Optical micromanipulations inside yeast cells
Leonardo Sacconi, Iva M. Tolić-Nørrelykke, Chiara Stringari,
Renzo Antolini, and Francesco S. Pavone
We present a combination of nonlinear microscopy and optical trapping applied to three-dimensional
imaging and manipulation of intracellular structures in living cells. We use Titanium-sapphire laser
pulses for nonlinear microscopy of the nuclear envelope and the microtubules marked with green
fluorescent protein in fission yeast. The same laser source is also used to trap small lipid granules
naturally present in the cell. The trapped granule is used as a handle to exert a pushing force on the cell
nucleus. The granule is moved in a raster-scanning fashion to cover the area of the nucleus and hence
displace the nucleus away from its normal position in the center of the cell. Such indirect manipulations
of an organelle (e.g., nucleus) can be useful when direct trapping of the chosen organelle is disadvantageous or inefficient. We show that nonlinear microscopy and optical manipulation can be performed
without substantial damage or heating of the cell. We present this method as an important tool in cell
biology for manipulation of specific structures, as an alternative to genetic and biochemical methods. This
technique can be applied to several fundamental problems in cell biology, including the mechanism of
nuclear positioning and the spatial coordination of nuclear and cell division. © 2005 Optical Society of
America
OCIS codes: 140.7010, 170.1420, 180.5810, 190.0190.
1. Introduction
In the last few years there has been a growing interest toward the use of nonlinear optical techniques for
the imaging of biological samples.1,2 With respect to
conventional fluorescence, these techniques offer several advantages, including higher spatial resolution,
reduced photodamage of the living sample, reduced
photobleaching of the dye, and increased penetration
depth. For example, nonlinear microscopy is well
suited for high-resolution imaging of intrinsic molecular signals in living cells and tissues,3 cell motility,4
L. Sacconi ([email protected]) and R. Antolini are with the
University of Trento, via Sommarive 14, 38050 Povo (Trento), Italy. I. M. Tolić-Nørrelykke and F. S. Pavone are with the European
Laboratory for Non-linear Spectroscopy (LENS), via Nello Carrara
1, 50019 Sesto Fiorentino (Florence), Italy. I. M. Tolić-Nørrelykke
is also with Rugjer Bošković Institute, Bijenika 54, 10000 Zagreb,
Croatia. F. S. Pavone is also with the Department of Physics,
University of Florence and Istituto Nazionale per la Fisica della
Materia Sezione di Firenze, via Sansone 1, 50019 Sesto Fiorentino
(Florence), Italy. C. Stringari is with the Department of Physics,
University of Bologna, viale C. Berti Pichat 6兾2, 40217 Bologna,
Italy.
Received 19 July 2004; revised manuscript received 2 November
2004; accepted 19 November 2004.
0003-6935/05/112001-07$15.00/0
© 2005 Optical Society of America
and the distribution of a neurotransmitter in living
cells.5
On the other hand, optical manipulation techniques have offered new opportunities for applied research in biophysics and nanotecnology.6 Many of the
most powerful optical manipulation techniques are
derived from single-beam optical traps known as optical tweezers.7,8 Optical tweezers have been used for
the manipulation of viruses and cells,9,10 as well as
for displacement of intracellular organelles.9,11–13 Recently, Ketelaar et al.14 used optical tweezers to trap
and stop the movement of the nucleus of root cells, in
order to study how cell growth depends on the location of the nucleus. Single molecules such as DNA15
and motor proteins16 have been also studied by use of
optical tweezers.
Here we report an integration of optical tweezers
with nonlinear microscopy. We show that it is possible to manipulate intracellular structure inside fission yeast cells, and we analyze the trapping of lipid
granules in the cytoplasm. We show that trapping
does not induce cell death or heating of the cell. Finally, we describe an optical manipulation procedure
designed to displace the cell nucleus away from its
normal position in the center of the cell. This technique can be used to study the mechanism of nuclear
positioning, as well as the spatial coordination of nuclear events (chromosome segregation) and cortical
activities (cytokinesis) during cell division.
10 April 2005 兾 Vol. 44, No. 11 兾 APPLIED OPTICS
2001
Fig. 2. Trapping of a lipid granule. A sequence of bright-field
images of a fission yeast cell, in which a lipid granule (arrow) is
optically trapped and moved through the cytoplasm.
Fig. 1. Experimental setup.
2. Materials and Methods
A.
