Abstract

Femtosecond laser pulses can be used to selectively disrupt and dissect intracellular organelles. We report on disruption of mitochondria in living HeLa cells using a femtosecond laser oscillator with a repetition rate of 76 MHz. We studied the laser parameters used for disruption. The long-term viability of the cells after disruption of a single mitochondrion was confirmed by the observation of cell division, indicating that intracellular disruption of organelles using a femtosecond laser oscillator can be performed without compromising the long-term cell viability.

© 2005 Optical Society of America

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Biophys. J.

E. L. Botvinick, V. Venugopalan, J. V. Shah, L. H. Liaw, and M. W. Berns, “Controlled ablation of microtubules using a picosecond laser,” Biophys. J. 87, 4203-4212 (2004).
[CrossRef] [PubMed]

Cell. Motil. Cytoskeleton

A. Khodjakov, R. W. Cole, and C. L. Rieder, “A synergy of technologies: Combining laser microsurgery with green fluorescent protein tagging,” Cell. Motil. Cytoskeleton 38, 311-317 (1997).
[CrossRef] [PubMed]

Chem. Phys. Lett.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350, 71-77 (2001).
[CrossRef]

Curr. Biol.

C. L. Rieder and R. Cole, “Microtubule disassembly delays the G2-M transition in vertebrates,” Curr. Biol. 10, 1067-1070 (2000).
[CrossRef] [PubMed]

Exp. Cell Res.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. Halbhuber, “Femtosecond near-infrared laser pulse elicit generation of reactive oxygen species in mammalian cells leading to apotosis-like death,” Exp. Cell Res. 263, 88-97 (2001).
[CrossRef] [PubMed]

Histochem. Cell Biol.

K. König, “Laser tweezers and multiphoton microscopes in life sciences,” Histochem. Cell Biol. 114, 79-92 (2000).
[PubMed]

Int. Rev. Cytol.

M. W. Berns, W. H. Write, and R. W. Steubing, “Laser microbeam as a tool in cell biology,” Int. Rev. Cytol. 129, 1-44 (1991).
[CrossRef] [PubMed]

J. Biol. Chem.

D. Arnoult, A. Grodet, Y. -J. Lee, J. Estaquier, and C. Blackstone, “Release of OPA1 during apoptosis participates in the rapid and complete release of cytochrome c and subsequent mitochondrial fragmentation,” J. Biol. Chem. 280, 35742-35750 (2005).
[CrossRef] [PubMed]

J. Biomed. Opt.

L. Sacconi, I. M. Tolić-Nørrelykke, R. Antolini, and F. S. Pavone, “Combined intracellular three-dimensional imaging and selective nanosurgery by a nonlinear microscope,” J. Biomed. Opt. 10, 014002 (2005).
[CrossRef]

J. Biosci. Bioeng.

T. Higashi, E. Nagamori, T. Sone, S. Matsunaga, and K. Fukui, “A novel transfection method for mammalian cells using calcium alginate microbeads,” J. Biosci. Bioeng. 97, 191-195 (2004).

J. Cell. Biol.

C. L. Rieder and R. W. Cole, “Entry into mitosis in vertebrate somatic cells is guarded by a chromosome damage checkpoint that reverses the cell cycle when triggered during early but not late prophase,” J. Cell. Biol. 142, 1013-1022 (1998).
[CrossRef] [PubMed]

J. Exp. Biol.

T. J. Collins and M. D. Bootman, “Mitochondria are morphologically heterogeneous within cells,” J. Exp. Biol. 206, 1993-2000 (2003).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B.

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophore with data from 690 to 1050nm,” J. Opt. Soc. Am. B. 13, 481-491 (1996).
[CrossRef]

J. Raman Spectrosc.

V. V. Yakovlev, “Advanced instrumentation for non-linear Raman microscopy,” J. Raman Spectrosc. 4, 957-964 (2003).
[CrossRef]

Mech. Chem. Biosyst.

N. Shen, D. Datta, C. B. Schaffer, P. LeDuc, D. E. Ingber, and E. Mazur, “Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor,” Mech. Chem. Biosyst. 2, 17-25 (2005).

Nat. Biotechnol.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17, 763-767 (1999).
[CrossRef] [PubMed]

Nat. Rev. Mol. Cell Biol.

A. Musacchio and K. G. Hardwick, “The spindle checkpoint: structural insights into dynamic signaling,” Nat. Rev. Mol. Cell Biol. 3, 731-741 (2002).
[CrossRef] [PubMed]

Nature

U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418, 290-291 (2002).
[CrossRef] [PubMed]

M. F. Yanik, H. Cinar, H. N. Cinar, A. D. Chisholm, Y. Jin, and A. Ben-Yakar, “Functional regeneration after laser axotomy,” Nature 432, 822 (2004).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Phys. Rev. Lett.

M. Lenzner, J. Krüger, S. Sartania, Z. Cheng, Ch. Spielmann, G. Mourou, W. Kautek, and F. Krausz, “Femtosecond optical breakdown in dielectrics,” Phys. Rev. Lett. 80, 4076-4079 (1998).
[CrossRef]

A. Ashkin, “Acceleration and trapping of particles by radiation pressure,” Phys. Rev. Lett. 24, 156-159 (1970).
[CrossRef]

V. Venugopalan, A. Guerra III, K. Nahen, and A. Vogel, “Role of laser-induced plasma formation in pulsed cellular microsurgery and micromaniplation,” Phys. Rev. Lett. 88, 078103 (2002).
[CrossRef] [PubMed]

Poc. Natl. Acad. Sci.

