Journal of Photochemistry and Photobiology B: Biology 121 (2013) 27–31
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Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Optical trapping of microalgae at 735–1064 nm: Photodamage assessment
Z. Pilát a,⇑, J. Ježek a, M. Šerý a, M. Trtílek b,c, L. Nedbal b,c, P. Zemánek a
a
Institute of Scientific Instruments of the ASCR, v.v.i., Královopolská 147, 612 64 Brno, Czech Republic
Photon Systems Instruments, Spol. s r.o., Drásov 470, 664 24 Drásov, Czech Republic
c
Global Change Research Centre of the ASCR, v.v.i., Bělidla 4a, 603 00 Brno, Czech Republic
b
a r t i c l e
i n f o
Article history:
Received 22 October 2012
Received in revised form 15 January 2013
Accepted 4 February 2013
Available online 19 February 2013
Keywords:
Optical trapping
Photodamage
Microalgae
PAM fluorescence microspectroscopy
a b s t r a c t
Living microalgal cells differ from other cells that are used as objects for optical micromanipulation, in
that they have strong light absorption in the visible range, and by the fact that their reaction centers
are susceptible to photodamage. We trapped cells of the microalga Trachydiscus minutus using optical
tweezers with laser wavelengths in the range from 735 nm to 1064 nm. The exposure to high photon flux
density caused photodamage that was strongly wavelength dependent. The photochemical activity
before and after exposure was assessed using a pulse amplitude modulation (PAM) technique. The photochemical activity was significantly and irreversibly suppressed by a 30 s exposure to incident radiation
at 735, 785, and 835 nm at a power of 25 mW. Irradiance at 885, 935 and 1064 nm had negligible effect at
the same power. At a wavelength 1064 nm, a trapping power up to 218 mW caused no observable
photodamage.
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
Microbial cultures have been exploited by mankind for millennia [1]. The ever-increasing number of usable metabolites produced by microbes has driven the recent rapid development of
microbial biotechnology [2,3]. Optical non-contact micromanipulation offers an attractive opportunity to select, handle and sort
individual microbial cells. Optical tweezers operate by employing
transfer of momentum from tightly focused laser beam to a micro-object, resulting in its spatial confinement [4], and have been
used to manipulate heterotrophic cells in broad areas of biology
[4,24,28–30,36,37]. In contrast, the use of optical tweezers for photoautotrophic microorganisms has been marginally explored.
However, photoautotrophic organisms have lately become a focus
of enormous interest because of their ability to utilize solar energy
for production of biofuels, high-value compounds and for assimilation of atmospheric CO2 [5,6]. The ability to sort such cells, and the
potential effects of sorting techniques on the cells is, therefore, of
considerable interest.
Algae and cyanobacteria are susceptible to photoinhibition even
when exposed to moderate photon flux densities [7]. Potentially,
this hinders the application of optical micromanipulation. The
absorption of light is increased by excitonic energy transfer from
hundreds of pigment molecules to the reaction centers of two
photosystems [8]. Photosystem II is particularly sensitive to photoinhibition during which overexcitation can result in production
⇑ Corresponding author.
E-mail address: [email protected] (Z. Pilát).
1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2013.02.006
of harmful oxygen radicals, and other reactive species, that destroy
the photosynthetic capacity of the cell and may lead to cell death
[9]. To avoid this, optical trapping may employ wavelengths in
the near infrared wavelength range beyond the red absorption
edge of chlorophyll [10]. However, several reports have pointed
out that reaction centers may have activity beyond the red edge
of chlorophyll absorption [11–13]. This study aims to elucidate
the effect of trapping on photosynthetic cells across a range of
wavelengths and incident energy levels.
Current optical micromanipulation techniques utilize many
tools and methods that enable spatial confinement and manipulation of microscopic objects, including living cells [14–18]. A single
laser beam focused to a diffraction limited spot acts as the optical
tweezers, and allows three-dimensional (3D) manipulation [19] of
a single object ranging in size from tens of nanometers to tens of
micrometers. Incident laser power may be used in the range from
extremely low levels up to hundreds of milliWatts (mW). However,
the power densities at these irradiance levels can reach MW cm 2,
exceeding by more than six orders of magnitude the power density
of sunlight incident at the earth’s surface (approx. 100 mW cm 2).
