Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor.

Analysis of cell regulation requires methods for perturbing molecular processes within living cells with spatial discrimination on the nanometer-scale. We present a technique for ablating molecular structures in living cells using low-repetition rate, low-energy femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we ablate cellular material inside the cell through nonlinear processes. We selectively removed sub-micrometer regions of the cytoskeleton and individual mitochondria without altering neighboring structures or compromising cell viability. This nanoscissor technique enables non-invasive manipulation of the structural machinery of living cells with several-hundred-nanometer resolution. Using this approach, we unequivocally demonstrate that mitochondria are structurally independent functional units, and do not form a continuous network as suggested by some past studies.

[1]  U. Parlitz,et al.  Energy balance of optical breakdown in water at nanosecond to femtosecond time scales , 1999 .

[2]  Guenther Paltauf,et al.  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 , 2002, SPIE LASE.

[3]  Sandu Popescu,et al.  OSA trends in optics and photonics , 2003 .

[4]  U. Parlitz,et al.  Shock wave emission and cavitation bubble generation by picosecond and nanosecond optical breakdown in water , 1996 .

[5]  Kazuyoshi Itoh,et al.  Femtosecond laser disruption of subcellular organelles in a living cell. , 2004, Optics express.

[6]  R. Siebert,et al.  An Easy and Reliable Procedure of Microdissection Technique for the Analysis of Chromosomal Breakpoints and Marker Chromosomes , 2004, Chromosome Research.

[7]  K König,et al.  Nanodissection of human chromosomes with near-infrared femtosecond laser pulses. , 2001, Optics letters.

[8]  Perry,et al.  Nanosecond-to-femtosecond laser-induced breakdown in dielectrics. , 1996, Physical review. B, Condensed matter.

[9]  Vasan Venugopalan,et al.  Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation. , 2002, Physical review letters.

[10]  A. Periasamy Methods in Cellular Imaging , 2001, Methods in Physiology.

[11]  A Khodjakov,et al.  A synergy of technologies: combining laser microsurgery with green fluorescent protein tagging. , 1997, Cell motility and the cytoskeleton.

[12]  James G. Fujimoto,et al.  Time-resolved measurements of picosecond optical breakdown , 1989 .

[13]  A Vogel,et al.  Cavitation bubble dynamics and acoustic transient generation in ocular surgery with pulsed neodymium: YAG lasers. , 1986, Ophthalmology.

[14]  J. Fujimoto,et al.  Time-resolved studies of Nd:YAG laser-induced breakdown. Plasma formation, acoustic wave generation, and cavitation. , 1985, Investigative ophthalmology & visual science.

[15]  M W Berns,et al.  Controlled ablation of microtubules using a picosecond laser. , 2004, Biophysical journal.

[16]  Lawrence M. Lifshitz,et al.  Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. , 1998, Science.

[17]  E. Mazur,et al.  Dynamics of femtosecond laser-induced breakdown in water from femtoseconds to microseconds. , 2002, Optics express.

[18]  Gerd Weber,et al.  The light microscope on its way from an analytical to a preparative tool , 1992 .

[19]  M. Berns,et al.  Laser microirradiation of kinetochores in mitotic PtK2 cells , 1980, Cell Biophysics.

[20]  A. Quantock,et al.  Ultrastructure of picosecond laser intrastromal photodisruption. , 1996, Journal of refractive surgery.

[21]  F. Ichas,et al.  Electrical coupling and plasticity of the mitochondrial network. , 2000, Cell calcium.

[22]  K J Halbhuber,et al.  Intracellular nanosurgery with near infrared femtosecond laser pulses. , 1999, Cellular and molecular biology.

[23]  E. Mazur,et al.  Bulk heating of transparent materials using a high-repetition-rate femtosecond laser , 2003 .

[24]  G. Kastis,et al.  Time‐resolved observations of shock waves and cavitation bubbles generated by femtosecond laser pulses in corneal tissue and water , 1996, Lasers in surgery and medicine.

