium was then sandwiched between
two cover glasses and the space between the two glasses was 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
Fig. 7 (a) Cross-sectional drawing of the micro-aquarium and surroundings. The set-up can sustain live Euglena cells for > 2 weeks. (b) Examples
of Euglena motion. The three traces on the left show straight line forward swimming with slight curvature under red light, and the three on the
right show rotational tumbling under blue light. In each case, the movement duration was 9.4 s in the direction from green to yellow. Scale
bar = 200 mm. (c) Schematic illustration of the experimental system, including the Euglena micro-aquarium (E), objective lens (OL), reduction
lenses (RL1, RL2), filters (F1, infrared light cut-off; F2, blue light cut-off) 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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swimming activity of Euglena cells. Three types of transient
behaviors at the time the blue light was turned 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 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, as yet unidentified, is produced
by light illumination and promotes the smooth switching of
the mode of flagellum movement from in situ rotation
to straightforward swimming. The analysis of transient freezing
is needed to determine the signal path responsible for the
Plant Cell Physiol. 55(10): 1704–1712 (2014) doi:10.1093/pcp/pcu101
Funding
This work was supported by the Ministry of Education, Science,
Sports and Culture [Grant-in-Aid for Scientific Research (B)
grant Nos. 21360192 (2009–2012), 25280092 (2013–2016)];
the Ministry of Education, Science and Technology [National
Research Foundation of Korea (NRF) grant Nos.
2013R1A2A2A01014234 and 2012R1A6A1029029].
Acknowledgments
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.
Disclosures
The authors have no conflicts of interest to declare.
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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 >2 weeks in these evaporation-resistant surroundings. The photophobic response in this study was also observed even 2
weeks after transferring the cells from the microtube into the microchip. The
increase in the cell number can be ignored for the time periods used in this
study.
Transient behavioral responses of E. 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 microaquarium was placed on the microscope stage and illuminated from 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 cm 2) 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
140 mm deep, the swimming directions of all Euglena cells were perpendicular to
the 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 5 objective lens (MPLFLN5X;
Olympus) at intervals of approximately 0.1 s at a resolution of 200 pixels
mm 1 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 = 3–10) 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 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 E. gracilis in a straight line (termed ‘straightforward swimming’) under
red light and the ‘rotational tumbling’ motion under blue light are presented
in Fig. 7b.
The results described herein have been reproduced for three independent
cell samples of Euglena gracilis Z strain supplied from three different
organizations
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