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Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 2, Iss. 7 — Jul. 16, 2007
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Real-time actin-cytoskeleton depolymerization detection in a single cell using optical tweezers

Anna Chiara De Luca, Giovanni Volpe, Anna Morales Drets, Maria Isabel Geli, Giuseppe Pesce, Giulia Rusciano, Antonio Sasso, and Dmitri Petrov  »View Author Affiliations


Optics Express, Vol. 15, Issue 13, pp. 7922-7932 (2007)
http://dx.doi.org/10.1364/OE.15.007922


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Abstract

The cytoskeleton provides the backbone structure for the cellular organization, determining, in particular, the cellular mechanical properties. These are important factors in many biological processes, as, for instance, the metastatic process of malignant cells. In this paper, we demonstrate the possibility of monitoring the cytoskeleton structural transformations in optically trapped yeast cells (Saccharomyces cerevisiae) by tracking the forward scattered light via a quadrant photodiode. We distinguished normal cells from cells treated with latrunculin A, a drug which is known to induce the actin-cytoskeleton depolymerization. Since the proposed technique relies only on the inherent properties of the optical trap, without requiring external markers or biochemical sensitive spectroscopic techniques, it can be readily combined with existing optical tweezers setups.

© 2007 Optical Society of America

1. Introduction

There are only few experimental techniques which are able to give information about cellular mechanical properties. Historically, the prevalent technique has been micropipette aspiration [5

5. R. M. Hochmuth, “Micropipette aspiration of living cells,” J. Biomech. 33, 15–22 (2000). [CrossRef]

]. More recently, micro-rheological methods have been used to make local measurements of viscoelastic properties inside cells [6

6. I. Y. Wong, M. L. Gardel, D. R. Reichman, E. R. Weeks, M.T. Valentine, A. R. Bausch, and D. A. Weitz, “Anomalous diffusion probes microstructure dynamics of entangled F-Actin natworks,” Phys. Rev. Lett. 92, No.17, 178101–4 (2004). [CrossRef] [PubMed]

, 7

7. I. M. Tolic-Norrelykke, E-L. Munteanu, G. Thon, L. Oddershede, and K. Berg-Sorensen, “Anomalous diffusion in living yeast cells,” Phys. Rev. Lett. 93, 0781021–0781024 (2004). [CrossRef]

].

In this work, we propose and demonstrate a simple, fast and reliable method to monitor the actin-cytoskeleton structural transformations in optically trapped yeast cells (Saccharomyces cerevisiae) by tracking the forward scattered light via a quadrant photodiode. This technique is similar to the photonic force microscope [12

12. L. P. Ghislain and W. W. Webb, “Scanning-force microscope based on an optical trap,” Opt. Lett. 18, 1678–1680 (1993). [CrossRef] [PubMed]

, 13

13. E. L. Florin, A. Pralle, E. H. Stelzer, and J. K. H. Horber, “Photonic forcemicroscope calibration by thermal noise analysis,” Appl. Phys. A 66, 71–73 (1998). [CrossRef]

] and has been applied for living cells in [7

7. I. M. Tolic-Norrelykke, E-L. Munteanu, G. Thon, L. Oddershede, and K. Berg-Sorensen, “Anomalous diffusion in living yeast cells,” Phys. Rev. Lett. 93, 0781021–0781024 (2004). [CrossRef]

, 14–16

14. E. Helfer, S. Harlepp, L. Bourdieu, J. Robert, F. C. MacKintosh, and D. Chatenay, “Microreology of Biopolymer-Membrane Complexes,” Phys. Rev. Lett. 85, No.2 457–460 (2000). [CrossRef] [PubMed]

]. The non invasive character of Optical Tweezers allows the observation of cellular response to stress without interference due to non physiological handling [17–21

17. M. T. Wei and A. Chiou, “Three-dimensional tracking of Brownian motion of a particle trapped in optical tweezers with a pair of orthogonal tracking beams and the determination of the associated optical force constants,” Opt. Express 13, No.15 5798–5806 (2005). [CrossRef] [PubMed]

], which might lead to measurement artefacts. In addition, since this technique relies only on the inherent properties of the optical trap, without requiring external markers or biochemical sensitive spectroscopic techniques, it can be readily combined with existing optical tweezers set-ups.