[25]  Eric Mazur,et al.  Photodisruption in biological tissues and single cells using femtosecond laser pulses , 2001, CLEO 2001.

[26]  M W Berns,et al.  Laser microsurgery in cell and developmental biology. , 1981, Science.

[27]  Sverre Myhra,et al.  Imaging and nano‐dissection of tobacco mosaic virus by atomic force microscopy , 1995 .

[28]  A. Niendorf,et al.  A new method for histological microdissection utilizing an ultrasonically oscillating needle: demonstrated by differential mRNA expression in human lung carcinoma tissue. , 2001, The American journal of pathology.

[29]  D. Ingber,et al.  Mechanical behavior in living cells consistent with the tensegrity model , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[30]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[31]  N. Bloembergen,et al.  Laser-induced electric breakdown in solids , 1974 .

[32]  E Mazur,et al.  Minimally disruptive laser-induced breakdown in water , 1997, CLEO '97., Summaries of Papers Presented at the Conference on Lasers and Electro-Optics.

[33]  G. Mourou,et al.  Applications of femtosecond lasers in corneal surgery , 2000 .

[34]  D. Jay,et al.  Chromophore-assisted laser inactivation (CALI) to elucidate cellular mechanisms of cancer. , 1999, Biochimica et biophysica acta.

[35]  Gary D. Noojin,et al.  Laser-induced breakdown in the eye at pulse durations from 80 ns to 100 fs , 1998, Photonics West - Biomedical Optics.

[36]  A. Vogel,et al.  Influence of pulse duration on mechanical effects after laser-induced breakdown in water , 1998 .

[37]  G. Mourou,et al.  Corneal refractive surgery with femtosecond lasers , 1999 .

[38]  G. Mourou,et al.  Femtosecond Optical Breakdown in Dielectrics , 1998 .

[39]  Y. Zhang,et al.  Proceedings of the 20th Annual International Conference of the IEEE Engineering in Medicine and Biology Society , 2000 .

[40]  N. Nakatsuji,et al.  In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene , 2000, FEBS letters.

[41]  E. Mazur,et al.  Tissue ablation with 100-fs and 200-ps laser pulses , 1998, Proceedings of the 20th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. Vol.20 Biomedical Engineering Towards the Year 2000 and Beyond (Cat. No.98CH36286).

[42]  C. Sacchi Laser-induced electric breakdown in water , 1991 .

[43]  G. Steding,et al.  Experimental study on the significance of abnormal cardiac looping for the development of cardiovascular anomalies in neural crest-ablated chick embryos , 1996, Anatomy and Embryology.

[44]  Peter Lipp,et al.  Mitochondria are morphologically and functionally heterogeneous within cells , 2002, The EMBO journal.

[45]  R R Krueger,et al.  The picosecond laser for nonmechanical laser in situ keratomileusis. , 1998, Journal of refractive surgery.

[46]  G. Krockmalnic,et al.  Imaging cytoskeleton--mitochondrial membrane attachments by embedment-free electron microscopy of saponin-extracted cells. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[47]  Charles G. Durfee,et al.  Ultrafast laser and amplifier sources , 1997 .

[48]  Gerard Mourou,et al.  Laser‐induced breakdown by impact ionization in SiO2 with pulse widths from 7 ns to 150 fs , 1994 .

[49]  M. Ashby,et al.  Perinuclear, perigranular and sub‐plasmalemmal mitochondria have distinct functions in the regulation of cellular calcium transport , 2001, The EMBO journal.

[50]  C. Siegerist,et al.  Reproducible Imaging and Dissection of Plasmid DNA Under Liquid with the Atomic Force Microscope , 1992, Science.

[51]  G. Mourou,et al.  Photodisruption in the human cornea as a function of laser pulse width. , 1997, Journal of refractive surgery.

[52]  Charles G. Durfee,et al.  High power ultrafast lasers , 1998 .

[53]  Alfred Vogel,et al.  Numerical simulation of optical breakdown for cellular surgery at nanosecond to femtosecond time scales , 2001, European Conference on Biomedical Optics.