A. P. Joglekar, H. H. Liu, E. Meyhöfer, G. Mourou, and A. J. Hunt, ”Optics at critical intensity: Applications to nanomorphing,” Poc. Natl. Acad. Sci. USA 101, 5856-5861 (2004).
[CrossRef]

Proc. Natl. Acad. Sci.

W. Supatto, D. Débarre, B. Moulia, E. Brouzés, J. -L. Martin, E. Farge, and E. Beaurepaire, “In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses,” Proc. Natl. Acad. Sci. USA 102, 1047-1052 (2005).
[CrossRef] [PubMed]

Proc. SPIE

A. Vogel, J. Noack, G. Huettmann, and G. Paltauf, “Femtosecond-laser-produced low-density plasmas in transparent biological media: a tool for the creation of chemical, thermal, and thermomechanical effects below the optical breakdown threshold,” Proc. SPIE 4633, 23-37 (2002).
[CrossRef]

Rev. Sci. Instrum.

J. Colombelli, S. W. Grill, and E. H. K. Stelzer, “Ultraviolet diffraction limited nanosurgery of live biological tissues,” Rev. Sci. Instrum. 75, 472-478 (2004).
[CrossRef]

Scanning

H. Oehring, I. Riedmann, P. Fisher, K. J. Halbhuber, and K. König, “Ultrastructure and reproduction behaviour of single CHO-K1 cells exposed to near infrared femtosecond laser pulses,” Scanning 22, 263- 270 (2000).
[CrossRef] [PubMed]

Science

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73-76 (1990).
[CrossRef] [PubMed]

R. L. Amy and R. Storb, “Selective mitochondrial damage by a ruby laser microbeam - an electron microscopic study,” Science 150, 756-757 (1965).
[CrossRef] [PubMed]

Traffic

J. Colombelli, E. G. Reynaud, J. Rietdorf, R. Pepperkok, E. H. K. Stelzer, “In vivo selective cytoskeleton dynamics quantification in interphase cells induced by pulsed ultraviolet laser nanosurgery,” Traffic 6, 1093-1102 (2005).
[CrossRef] [PubMed]

Other

B. Alberts, D. Bray, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Essential Cell Biology (Taylor and Francis, New York, 1997).

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Figures (6)

Fig. 1.
Fig. 1.

Schematic diagram of the experimental setup. FI, Faraday isolator; P, SF10 prism; M mirror; L, lens; ND, neutral density filter; DM, dichroic mirror; OB, objective lens; GM, pair of galvanometer mirrors; PMT, photomultiplier tube

Fig. 2.
Fig. 2.

Stacked three-dimensional confocal images (a) before and (b) after femtosecond laser irradiation with 0.39 nJ/pulse (exposure time: 32 ms). Stacked images were obtained by translating the objective lens by 1 μm in the depth direction in steps of 250 nm. Yellow fluorescence shows mitochondria of HeLa cells visualized by EYFP. A target mitochondrion is indicated by a red arrow. Scale bar: 10 μm. Confocal cross-sectional images at different depths (c) before and (d) after irradiation of the femtosecond laser pulses. The femtosecond laser pulses were focused at a depth of z = 0. Scale bar: 3 μm.

Fig. 3.
Fig. 3.

Mitochondrial fragmentation in HeLa cells induced by femtosecond laser pulses. Stacked three-dimensional confocal images obtained (a) before and (b) after irradiation with 0.53 nJ/pulse (exposure time: 32 ms). The red arrow indicates the irradiation point. Scale bar: 10 μm. (c) Time-lapse confocal images after irradiation. Scale bar: 5 μm.

Fig. 4.
Fig. 4.

Dependence of disruption of mitochondria with EYFP and MitoTracker Red on femtosecond laser pulse energy and irradiation time. (a–c) The rate of disruption and fragmentation of the EYFP-mitochondria at irradiation times of (a) 32 ms, (b) 16 ms, and (c) 8 ms for various laser energies. (d) The rate of disruption and fragmentation of the mitochondria stained with MitoTracker Red at an irradiation time of 32 ms for various laser energies. The number of measurements was 10 for each pulse energy and irradiation time.

Fig. 5.
Fig. 5.

Cell division after femtosecond laser disruption of a mitochondrion labeled with EYFP. Confocal images (a) before and (b) after femtosecond laser irradiation with 0.39 nJ/pulse (exposure time: 32 ms). The red arrow indicates the irradiation point. (c–f) Time-lapse confocal images. The process of cell division finished successfully 12 h after laser irradiation (c–d). The migration of daughter cells was observed from 12 to 16.5 h after laser irradiation (f). Scale bar: 10 μm.

Fig. 6.
Fig. 6.

Mitotic events of cell division after disruption of mitochondria in the histone EGFP-H1 expressed cell. The disruption of a single mitochondrion by femtosecond laser irradiation had no influence on cell division or cell activity. The cell nuclei and mitotic chromosomes in HeLa cells were visualized using histone EGFP-H1. Mitochondria were stained with MitoTracker Red. Confocal fluorescence image and transmission image (a) before and (b) after femtosecond laser irradiation with 0.39 nJ/pulse (exposure time: 32 ms). The yellow arrow indicates the irradiation point. (c)–(f) Time-lapse confocal images and transmission images. The mitotic events of cell division in the irradiated cells proceeded normally. Scale bar: 20 μm.

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