2. Experimental setup

Figure 1 illustrates the main components of our experimental setup. The trapping beam is given by a semiconductor laser (785nm, Monocrom, Barcelona, Spain) with a monomode fiber output that generates a Gaussian beam and emits a maximum power of 15mW. It is tightly focused into the sample by an Olympus oil-immersion infinity corrected objective lens (100X, 1.25 N.A.). A telescope, formed by lenses L 1 (f 1 = 10cm) and L 2 (f 2 = 30cm), assures a collimated beam overfilling at the objective input pupil. The forward scattered light from the trapped object is collected by a second objective lens (40X, 0.75 N.A.) positioned over the sample and projected onto a quadrant photodiode (QPD), to track the Brownian motion of the trapped cell organelles. The resulting signals are, then, transferred through an analog to digital conversion card to a computer for the analysis. The light from a LED, focused on the sample by the same 40X objective, was used to illuminate the sample; an image of the trapped cell was obtained by using a CCD camera, coupled to the microscope. The cells are suspended in physiological solution and contained in an home-made sample holder, constituted by two sandwiched 80μm coverslips separated by a 100μm spacer and sealed with water-insoluble silicone vacuum grease to prevent sample evaporation.

For this experiment, we use a kind of yeast, Saccharomyces cerevisiae, 3 – 5μm diameter. It is an excellent model organism for research in cellular and molecular biology as many fundamental cellular processes are conserved from yeast to human cells. Moreover, they are non-toxic, easily available and easy to grow. To examine the effects of the actin cytoskele-ton depolymerization we have treated the cell with latrunculin A (LAT-A), a monomeric actin sequestering drug that depolymerizes the actin cytoskeleton in numerous cell type, including yeast cell [22

22. K. R. Ayscough, J. Stryker, N. Pokala, M. Sanders, P. Crews, and D. G. Drubin, “High rates of actin filaments turnover in budding yeast and roles for actin in extablishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin A,” J. Cell. Biol. 137, 399–416 (1997). [CrossRef] [PubMed]

, 23

23. M. Cou, S. L. Brenner, I. Spector, and E. D. Korn, “Inhibition of actin polymerization by latrunculin A,” FEBS Lett. 213, 316–318 (1987). [CrossRef]

]. The cells were grown to exponential phase, which was checked by measuring the Optical Density (OD) of the cell culture (1.4 at 600nm). LAT-A was added from a 10mM dimethyl sulfoxide (DMSO) stock to a final concentration of 200mM, at a temperature of 25°C. After the alignment of the QPD, a single yeast cell, i.e. which does not have a bud, is trapped and the sum (z) and differential (x and y) signals from the QPD are monitored. This technique is extremely sensitive to the distance of the trap from the coverslip, which was always set to 15μm for the reproducibility of the experiment. By studying Brownian motion of granules inside them we distinguished healthy cells (in DMSO) from cells treated with LAT-A.

Fig. 1. Experimental setup: L, lens; M, mirror; DM, dichroic mirror; QPD, quadrant pho-todiode.

3. Results and discussion

Single yeast cells can be trapped by using moderate laser power (P ≃ 1.5mW on the sample). At this power level, the heating and photodamage can be considered negligible. Indeed, for an aqueous sample irradiated by a focused laser beam (λ = 1064nm), the heating rate is about 10°C/W [24

24. S. C. Kuo, “A single assay for local heating by optical tweezers,” Methods Cell Biol. 55, 43–45 (1998). [CrossRef]

, 25

25. E. J. G. Peterman, F. Gittes, and C. F. Schmidt, “Laser-induced heating in optical traps,” Biophys. J. 84, 1308–1316 (2003). [CrossRef] [PubMed]

] and yeast cells have been shown to progress in their cell-cycle under such trapping conditions [26

26. G. P. Singh, G. Volpe, C. M. Creely, H. Grötsch, I. M. Geli, and D. Petrov, “The lag phase and G1 phase of a single yeast cell monitored by Raman microspectroscopy,” J. Raman Spectrosc. 37, 858–864 (2006). [CrossRef]

].

Fig. 2. (A), yeast cell under zero-dragging condition (v = 0): the trapped organelle is in the cell center. (B), yeast cell under dragging condition (v = v 1) the whole cell is translated while the organelle remains fixed. (C),increasing the dragging force (v 2 > v 1) trapped organelle is almost in contact with the cell membrane.
Fig. 3. The optical potential well U(x) for a healthy yeast cell. The solid line represent the best fit performed with a parabolic function.