Cell Preparations
The Schizosaccharomyces pombe strain SP837 (h90
leul-32 ura4-D18 ade6-216) was transformed with
the green fluorescent protein (GFP)–fusion construct
D81717 to visualize the nuclear envelope. The cells
were cultured at 30 °C on a synthetic drop-out medium lacking leucine (AA-leu). Visualizing the mitotic spindle was possible with the above strain being
transformed with the GFP-␣2-tubulin fusion construct pDQ10518 and cultured at 30 °C on the AA-leu
medium plus 2 ␮M thiamine. Before microscopy, the
cells were resuspended in liquid AA medium and
mounted on a coverslip covered with lectin BS-1
(2 mg兾ml, Sigma). Experiments were performed at
room temperature (21 °C–22 °C).
B.
Experimental Setup
A scheme of the experimental setup is shown in Fig.
1. The expanded beam of a mode-locked Titaniumsapphire laser (Mira 900F, Coherent, 100-fs pulse
duration, 80-MHz repetition rate) passes through the
scanning system. This system is made of two galvomirrors (VM500, GSI Lumunics) and a telescopic lens
pair (L1 and L2) that pivot the laser beam from the
first galvomirror (GX) to the second one (GY). L3 and
TL (see Fig. 1) pivot the laser beam in the back focal
plane of the microscope objective (Plan Apo 60X兾1.40
Oil, Nikon). The highly focalized laser beam can be
used for nonlinear microscopy with the laser in the
mode lock (ML) configuration, or it can be used as
optical tweezers with the laser in a continuous-wave
(cw) configuration. In the nonlinear microscope configuration, the optical sectioning (Z direction) of the
sample is achieved with a piezoelectric translator (P721, Physik Intrumente). The two-photon emission of
the specimen is extracted from the backscattered sig2002
nal by a dichroic mirror (DM), and the signal is filtered by an IR blocking filter (BF), focalized, and
collected by an avalanche photon diode (APD). In the
optical tweezers configuration, obtaining a strong
trap requires that the laser beam be aligned with the
optical axis of the objective by use of galvomirrors. In
this case we use a XY piezoelectric translator (P-500,
Physik Intrumente) to move the sample with respect
to the optical trap. Galvomirrors and the XYZ piezoelectric translator are computer-controlled during
microscopy and optical manipulation experiments by
a software written in LabVIEW (National Instruments).
A typical experiment was performed as follows.
First, a three-dimensional image of the cell was taken
by two-photon microscopy.19,20 Then, the laser parameters were changed from the imaging configuration (ML mode, ␭ ⫽ 880 nm, power in the sample
⬃1 mW) to the trapping configuration (cw mode, ␭
⫽ 830 nm, power in the sample ⬃200 mW). After the
optical manipulation, the laser parameters were
changed back to those for microscopy and a timelapse sequence of three-dimensional images was acquired to visualize the effect of the manipulation and
the subsequent behavior of the cell.
APPLIED OPTICS 兾 Vol. 44, No. 11 兾 10 April 2005
3. Results and Discussion
A.
Trapping of Lipid Granules
Using the experimental setup described above, we
could easily trap small lipid granules naturally
present in the cell (Fig. 2). We set the laser in cw
mode to avoid nonlinear absorption of the GFP in the
cell. We chose to use a laser wavelength of 830 nm to
reduce photodamage,21 and a laser power of
⬃200 mW in the sample to obtain a sufficient force
for trapping and moving organelles inside the cytoplasm. The trap location was fixed, while the sample
(cells immobilized on a glass coverslip) were moved
with the piezoelectric stage. Thus, the cell was moved
with respect to the trapped object, but we will refer to
this process simply as moving an object through the
cell. After establishing the trapping of lipid granules,
the next step was to analyze physical aspects of trapping and its influence on cell behavior.
1. Maximum Trapping Force
When a particle is moved at a high velocity, the drag
force between the particle and its surroundings is
higher than the force exerted by the optical tweezers,
hence the particle escapes from the trap.22 The maximum force exerted by optical tweezers on a lipid
granule was estimated as the drag force at the maximum velocity (␯max) with which a trapped granule
could be moved without escaping from the trap. The
granule was moved at velocities from 10 to 30 ␮m兾s,
and the escape velocity was found to be greater than
25 ␮m兾s. The apparent viscosity of the cytoplasm
was previously measured to be ␩ ⫽ 0.1–0.8 Pa s.23,24
The radius of the granules (r) was estimated to be 150
nm from electron micrographs of fission yeast cells.25
We estimated the force assuming Stoke’s law, F
⫽ 6␲r␩␯max ⫽ 10–60 pN. This value has to be taken
with reservation because (i) the cytoplasm is viscoelastic and not purely viscous, though the viscous
response dominates at time scales of 0.1 ms to 10 s,26
(ii) the Stoke’s law assumption of an infinite medium
does not hold in a ⬃4 ⫻ 12 ␮m cell surrounded by a
stiff wall, and the cell wall proximity implies that the
real magnitude of the force is higher than the estimated value, and (iii) the cytoplasm is inhomogeneous and the escape force may be affected by the
presence of internal membranes and organelles.