In the case of organisms having cellular pigments at low concentrations (some bacteria, heterotrophic microorganisms) several
earlier studies investigated the heating of the cells resulting from
absorption of the beam from the continuously working laser [20–
24] and concluded that there exists a window of usable wavelengths between 750 and 1200 nm that has a lesser effect on the
cells. This wavelength range occurs between the absorption range
of proteins at visible wavelengths, and the absorption of water in
the infrared region [25]. Investigations performed with Chinese
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Z. Pilát et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 27–31
hamster ovary cells (CHO) showed that a temperature rise close to
1 K per 100 mW of incident trapping power occurred with these
cells [21,22]. An independent study performed on silica or polystyrene micrometer spheres [26] indicated that such a temperature
rise is mainly caused by absorption of the trapping beam in solution (water).
Investigation of cloning efficiency of CHO cells [23] revealed
that this had strong spectral dependence in the range from
700 nm to 1064 nm. Maximum clonability was observed between
950 and 990 nm and least clonability between 740 and 900 nm.
Cloning efficiency reached 100% in cells trapped for 1 min with
an incident power of 176 mW at the optimal trapping wavelengths. Similar spectral dependence was observed when changes
in rotation of Escherichia coli were investigated in the optical trap
[24]. Moreover, this study observed a dramatic decrease in photodamage under anaerobic conditions, implicating oxygen in the
photodamage pathway. More recent studies performed on fibroblasts [27], E. coli and Listeria [28–30], CHO cells [31], T cells
[32], nerve cells [33–35], Saccharomyces cerevisiae [36], stem cells,
and Caenorhabditis elegans [37] indicate that if the laser is focused
down to diffraction-limited spot of a diameter comparable to the
trapping wavelengths (1064 nm in vacuum), with an incident
power less than 30 mW, this has little deleterious effect on the cell
during an irradiation period of less than a minute. However, few
studies have been made concerning the effects of optical trapping
techniques performed on phototrophic organisms such as algae or
cyanobacteria.
Experiments with laser irradiation and trapping of algal cells
were conducted by Ashkin and Dziedzic [38] on Spirogyra and
Hydrodicton at a laser wavelength of 1064 nm. The authors manipulated organelles and visually evaluated any observed damage.
Similar laser manipulations of Spirogyra [10] Micrasterias, Pleurenterium, and Closterium [39] at 1064 nm were performed, and were
mainly focused on the anatomical disturbances caused by the laser
beam. In contrast, another study [40] evaluated the absorption
spectra and viability of Chlorella pyrenoidosa cells after irradiation
with lasers at wavelengths of 1060 nm, 530 nm, and 694.3 nm.
The spectral response of Chlorella after irradiation with 633 nm
was also studied [41]. These investigations clearly demonstrated
that there is a relation between the incident laser wavelength
and the extent of the cell damage. Substantial damage occurred
when using wavelengths in the visible range, even after a short
exposure, while no damage was observed when using wavelengths
in the near-infrared wavelength (1064 nm). However, none of the
studies concentrated on finding the optimum wavelength region
for optical manipulation of photosynthetic cells.