In our experiment, by tracking Brownian motion of the cellular organelles, we monitor the structural evolution of the cytoskeleton. It is important to underly that the beam waist at the focal plane is of the order of the wavelength in the medium (λ ≃ 0.6μm in our case) and is much less than the cell size. In these conditions, while a single organelle is trapped, all the other cellular components continue to move freely. To illustrate this, we applied a drag force to the trapped cell. That was achieved by translating the stage with the sample at a given velocity. The frame A of Fig.2 corresponds to zero-dragging condition (v = 0): it can be seen that the granules are confined in the optical trap. When the drag was on (v = v 1), the whole cell except the trapped granules was translated (see frame B). Increasing the dragging (v = v 2 > v 1), the cell displaced even more so that the trapped granules are almost in contact with the cell membrane (see frame C).

The fact that the granules are the trapped part of the cell represents a big advantage because that allows to study the mechanical response of the cytoskeleton. That was done by tracking the Brownian motion of the trapped granules by means of the forward scattering light and a position detector. The position of the trapped granule was measured in terms of the signal provided by the QPD (Vx, expressed in Volts) and for small displacement from the laser focus the optical potential well U(Vx) formed by the optical beam along the axis x perpendicular to the laser propagation axis can be written as:

U(Vx)=12kxβ2Vx2
(1)

where kx is the trap stiffness β = x/Vx is the voltage-to-displacement conversion coefficient. In Fig. 3 we show a typical potential well obtained by trapping a healthy yeast cell. As it can be seen, although we deal with a non-spherical particle, for small displacements the potential shape approximates quite well a parabola. The shape of the potential along the axis y looks quite similar. In some cases, we found a strong deviation of the potential well from the parabolic behavior; this occurs, in particular, when more than one particle was trapped. In these cases, the measurement was rejected. From the fit of the experimental data we could estimate the term kx β 2 in Eq. 1. As it will better clarified later, for the purpose of our experiment it is sufficient to express the stiffness not in absolute units but in terms of QPD calibration factor β. We have repeated the same procedure trapping different healthy yeast cells (N ≃ 30) and calculated the average of the values kxβ 2:

<kxβ2>=(2.9±0.8)1013pNmV2
(2)

Afterwards we studied cells treated with LAT-A.

For these cells, we tracked the Brownian motion at different incubation time. Data were collected between 15min and 2h after the application of the LAT-A, when the effect of this drug reached its saturation. In Fig. 4 we report the optical potential at the beginning and at the end of this process. From a parabolic fit we found kxβ 2 = (2.5 ± 0.2) · 10-13 pNm/V 2 at the beginning of the LAT-A application. This value is consistent with the average value obtained from the statistic analysis of healthy cells shown above. The final effect of LAT-A is quite evident if we look at the potential well 2h after the application of the drug (see Fig. 4).

Fig. 4. The optical potential well for a LAT-A treated cell after a incubation time of 15 min (a) and 2 h (b).

The time evolution of kxβ 2 during the LAT-A action is shown in Fig.5. In particular, after 2h from the application of LAT-A kxβ 2 = (19.2 ± 0.4) ∙ 10-13 pNm/V 2, i.e. a value rather different from that of healthy yeast cells. Therefore, if we assume that the calibration factor β is independent on the status of the cell (i. e. LAT-A does not affect the granules), we can conclude that the trap stiffness increases for the yeast cells treated by LAT-A.

In order to obtain more specific information concerning the medium inside the cell we subsequently analyzed the stochastic signal derived from the granules Brownian motion. According to the solution of the Langevin equation for each cartesian coordinate the theoretical Power Spectral Density (PSD) of the Brownian motion for a trapped particle is given by the function [12

12. L. P. Ghislain and W. W. Webb, “Scanning-force microscope based on an optical trap,” Opt. Lett. 18, 1678–1680 (1993). [CrossRef] [PubMed]

, 13

13. E. L. Florin, A. Pralle, E. H. Stelzer, and J. K. H. Horber, “Photonic forcemicroscope calibration by thermal noise analysis,” Appl. Phys. A 66, 71–73 (1998). [CrossRef]

]:

P(f)=kBTπ2γ1(fc2+f2)
(3)

fc=ki2πγ
(4)

where ki is the trap stiffness constant along the ith direction, γ = 6πηa is the hydrodynamic coefficient, η the fluid viscosity and a the radius of optically trapped particle. To test our apparatus we trapped first a spherical dielectric particle in water. In Fig. 6 trace (a), we show the PSD of a 4.5 μm diameter polystyrene bead in water; the experimental data fit with a Lorentzian profile.