2. Cell Viability
To test for potential photodamage to the cell, we
trapped a lipid granule in the cell for 5 min and
observed the subsequent behavior of the cell. Trapping did not induce any visible damage to the cell.
Yet, when the granules were trapped at a wavelength
of 880 nm (used in two-photon microscopy) instead of
830 nm as usual, we observed photodamage effects (a
bubble forming near the trapped granule) after a few
minutes of trapping. This observation is consistent
with the finding of a photodamage minimum at 830
nm and a maximum at 870 nm21 in Escherichia coli,
suggesting that photodamage may be caused by a
similar mechanism in different cell types. A minor
photodamage can be expected even when a wavelength of 830 nm is used.
We found that the cells were alive for 15 h after
trapping, by observing the characteristic motion of
the granules similar to that in intact cells (⬎95% of
more than 50 cells),26 and by observing that the cells
continued growing and dividing. However, the cells
with trapped granules grew and divided with a delay
of 3–7 h with respect to intact cells (n ⫽ 4 cells, Fig.
3A), which indicates that trapping generated stress
on the cells. The mechanism of laser-induced damage
can be local heating, photochemical processes, or
both, creating reactive chemical species. Inasmuch as
we did not detect substantial heating of the cell (see
Subsection 3.A.3), we assume that the observed delay
in cell growth is due to stress caused by the creation
of reactive chemical species, e.g., singlet oxygen.21
We propose biological applications of trapping to
study microtubule-based processes such as positioning of the nucleus by microtubule forces, as described
Fig. 3. (A) Cells are alive, growing, and dividing after laser trapping. Two sister cells were chosen (inset); a granule was laser
trapped for 5 min in the cell marked L⫹, whereas the cell marked
L⫺ was intact. Cell length is shown as a function of time. Both cells
grew and divided (marked with an asterisk), but the cell with
trapped granules showed a delay of ⬃7 h with respect to the intact
cell. This delay indicates a moderate laser-induced stress, which
the cell can manage. (B) Microtubules are not significantly affected
by trapping. A three-dimensional rendering of the interphase microtubule array before (1) and (2) after laser trapping. (C) Trapping
does not perturb progression through mitosis. Selected images of a
cell in anaphase B without laser trapping (L⫺) and with trapping
(L⫹). The images marked 1 were taken in midanaphase B,
whereas the images marked 2 were taken 7 min later, at the end
of anaphase B. In the L⫹ cell, a granule was trapped close to the
cell center between the acquisition of the two images for 5 min.
Note that the behavior of the two spindles is very similar. (D) and
(E) Trapping does not induce a detectable heating of the cell. (D)
Spindle elongation rate (mean ⫾ s.t.d.) in cells in which a granule
was trapped for 5 min was not significantly different from that in
cells without laser trapping. (E) Spindle elongation rate as a function of temperature. Measurements (circles) are taken from Refs.
28 (data at 20 °C and 36 °C) and 29 (data at 24.5 °C). The shaded
area shows our measurements of the spindle elongation rate in
cells with laser trapping at room temperature (horizontal lines,
mean ⫾ s.t.d.) and the corresponding temperature range (vertical
lines). These measurements were performed at room temperature
(21 °C–22 °C) and correspond to a range of 17.5 °C–21.5 °C on the
graph, suggesting that the cell was not substantially heated by the
laser trap. Scale bar in (B) and (C), 2 ␮m.
10 April 2005 兾 Vol. 44, No. 11 兾 APPLIED OPTICS
2003
in Subsection 3.B. We therefore tested whether trapping affects microtubules. When the trapping was
performed in interphase cells, a typical interphase
array of microtubules was visible after trapping (n
⫽ 3 cells, Fig. 3B). The microtubules showed normal
dynamics of growth and shrinking. When the trapping was performed in cells that were dividing, the
mitotic spindle continued elongating at a normal rate
(n ⫽ 10 cells, Figs. 3C and 3D) and the process of cell
division finished as usual. We conclude that trapping
does not affect the dynamics of interphase microtubules and of the mitotic spindle, and hence it can be
used to study microtubule-related processes, including those during cell division.