Photodamage in the photosynthetic apparatus typically occurs
in the reaction centers of Photosystem II [42]. The loss of Photosystem II activity can be measured [43,44] as a loss of variable fluorescence yield Fv that is defined as the difference Fm–F0 between the
maximum fluorescence yield Fm and the constant fluorescence
yield F0. F0 is independent of the photochemical activity of the
reaction centers, whereas Fm may be increased by eliminating
the photochemical quenching of Photosystem II by applying a saturating flash of light to the sample. The ratio Fv/Fm in healthy higher plants is slightly over 0.8, a value that is interpreted as the
maximum quantum yield of photochemistry in the Photosystem
II reaction centers [45]. Photoinhibition of the reaction centers is
reflected by a decrease of Fv/Fm. The ratio Fv/Fm in healthy and unstressed algae and cyanobacteria is typically lower than in plants,
and its interpretation as the maximum quantum yield is not possible because of significant contribution of Photosystem I and antenna systems to F0 [46]. However, the ratio is often used as a
convenient surrogate for activity of Photosystem II [44]. In this
study we measured changes in Fv/Fm resulting from optical trapping by focused laser radiation of different wavelengths. In accor-
dance with available studies on other cells discussed above, we
chose a moderate trapping power of 25 mW with duration of the
optical confinement of the microorganisms set to 30 s. In accordance with our previous investigations [47–50] we chose the
microalga Trachydiscus minutus for this study. T. minutus contains
chlorophyll a, which is the central photosynthetic pigment of the
vast majority of the other eukaryotic algae, as well as all green
plant. This makes it good universal candidate for photodamage
experiments.
2. Materials and methods
The experimental setup (Fig. 1) was based on a Micro-FluorCam
system (Photon Systems Instruments, Brno, CZ). The microscope
was modified by an extra input port for the trapping beam (gray
arrows). A Mira HP (Coherent) optically pumped by a Verdi V10
(Coherent) laser continuously emitted the trapping beam that
was linearly polarized. The emission was tuned in the experiments
to five wavelengths: 735, 785, 835, 885, and 935 nm. The trapping
beam from Mira was first coupled to an optical fiber. The beam was
then collimated by the L2 lens (Thorlabs, AC 254-030-B) and
passed through a rotating half-wave plate WP (Thorlabs,
AHWP05M-980) that rotated the beam polarization before passing
it through a fixed polarizer (realized by polarizing beam splitter
PBS, Thorlabs, PBS202). The power of the beam was adjusted by
relative orientation of the beam polarization and the polarizer
(minimal if crossed) before entering the Micro FluorCam. The trapping beam was reflected at the dielectric mirrors M4 and M3, and
passed through filter F before being focused by an IR-optimized
water-immersion objective (Olympus, UPLSAPO, 60, NA 1.20)
into a diffraction limited spot. The resulting tightly focused beam
was used as an optical trap to which the micro-object was confined. The same objective was used for collection of the cell autofluorescence and imaging the sample to the CCD camera. For
optical trapping at a wavelength of 1064 nm, an Ytterbium fiber laser (YLM-10-1064-LP, IPG Laser) was coupled to the fluorescent
microscope in the same way as described above for Mira. The
transmittance data of all the components in the optical path were
obtained from their manufacturers. The total transmittance of the
system was calculated and used to set equal power of the trapping
beam at all the given wavelengths. The transmittance of the objective at 1064 nm was not listed by the manufacturer and was determined using the dual objective method [51,14]. At each trapping
wavelength the laser power incident at the sample plane was kept
constant at 25 mW. The total energy delivered to the sample plane
within 30 s of trapping was 0.75 J. The trapping capability of the laser beam was tested before each experimental run by trapping a
cell in the sample, followed by dragging of the confined cell over
a distance of several cell diameters. The cell motion relative to
the medium was achieved by moving the micropositioning stage
of the Micro-FluorCam.
T. minutus (Bourrelly) Ettl, CCALA was obtained from the Culture Collection of Autotrophic Organisms, CCALA (Institute of Botany of the ASCR, v.v.i.). T. minutus was grown in 500 mL bubbled
batch cultures at room temperature (approx. 22 °C), in a medium
containing (in mg L 1) N 150; P2O5 50; K2O 300; MgO 30; SO3
75; B 0.3; Cu 0.1; Fe 0.7; Mn 0.4; Mo 0.04; Zn 0.3, dissolved in
tap water (pH 7.2–7.7; hardness 2.4–2.6 mmol L 1) that supplied
calcium. Cultures were cultivated in sunlight. The light cycle was
approximately 12 h light/12 h dark, incident PAR flux density fluctuated between 100 and 300 lmol photons m 2 s 1 during daytime, which translates to an optical intensity of about 5–
16 mW cm 2 [52]. Experimental cultures were used when stationary and 1 week after inoculation.