Our analysis of the PSD obtained from the yeast cells started from healthy cells. Measurements were performed keeping the trapped cell under observation for about two hours. In Fig.6 trace (b), we show a typical PSD signal for a healthy yeast cell. As it is possible to see, although the Langevin equation which governs the Brownian motion of a over-damped rigid sphere in a harmonic potential well does not strictly apply to the granules, the experimental data fit sufficiently well with the function of Eq. 3. From the fitting of the experimental PSD we could estimate the corner frequency. In Fig. 7, trace (a) we report the corner frequency value for a single healthy cell as function of observation time. The fcx and fcy values resulted consistent and uniformly distributed around their mean value. The error on the single determination was estimated from the fitting procedure. It is important to point out that no detectable transformation was induced by the presence of the trap, in accordance with reference [26

26. G. P. Singh, G. Volpe, C. M. Creely, H. Grötsch, I. M. Geli, and D. Petrov, “The lag phase and G1 phase of a single yeast cell monitored by Raman microspectroscopy,” J. Raman Spectrosc. 37, 858–864 (2006). [CrossRef]

]. The average value for fc calculated on N = 30 repeated measurements is reported in Tab.1.

Finally, we have applied this method to determine the effect of LAT-A on actin cytoskeleton in yeast cells. Again we started our observation after 15 min from the LAT-A application while the duration was of about 2 hours. A comparison of the fc at different incubation time of LAT-A treated cells is shown in Fig. 7, trace (b): the frequency corner increases with time and reaches a plateau value (fc ≃ 3.5Hz) after two hours. For completeness the measured fc at the beginning (t = 15min) and after 2h from the LAT-A application are reported in Tab.1.

Table 1. A comparison of the trap stiffness, corner frequency and their ratio R for different conditions of yeast cell.

table-icon
View This Table

Cytoskeleton depolymerization, induced by LAT-A, leads to a change of both the trap stiffness (kxβ 2) and corner frequency (fc). The former change can be ascribed to a variation of the refractive index of the depolymerized actin network. Indeed, the trap stiffness depends on the ratio between the refractive index of the trapped object and the surrounding medium. Less direct is the interpretation of the latter change. The corner frequency fcx = kx/(2πγ) depends both on the stiffness and on the viscosity. Therefore, it seems convenient to calculate their ratio R:

R=kxβ2fcxη.
(5)

In this way, we can obtain direct information about the viscosity. These data are plotted in Fig. 8 as function of the LAT-A action time, while the values at beginning and at the end of this process are listed in Tab. 1. As it can be seen in Fig. 8, it seems that the depolymerization of the Actin-cytoskeleton network increases the viscous character of the intracellular environment.

It is important to emphasize that the goal of this investigation is not to study cellular vis-coelasticity. This kind of measurements would require the determination of the complex shear modulus G *(f), which is the physical quantity commonly employed to quantify the viscoelas-ticity of a medium [33

33. K. M. Addas, C. F. Schmidt, and J. X. Tang, “Microrheology of solutions of semiflexible biopolymer filaments using laser tweezers interferometry,” Phys. Rev. E 70, 021503 1–16 (2004). [CrossRef]

]. This approach requires a more sophisticated data analysis which is beyond the purpose of this work. On the contrary, our approach provides a fast and reliable method to sort cells in different physiological states, otherwise undistinguishable by using an optical microscope.

Fig. 5. Behavior of kxβ 2 for a LAT-A treated cell versus the observation time.
Fig. 6. Experimental PSD from the x signal for a trapped polystyrene bead (trace a) and for a trapped normal yeast cell (trace b). The Lorentzian fitting curves are also shown.
Fig. 7. Measured frequency corner for a living cell (a) and a LAT-A treated cell (b) during 2 hours. In the second case, the corner frequency increases with time.
Fig. 8. Behavior of η for a LAT-A treated cell under 2 h.

4. Conclusion

The results shown demonstrate the possibility of monitoring the cytoskeleton structural transformations in optically trapped cells by tracking the forward scattered light via a quadrant photodiode. The analysis of its PSD allows us to distinguish healthy yeast cells from the ones whose cytoskeleton is depolymerized and it also permits us to track the depolymerization process in real-time. In particular, the F-actin cytoskeleton depolymerization, induced by treatment with LAT-A, results in a progressive increased corner frequency by a factor of 1.7. Since F-actin plays an important role in cellular mechanics, these changes alter the Brownian motion of the cellular organelles; therefore, this can be seen as an inherent cell marker that offers an alternative to traditional techniques and opens the door to a new and cheap tool for cell characterization.

