Nanoscale Operation of a Living Cell Using an Atomic Force

ABSTRACT. We have developed a tool for performing surgical operations on living cells at nanoscale resolution using atomic force microscopy (AFM) and...
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NANO LETTERS

Nanoscale Operation of a Living Cell Using an Atomic Force Microscope with a Nanoneedle

2005 Vol. 5, No. 1 27-30

Ikuo Obataya,† Chikashi Nakamura,*,†,‡ SungWoong Han,‡ Noriyuki Nakamura,†,‡ and Jun Miyake†,‡ Research Institute for Cell Engineering (RICE), National Institute of AdVanced Industrial Science and Technology (AIST), 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan, and DiVision of Biotechnology and Life Science, Tokyo UniVersity of Agriculture and Technology (TUAT), 2-24-16 Naka-cho, Koganei, Tokyo 184-8588 Japan Received September 7, 2004; Revised Manuscript Received November 11, 2004

ABSTRACT We have developed a tool for performing surgical operations on living cells at nanoscale resolution using atomic force microscopy (AFM) and a modified AFM tip. The AFM tips are sharpened to ultrathin needles of 200−300 nm in diameter using focused ion beam etching. Force− distance curves obtained by AFM using the needles indicated that the needles penetrated the cell membrane following indentation to a depth of 1−2 µm. The force increase during the indentation process was found to be consistent with application of the Hertz model. A threedimensional image generated by laser scanning confocal microscopy directly revealed that the needle penetrated both the cellular and nuclear membranes to reach the nucleus. This technique enables the extended application of AFM to analyses and surgery of living cells.

There is great demand for single-cell manipulation in current biotechnology because it is significantly important to investigate where and when molecules display their functions in controlling the activity of cells. If the molecules of interest such as nucleic acids, proteins and chemicals can be transferred to the inside of a living cell at a known time, we can modulate the cell activity while monitoring the reaction of the cell in real-time. Such techniques will be applied not only for investigation of cell activity but also in controlled differentiation or therapy of living cells. Microinjection of proteins, peptides, and genetic materials into a living cell is widely practiced using microcapillaries.1,2 The technique plays an important role in the transplantation of nuclei or introduction of nucleic acids for tracking the protein expression. However, fatal damage from using microcapillaries due to the shape of the capillaries and inaccuracy of the displacement is often problematic for manipulation of small cells. To overcome these problems, fundamental changes in controlling devices for displacement of inserting materials and optimization of the size and shape of the inserting materials are necessary. Recently, atomic force microscopy (AFM) has been used for imaging cell surfaces because the apparatus allows * Corresponding author. Phone: +81-6-6494-7858, Fax: +81-6-64947862, E-mail: [email protected]. † RICE/AIST. ‡ TUAT. 10.1021/nl0485399 CCC: $30.25 Published on Web 11/25/2004

© 2005 American Chemical Society

researchers to observe living cells in physiological conditions.3,4 Besides surface observation, the AFM probe can act against a cell surface. For example, the elastic5-7 or viscoelastic8 properties of the cell surface have been estimated for various cells by contacting and indenting the surface with an AFM probe. Since the surface of an AFM probe can be chemically modified, probes have been used for the chemical modification of the surfaces of bacteria.9 Moreover, it is considered that an atomic force microscope is suitable as a controlling device for cell manipulation in order to access the interior of living cells. Investigation of the localization of mRNA by extraction of mRNA with an AFM tip from living cells has been reported.10 The displacement of the cantilever can be controlled at nanoscale resolution by piezo devices while monitoring the exerting force on the cantilever. Since the force should reflect the situation of cantilever (contacting, indenting, or penetrating), the position of tip is predictable. This feature is essentially superior to the conventional micromanipulation techniques with respect to precise displacement and monitoring. To insert a solid material, the size and shape of the tip should be improved for certainty of insertion and minimization of damage to a living cell. Therefore, we have attempted to develop a system and materials for a single-cell operation using AFM and have fabricated AFM probes in the shape of an ultrathin needle, called a nanoneedle.11,12 In this paper, we present the

Figure 1. (A) Schematic representation of a nanoneedle on the AFM tip over a living cell. (B) Electron microscopic image of nanoneedle with a scale bar of 3 µm.