Z. Pilát et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 27–31
29
Fig. 1. Experimental setup. Fluorescence microscope system (Micro-FluorCam) modified with tunable laser trap (functional details described in the text).
The measurement of the chlorophyll fluorescence was done as
described in [53]. Measuring flashes lasting 10 ls with an intensity
0.03 lmol photons m 2 s 1 were given every 400 ms for 4 s to
measure the F0 value of dark adapted cells. The oxidation of the
plastoquinone pool during the F0 measurement was checked by a
1 s pre-illumination by far-red light. For measurement of Fm, a saturating flash lasting 800 ms was used to reduce the primary quinone acceptor in all active Photosystem II reaction centers.
Measurements of Fm and F0 were taken for each pixel within the
manually selected cell area and averaged. Subsequently, the variable fluorescence Fv = Fm–F0, and the ratio Fv/Fm, was calculated
by the FluorCam software. The first Fv/Fm value for every selected
cell was recorded prior to laser trapping (time stamp: t 30). After
the fluorescence measurement, the trapping laser tuned to a specified wavelength was switched on for 30 s. Two measurements of
Fv/Fm were recorded 15 s (t + 15) and 60 s (t + 60) after switching
off the trapping laser. Control cells were treated in exactly the
same way, except the trapping laser remained off. Ten cells per
treatment were used and their Fv/Fm values were averaged. The
procedure was repeated for each tested laser wavelength. Measurement of the cell’s response to varying trapping power (in four
steps from 25 to 218 mW), was conducted at a wavelength
1064 nm. The measurement protocol was identical, except the
t + 60 data-point was omitted.
3. Results and discussion
Fig. 2 shows the optical trapping of a single algal cell using the
optical tweezers at 1064 nm. The micropositioning stage was used
to move the microscope slide (with two adhered darker cells) with
respect to the optically trapped bright cell that stayed static in the
field of view.
Fig. 3 shows the Fv/Fm ratio in cells before and after 30 s of optical trapping by different laser wavelengths. Prior to trapping, the
Fv/Fm ratio was between 0.3 and 0.5 which is a value significantly
lower than in higher plants but typical for algae and cyanobacteria
[54]. The 735 nm optical trap profoundly reduced the variable fluorescence of the trapped cells. No recovery was recorded after 15 s
and 60 s of darkness. Only about 50% of the original Fv/Fm ratio
was measured after trapping at 785 nm, and somewhat more after
trapping at 835 nm. The Fv/Fm ratio before and after the trapping
did not change significantly at wavelengths of 935 and 1064 nm.
Fig. 4 shows that a trapping wavelength of 1064 nm had no
damaging effect on cells with a laser power from 25 to 218 mW.
Fv/Fm remained approximately the same before (blue bars1) and
after the trapping (red bars). The small variability between the values of Fv/Fm in the individual experiments was due to biological variability of the cells. It was surprising to find that photosynthetic cells
are insensitive to irradiance at 1064 nm at a power that has been
shown to exert effects on heterotrophic cells (most likely by local
heating due to absorption by water). One can speculate that the local
heating dissipation mechanisms reported for photosynthetic membranes [55] can be an important feature contributing to their observed resistance to photodamage at these wavelengths.
Photosystem II does not absorb wavelengths longer than
700 nm [8]. However, Photosystem II photochemistry has been observed at wavelengths significantly longer than 700 nm [11,12]
with an explanation proposed by Thapper et al. [13]. Albeit of a
low yield, the oxygen evolution, variable fluorescence, and electron
paramagnetic resonance signals elicited by these photons are similar to respective phenomena observed in visible light. Considering
that the photon flux density in the optical tweezers is by orders of
magnitude higher than in sunlight, the low yield photochemical
activity driven by these low energy photons could have contributed to the photodamage we observed in Fig. 3 between 735 nm
and 885 nm.