Acknowledgments

A. C. De Luca acknowledges the financial support of the Italian National Research Council (CNR, International Short-term Mobility Program). This research was carried out in the framework of ESF/PESC (Eurocores on Sons), through grant 02-PE-SONS-063-NOMSAN, and with the financial support of the Spanish Ministry of Science and Technology (FIS2005- 02129). It was also supported by the Departament d’Universitats, Recerca i Societat de la Informació and the European Social Fund. Finally, the authors thanks Isabel María Fernández-Golbano for providing the yeast cell.

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L. P. Ghislain and W. W. Webb, “Scanning-force microscope based on an optical trap,” Opt. Lett. 18, 1678–1680 (1993). [CrossRef] [PubMed]

13.

E. L. Florin, A. Pralle, E. H. Stelzer, and J. K. H. Horber, “Photonic forcemicroscope calibration by thermal noise analysis,” Appl. Phys. A 66, 71–73 (1998). [CrossRef]

14.

E. Helfer, S. Harlepp, L. Bourdieu, J. Robert, F. C. MacKintosh, and D. Chatenay, “Microreology of Biopolymer-Membrane Complexes,” Phys. Rev. Lett. 85, No.2 457–460 (2000). [CrossRef] [PubMed]

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23.

M. Cou, S. L. Brenner, I. Spector, and E. D. Korn, “Inhibition of actin polymerization by latrunculin A,” FEBS Lett. 213, 316–318 (1987). [CrossRef]

24.

S. C. Kuo, “A single assay for local heating by optical tweezers,” Methods Cell Biol. 55, 43–45 (1998). [CrossRef]

25.

E. J. G. Peterman, F. Gittes, and C. F. Schmidt, “Laser-induced heating in optical traps,” Biophys. J. 84, 1308–1316 (2003). [CrossRef] [PubMed]

26.

G. P. Singh, G. Volpe, C. M. Creely, H. Grötsch, I. M. Geli, and D. Petrov, “The lag phase and G1 phase of a single yeast cell monitored by Raman microspectroscopy,” J. Raman Spectrosc. 37, 858–864 (2006). [CrossRef]

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33.

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OCIS Codes
(110.0180) Imaging systems : Microscopy
(140.7010) Lasers and laser optics : Laser trapping
(170.1420) Medical optics and biotechnology : Biology
(170.4520) Medical optics and biotechnology : Optical confinement and manipulation

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: March 28, 2007
Revised Manuscript: May 11, 2007
Manuscript Accepted: June 5, 2007
Published: June 11, 2007

Virtual Issues
Vol. 2, Iss. 7 Virtual Journal for Biomedical Optics

Citation
Anna Chiara De Luca, Giovanni Volpe, Anna Morales Drets, Maria Isabel Geli, Giuseppe Pesce, Giulia Rusciano, Antonio Sasso, and Dmitri Petrov, "Real-time actin-cytoskeleton depolymerization detection in a single cell using optical tweezers," Opt. Express 15, 7922-7932 (2007)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-15-13-7922


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References

  1. B. Alberts, D. Bray, A. Johnson, J. Lewis, M. Raff, K. Roberts and P. Walter, Essential Cell Biology, (Garland, New York, 2002).
  2. K. M. Rao and H. J. Cohen, "Actin cytoskeleton network in aging and cancer," Mutat. Res. 256, 139-148 (1991). [PubMed]
  3. J. Guck, S. Schinkinger, B. Lincoln, F. Wottawah, S. Ebert, M. Romeyke, D. Lenz, H. M. Erickson, R. Ananthakrishnan, D. Mitchell, J. Kas, S. Ulvick, and C. Bilby, "Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence," Biophys. J. 88, 3689-3698 (2005). [CrossRef] [PubMed]
  4. K. A. Ward, W. I. Li, S. Zimmer and T. Davis, "Viscoelastic properties of transformed cells: role in tumor cell progression and metastasis formation," Biorheology 28, 301-313 (1991). [PubMed]
  5. R. M. Hochmuth, "Micropipette aspiration of living cells," J. Biomech. 33, 15-22 (2000). [CrossRef]
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