mechanical response during insertion of a nanoneedle and direct observation of a living cell and the nanoneedle inserted by the system. We developed a cell manipulation apparatus using a molecular force probe (MFP-1D, Asylum Research, Santa Barbara) as a force detecting instrument. The MFP is placed on the stage of a confocal fluorescence microscope FV-300/ IX71 (Olympus, Tokyo). This combination allows us to obtain phase contrast, fluorescent and confocal fluorescent images of cells, and a cantilever simultaneously. Ultrathin needles were prepared by focused ion beam (FIB) etching from pyramidal Si AFM tips (Nanosensors, Neuchatel) that have spring constants from 0.1 to 0.2 N/m. There are commercially available AFM tips with high aspect ratio, however, they are not long enough to reach inside the cell. Since the heights of living cells are more than 3 µm, the length of the tip body must be more than this height, otherwise the base of the tip bumps the cell surface and disturbs the force analyses. Furthermore, the tapered shapes of such etched tips are not favorable with respect to the probability of penetration and indentation depth before penetration as we reported previously.11 To fabricate the ultrathin needle, the FIB was sequentially focused on two areas of the pyramid tip so as to leave a thin area on the tip. Then, the resulting triangular plate on the tip was etched in the same manner after rotating the cantilever perpendicular to the original direction. The needle was tilted about 11° with respect to the center axis of the tip in order to compensate for the mounting angle of its holder (Figure 1A). The tips were etched into needles of 200-300 nm in diameter and 6-8 µm in length (Figure 1B). Force measurements were conducted on a human epidermal melanocyte (NHEM, Kurabo, Osaka) in a medium at 37 °C under 5% CO2 atmosphere. The tip was lowered over the center of the cell. Figure 2A shows a typical forcedistance curve during indenting and pulling of a normal pyramidal AFM tip. The force curve shows a continuous increase as the cantilever was lowered, implying that the tip indented the cell surface without penetration. It has been reported that an indenting force of no more than 10 nN is required when using a normal pyramidal AFM tip in order to penetrate the cell surface.13-15 But such a large indentation depth relative to the height of the cell may cause serious damage. Even if the cell could survive after indentation, the large deformation may cause undesirable mechanotransduc28

Figure 2. Force-distance curves during approach and retraction from a melanocyte using a normal pyramidal AFM tip (A) and the nanoneedle (B). The cantilever approached (blue arrow) and then retracted (red arrow).

tions.16 On the other hand, the force curve using a nanoneedle was quite different from that using a pyramidal tip (Figure 2B). The force increased until about 1 µm in the approaching process, but the force did not show a continuous increase as seen in the plot with a pyramidal tip. This force dropping during the approach process was considered to be the point of the penetration of the needle tip into the cell. Such force dropping or relaxation has been reported when erythrocytes were indented to penetrate the membrane using a pyramidal tip.13 However, both the force and the indentation depth for penetration were quite small when using the nanoneedle. It is important to maintain the condition or the activity of the cell for a living cell operation; therefore, in these respects we believe that the nanoneedle is superior to a normal pyramidal tip for penetration into a cell. After the indentation, the force fluctuated by 1 to 2 nN, suggesting that the needle was inserted through the cell membrane. The force might contain friction force and viscous resistance between the surface of the needle and the cell constituents. After this inserting section, the force increased continuously producing a steeper slope, suggesting that the base of needle made contact with the cell surface over a larger area. Since the first increase of the force curve was expected to be due to indentation of the cell surface, we analyzed the force curve by applying the Hertz model that describes the indentation of homogeneous elastic material by a stiff material with a defined geometry.6,17 In fact, living cells are not ideal samples for applying the model because of their anisotropy and heterogeneity of the surface; however, it should be worth rationalizing the force curve and comparing with reported values estimated by applying this model. We prepared two types of nanoneedles, varying the shapes of their tip (flat-ended cylinder and cone shape), for comparison. Figure 3 shows the typical force curves when using the needles. For the needle with a cone-shaped tip, the force curve showed a quadratic force increase after contacting the cell surface. Then the force increased with a linear function. The point of this functional change in the force increase corresponded to the point of the geometrical change from a cone to a cylinder. On the other hand, the force increase showed only a linear function with the cylindrical needle Nano Lett., Vol. 5, No. 1, 2005

Figure 3. Typical force-distance curves for a melanocyte using nanoneedles that have cone-like shapes (A) and cylindrical shapes (B) at the edges. Insets show magnified SEM images around the tip of the needle. Proposed situations of the nanoneedle tip and the cell surface are depicted under the plots.