3. Laser-Induced Heating in the Cell
In optical trap experiments an intense laser beam is
tightly focused at high intensities, which can induce
heating of the sample.27 The temperature increase in
the focus can reach 50 K兾W, depending on the properties of the trapped particle and its surroundings.27
For the application of optical tweezers in the study of
cell division, as in the current research, it is essential
to estimate the overall laser-induced heating of the
cell, instead of the heating in the focus. The overall
heating can affect the dynamics of cell division activities, whereas the extent of heating of the trapped
particle is of secondary importance, since this particle
is used merely as a handle, as described (see Subsection 3.B). We estimated the overall heating in the cell
by investigating a well-characterized temperaturedependent process: the elongation of the mitotic spindle in Anaphase B.28 To examine whether trapping
increases the spindle elongation rate, we used cells
with a mitotic spindle marked by GFP (Fig. 3C). We
chose those in midanaphase B (using the imaging
configuration of the setup), and trapped a granule
close to the cell center for 5 min (using the trapping
configuration). The 5-min interval was chosen because the application of this method (the displacement of the nucleus) required trapping of the granule
for up to 5 min (see Subsection 3.B). The measured
spindle elongation rate during the period of optical
trapping in the cell was 0.35 ⫾ 0.14 ␮m兾min
(mean ⫾ s.t.d., n ⫽ 10 cells). As a control, the spindle
elongation rate in intact cells at room temperature
was measured to be 0.36 ⫾ 0.11 ␮m兾min (n ⫽ 14).
The two groups of elongation rates (with and without
trapping) were not significantly different (t-test, p
⫽ 0.81, Fig. 3D). The previously published spindle
elongation rates are 0.38 ⫾ 0.1 ␮m兾min at 20 °C,28
0.67 ⫾ 0.18 ␮m兾min at 24 °C–25 °C,29 and 1.4
⫾ 0.2 ␮m兾min at 36 °C.28 The rates measured here in
the cells with laser trapping correspond to a temperature range of 17.5 °C–21.5 °C, considering elongation rate as a function of temperature (Fig. 3E). These
results indicate that the cell was not substantially
heated by the laser trap.
B.
Optically Induced Displacement of the Cell Nucleus
In fission yeast, the nucleus is positioned in the center of the cell. The site of cell division, which also
2004
APPLIED OPTICS 兾 Vol. 44, No. 11 兾 10 April 2005
Fig. 4. Scheme of the scanning-trap procedure for displacing the
cell nucleus. A lipid granule (small sphere) is optically trapped
close to the nucleus (large sphere). The granule is moved along a
raster-scanning trajectory in the y–z plane (small arrow), and simultaneously in the x direction to push the nucleus (large arrow).
coincides with the cell center, seems determined by
the position of the predivision nucleus.30 To study the
mechanism of nuclear centering and the coordination
between the location of the nucleus and of the division plane, we set out to displace the cell nucleus
away from the cell center. Using the experimental
setup described in Subsection 2.B, it was not possible
to optically trap the yeast nucleus, probably because
of an insufficient difference between the refractive
indices of the nucleus and the surrounding cytoplasm. Thus we trapped lipid granules in the cytoplasm, also as described in Subsection 3.A, and used
them as handles to push the nucleus.
We tried to displace the nucleus along the long cell
axis (x axis) by trapping a granule close to one side of
the nucleus and moving it toward the nucleus along
the x axis. This procedure, however, rarely resulted in
a detectable displacement of the nucleus. The nucleus
is soft and round, thus the trapped granule can deform the nucleus and pass without displacing it, especially if the starting position of the granule does not
coincide with the center of the nucleus along the y
axis (the short axis of the cell) and the z axis. Therefore, we designed a scanning-trap technique in which
the trapped granule is being moved along a rasterscanning trajectory over a 2 ␮m ⫻ 2 ␮m area in the
y–z plane, covering the projection of the nucleus on
the y–z plane. The trap moves simultaneously in the
x direction, as shown in Fig. 4. The speed of the trap
movement was 25 nm兾s in the x direction, 2.5 ␮m兾s
in the y, and 0.125 ␮m兾s in the z. We chose this set of
values as a compromise between a high efficiency of
nuclear displacement, which requires slow movements, and a short laser-irradiation time of the cell.