Although trapping laser wavelength can be chosen far from the
absorption maxima of cellular pigments in photosynthetic cells,
using wavelengths in the infrared region may have deleterious effects due to energy absorption by water. Since cells usually contain
over 70% of water by weight, thermal stress of the cell from laser
absorption by water becomes a concern during optical trapping.
Water absorption is lower for 735 nm than for 1064 nm [56,25].
Therefore, because we did not observe any effect on Fv/Fm after cell
trapping at 1064 nm, we considered the laser absorption by water
negligible. It is apparent that the laser power in the sample plane
was too low to cause considerable heating due to the water
absorption.
1
For interpretation of color in Fig. 4, the reader is referred to the web version of
this article.
30
Z. Pilát et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 27–31
Fig. 2. Optical trapping of Trachydiscus minutus cells in time lapse images A–C as the microscope stage is moved to the right at a velocity of approximately 30 lm s 1. The
optically trapped bright cell is static, while the two dark cells below, and adhered on a glass slide, follow the motion of the microscope stage. A laser wavelength of 1064 nm
was used to trap the cell. The incident power was 25 mW in the sample plane. The white scale bar corresponds to 10 lm.
0.5
Fv/Fm(t-30s)
Fv/Fm(t+15s)
Fv/Fm(t+60s)
Fv/Fm
0.4
0.3
0.2
0.1
0
735
785
835
885
935
Control1
1064
Control2
Wavelength [nm]
Fig. 3. The effect of trapping laser wavelength on the Fv/Fm ratio of Trachydiscus minutus cells. Fv/Fm was recorded immediately before the cell was optically trapped (t 30),
and at 15 (t + 15) and 60 s (t + 60) after the laser was switched off (t + 0). Ten samples were averaged for each data-point. The error-bars represent the 95% confidence level.
Fv/Fm(t-30s)
0.5
Fv/Fm(t+15s)
0.4
Fv/Fm
0.3
lengths for Raman microspectroscopy and other analytical
methods involving laser radiation, or other strong radiation
sources. Regardless of the purpose, it is necessary to choose the
wavelength that best minimizes stress and retains the viability of
the manipulated cells.
4. Conclusions
0.2
0.1
0
0
25
50
98
218
Power [mW]
Fig. 4. The effect of trapping laser power incident on the Fv/Fm ratio of Trachydiscus
minutus cells at a wavelength of 1064 nm. Fv/Fm was recorded before irradiation
(t 30) and 15 s after the irradiation (t + 15). Ten samples were averaged per datapoint at 0 and 25 mW, three samples were averaged per data point at 50, 98, and
218 mW. Error-bars represent the 95% confidence level.
When investigating the optical micromanipulation of T. minutus
cells suspended in culture medium pulse amplitude modulation
fluorescence spectroscopy showed that a significant decrease of
Fv/Fm occurred after irradiation by shorter wavelengths (735 nm),
while at longer wavelengths (935 nm and 1064 nm) the decrease
was negligible. This finding indicates that non-invasive optical
manipulation of living cells of photosynthetic microorganisms
can be performed using laser wavelengths longer than 935 nm.
We found that a trapping wavelength of 1064 nm caused no
changes in Fv/Fm even at an incident laser power exceeding
200 mW in the sample plane. This power range is sufficient for reliable 3D manipulation of cells even in a flowing medium. Our conclusions are equally valid for other laser treatments of algal cells,
e.g. laser spectroscopy.
Acknowledgements
The thermal damage in our system can be further decreased if
the optical trapping is done with lower laser power using optimized optical tweezers with stiffer optical trap [57]. The choice
of an optimal wavelength for optical manipulations of living microorganisms is a task parallel to the optimization of excitation wave-
The authors acknowledge support from the European Commission, Ministry of Education, Youth, and Sports of the Czech Republic, and Ministry of Industry and Trade of the Czech Republic,
projects ALISI No. CZ.1.05/2.1.00/01.0017, FR-TI1/433, and Czech-
Z. Pilát et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 27–31
Globe – Centre for Global Climate Change Impacts Studies Reg. No.
CZ.1.05/1.1.00/02.0073.
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