(Figure 3B). The Hertz model mechanics describes force F as a function of indentation depth I with a defined shape of the indenter with constant values co and cy. F ) coI2 (for cone-shaped indenter) F ) cyI (for cylindrical indenter) The force curves using the two types of the tip were reasonable with respect to the tip geometry by applying this mechanics. Estimated elastic moduli were 2 to 10 kPa for melanocytes in the experiments. The values were comparable to the elasticity for HUVEC cells (7.2 kPa)18 and fibroblast cells (3-5 kPa)19 using the same parameters for estimation, suggesting that the first force increase after contacting with the nanoneedles was also explained by the Hertz model. Furthermore, laser scanning confocal microscopy (LSM) was employed to observe directly the cell and the inserted needle. We used a nanoneedle fabricated from another type of AFM probe. Since the probe has a tetrahedral tip that

protrudes from the very end of the cantilever, the needle can be aimed precisely at the living cell of interest. The needles were modified with 3-(aminopropyl)triethoxysilane followed by fluorescein isothiocyanate (FITC). Human embryonic kidney (HEK293) cells expressing red-fluorescent protein DsRed2-NES were used in the experiments in order to distinguish the FITC-needle from cell body by LSM. The DsRed2-NES has a nuclear exporting signal (NES) at the C-terminus of DsRed2. The FITC nanoneedle and the cells were simultaneously excited at 488 nm by an Ar laser, and the emission signal was divided by a dichroic mirror into green (510-530 nm) and red (565-660 nm) signals through emission filters. The needle was lowered toward the HEK293 cell by controlling the piezo device while the force on the cantilever was monitored. Since the heights of HEK293 cells were 1020 µm, the needle was lowered 8 µm after the force drop was observed after indentation of the cell surface in order to reach the needle tip into the nucleus. Figure 4A shows a stack of all confocal images. Figure 4B shows vertical section images at the positions indicated by the white line in Figure 4A. The cross-section indicated clearly that the nanoneedle was inserted accurately in the nucleus. The piezo device for displacement and the force detection used here were accurate enough for needle insertion into the cell. When the force curve without a force drop after indentation was observed, the needle failed to insert into the cell body through the cell membrane (Figure 4C). Therefore, the results suggested that the force drop or force relaxation could be regarded as indication of successful insertion. In addition, the shape of the plasma membrane and nucleus remained the same as before insertion. If the needle just indented or dragged into the cell, the shape of red fluorescence of DsRed2-NES protein should be deformed. Therefore, the needle did not indent the membrane, but it penetrated through the membrane. Meanwhile, the pyramidal tip caused great deformation by indenting both the cell

Figure 4. (A) Stack of confocal slices of HEK293 cell expressing DsRed2-NES and FITC-labeled nanoneedle excited at 488 nm when the nanoneedle was inserted. (B) Cross-section images for green and red emission processed from confocal slices. (C) Images are from when the needles are failing to penetrate the cell surface. (D) Images when a pyramidal tip is indenting the cell surface. Scale bars in all images indicate 10 µm. Nano Lett., Vol. 5, No. 1, 2005

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did penetrate the cell and nucleus membranes vertically as we expected. Intrinsic advantages of this system are the capability of accurate displacement and sensing the force on the needle, as does a surgeon’s finger during surgery. By modifying the surface of a needle, it can load various molecules such as nucleic acids, proteins or chemicals through standard immobilization chemistries. The system should be one of a number of versatile apparatus for cell surgery and observation in the future.

Figure 5. A sequence of three-dimensional images reconstructed from confocal slices of the HEK293 cell and inserted nanoneedle with a schematic representation for each.