To measure the nuclear position, we used cells in
which the nuclear envelope was marked by GFP. After acquiring a two-photon image of the cell, we applied the scanning-trap technique for 3–5 min. An
example of a cell with the displaced nucleus is shown
Fig. 5. Optically induced displacement of the nucleus. (A) Left, a
singe optical section of the cell before the optical manipulation. The
nucleus is in the cell center. Middle, the cell after the manipulation. Right, superposition of the two images, showing the displacement of the nucleus. Scale bar, 2 ␮m. (B) Set of optical z sections of
the cell before (1) and after (2) the manipulation, showing that the
whole volume of the nucleus was displaced.
in Fig. 5A. Optical sections along the z axis (Fig. 5B)
indicate that the scanning-trap technique displaced
the whole nucleus, instead of deforming it without a
net displacement. The maximum displacement obtained in a group of more than 20 cells was 2 ␮m,
whereas movements of the center of nonmanipulated
nucleus in interphase do not exceed 0.5 ␮m.
The nuclear envelope consists of an inner and an
outer nuclear membrane, connected through nuclear
pores. The outer membrane is continuous with the
endoplasmatic reticulum (ER).31 Manipulation of the
nucleus most likely results in remodeling of this network of membranes. If the ER is attached to the cell
cortex in the vicinity of the nucleus, a larger force
may be needed to displace the nucleus. This kind of
attachment may be the reason the optical manipulation did not result in a detectable displacement of the
nucleus in ⬃30% trials.
After the nucleus had been displaced by the optical
trap, it spontaneously moved back toward its original
position at the cell center (Fig. 6). What is the mechanism that positions the nucleus in the cell center?
One can directly test models of nuclear positioning,
which typically involve microtubule-dependent forces,32 by optically displacing the nucleus and studying
its return toward the cell center using microscopy
under native or nonnative (drug treatments and兾or
mutant cell) conditions.33 Our observation that trapping does not affect microtubule dynamics supports
the use of optical trapping for studies of nuclear po-
Fig. 6. Nucleus moves spontaneously back to the cell center after
the optical manipulation. (A) Sequence of images of the cell before
manipulation (the first image) and after the manipulation (the rest
of the sequence). Numbers refer to time in minutes. The red lines
serve as guides to the eye. Scale bar, 2 ␮m. Bottom, nuclear position as a function of time. The nucleus was displaced by 1.2 ␮m,
after which it returned toward the cell center. The error on each
data point is ⬃50 nm.
sitioning. Furthermore, the central position of the
yeast nucleus coincides with the position of the cell
division plane. Coordination of the nuclear position
and the site of cell division is crucial for cell survival:
After separation of the divided nuclei, the cell has to
divide between them, for successful distribution of
the genetic material to the daughter cells to occur.
Coordination of the nuclear and the division plane
position can be probed directly by optical manipulations of the nuclear position at various stages of the
cell cycle.33 Optical trapping does not affect the dynamics of cell division and allows for observation of a
single cell both before and after the manipulation,
hence it is possible to know the exact stage of the cell
cycle at which the manipulation is performed. The
latter may be an important advantage of the trapping
method over more commonly used centrifugation,34
in studies in which timing is crucial during displacement of cell organelles.
4. Conclusions
Optical micromanipulations of cell organelles can be
performed even when the chosen organelle cannot or
should not be trapped directly, by trapping another
intracellular structure and using it as a handle to
exert force on the chosen organelle. This technique
does not induce gross damage or heating of the cell.
10 April 2005 兾 Vol. 44, No. 11 兾 APPLIED OPTICS
2005
After trapping, the cell continues growing and divides, though with a delay. This indicates that the
laser induces stress on the cell, to an extent the cell
can manage. If trapping is performed during cell division, the mitotic spindle elongates at a normal rate
and the division completes as usual, suggesting that
the dynamics of cell division is not affected by trapping; hence trapping can be used to study divisionrelated processes. Optical manipulations, combined
with two-photon microscopy and laser nanosurgery,20
can be used to directly test models of cell mechanics
involving forces and spatial order in a living cell.
Outstanding problems in cell biology, such as positioning of cell organelles, centering of the mitotic
spindle,35 and spatial coordination of nuclear and cell
division,33 can be addressed by these methods.
This research was supported by the European Laboratory for Nonlinear Spectroscopy (LENS) contract
HPRI-CT-1999-00111 CE and by the Ente Cassa di
Risparmio di Firenze. We thank Yasushi Hiraoka for
the GFP-fusion constructs, Genevieve Thon for cell
transformation, Marco Capitanio for making Fig. 4
and for discussions, and Francesco Vanzi for constructive comments on the manuscript.
14.
15.
16.
17.
18.
19.
20.
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10 April 2005 兾 Vol. 44, No. 11 兾 APPLIED OPTICS
2007