surface and the nucleus despite its high aspect ratio of 5:1 (Figure 4D). It has been reported that mechanical stress causes various changes in cell activity.16,20 Minimal deformation of cells is essential for cell manipulation because undesired mechanical responses may interfere with the result of manipulation. The results suggested that the deformation from using the nanoneedle was small because the distance of indentation before penetration was less than 1 µm. Figure 5 shows a sequence of schematic representations and reconstructed three-dimensional fluorescent images by processing data from all confocal slices with FLUOVIEW software (Olympus). The signal intensity of cell body is reduced for clarity purposes. To the best of our knowledge, the results demonstrated for the first time that solid material was inserted into a nucleus of such a small living cell with highly accurate positioning. The features of this technique are advantageous compared to microcapillary-based techniques. First, the displacement of the needle is accurate, therefore this technique is not particular about the size of cells. Second, the position can be estimated by monitoring the exerting force of the needle. The microcapillary-based techniques have a limit for thinner capillaries because of the limit of optical observation of the capillary tip as well as difficulty in the vertical observation of it. Third, the ultrathin needle does not cause fatal damage to several types of living cells (Han et al., manuscript in preparation). In contrast, capillary insertion caused 50% of the plated cells to die in our preliminary experiments. Low invasiveness of the inserting materials is an essential feature for the operation in living cells. In summary, we developed a system for cell operation by using AFM and fabricated probes shaped as ultrathin needles. The force analysis suggested that the nanoneedle indented the cell’s surface, and then it penetrated through the cell surface, accompanied by a drop in the exerting force on the needle. LSM observation confirmed directly that the needle

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Acknowledgment. We thank Industrial Technology Research Grant Program in ′03 from New Energy and Industrial Technology Development Organization (NEDO) of Japan. I.O. acknowledges Industrial Technology Fellowship Program from NEDO. Supporting Information Available: Construction of pDsRed2-NES plasmid. Description of the Hertz model. SEM image of nanoneedle fabricated from ATEC-CONT. Sequential reconstituted LSM images as a movie file. This information is available free of charge via the Internet at http://pubs.acs.org. References (1) Tsulaia, T. V.; Prokopishyn, N. L.; Yao, A.; Carsrud, N. D. V.; Carou, M. C.; Brown, D. B.; Davis, B. R.; Yannariello-Brown, J. J. Biomed. Sci. 2003, 10, 328-336. (2) King, R. Methods Mol. Biol. 2004, 245, 167-174. (3) Fotiadis, D.; Scheuring, S.; Muller, S. A.; Engel, A.; Muller, D. J. Micron. 2002, 33, 385-397. (4) Wojcikiewicz, E. P.; Zhang, X.; Moy, V. T. Biol. Proc. Online 2004, 6, 1-9. (5) Schaer-Zammaretti, P.; Ubbink, J. Ultramicroscopy 2003, 97, 199208. (6) Radmacher, M. IEEE Eng. Med. Biol. Mag. 1997, 16, 47-57. (7) Rotsch, C.; Braet, F.; Wisse, E.; Radmacher, M. Cell Biol. Int. 1997, 21, 685-696. (8) Haga, H.; Nagayama, M.; Kawabata, K.; Ito, E.; Ushiki, T.; Sambongi, T. J. Electron. Microsc. (Tokyo) 2000, 49, 473-481. (9) Firtel, M.; Henderson, G.; Sokolov, I. Ultramicroscopy 2004, in press. (10) Uehara, H.; Osada, T.; Ikai, A. Ultramicroscopy 2004, 100, 197201. (11) Obataya, I.; Nakamura, C.; Han, S.; Nakamura, N.; Miyake, J. Biosens. Bioelectron. 2004, in press. (12) Han, S.; Nakamura, C.; Obataya, I.; Nakamura, N.; Miyake, J. Biosens. Bioelectron. 2004, in press. (13) Hategan, A.; Law, R.; Kahn, S.; Discher, D. E. Biophys. J. 2003, 85, 2746-2759. (14) Le Grimellec, C.; Lesniewska, E.; Giocondi, M. C.; Finot, E.; Vie, V.; Goudonnet, J. P. Biophys. J. 1998, 75, 695-703. (15) Takeuchi, M.; Miyamoto, H.; Sako, Y.; Komizu, H.; Kusumi, A. Biophys. J. 1998, 74, 2171-2183. (16) Charras, G. T.; Horton, M. A. Biophys. J. 2002, 82, 2970-2981. (17) Weisenhorn, A. L.; Khorsandi, M.; Kasas, S.; Gotzos, V.; Butt, H.-J. Nanotechnology 1993, 4, 106-113. (18) Mathur, A. B.; Truskey, G. A.; Reichert, W. M. Crit. ReV. Biomed. Eng. 2000, 28, 197-202. (19) Rotsch, C.; Jacobson, K.; Radmacher, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 921-926. (20) Hochmuth, R. M. J. Biomech. 2000, 33, 15-22.

NL0485399

Nano Lett., Vol. 5, No. 1, 2005