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Cell imaging and manipulation by nonlinear optical microscopy

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Abstract

Advances in the technologies for labeling and imaging biological samples drive a constant progress in our capability of studying structures and their dynamics within cells and tissues. In the last decade, the development of numerous nonlinear optical microscopies has led to a new prospective both in basic research and in the potential development of very powerful noninvasive diagnostic tools. These techniques offer large advantages over conventional linear microscopy with regard to penetration depth, spatial resolution, three-dimensional optical sectioning, and lower photobleaching. Additionally, some of these techniques offer the opportunity for optically probing biological functions directly in living cells, as highlighted, for example, by the application of second-harmonic generation to the optical measurement of electrical potential and activity in excitable cells. In parallel with imaging techniques, nonlinear microscopy has been developed into a new area for the selective disruption and manipulation of intracellular structures, providing an extremely useful tool of investigation in cell biology. In this review we present some basic features of nonlinear microscopy with regard both to imaging and manipulation, and show some examples to illustrate the advantages offered by these novel methodologies.

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References

  1. Shimomura, O., Johnson, F. H., and Saiga, Y. (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan. Aequorea. J. Cell Comp. Physiol. 59, 223–239.

    Article  PubMed  CAS  Google Scholar 

  2. Shimomura, O. (1998) The discovery of green fluorescent protein. In Green Fluorescent Protein: Properties, Applications, and Protocols (Chalfie, M., and Kain, S., eds.), Wiley-Liss, New York, pp. 1–14.

    Google Scholar 

  3. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.

    Article  PubMed  CAS  Google Scholar 

  4. Rizzo, M. A. and Piston, D. W. (2005) Fluorescent protein tracking and detection. In Live Cell Imaging. A Laboratory Manual (Goldman, R. D. and Spector, D. L., eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 3–24.

    Google Scholar 

  5. Isenberg, G., Bielser, W., Meier-Ruge, W., and Remy, E. (1976) Cell surgery by laser micro-dissection: a preparative method. J. Microsc. 107, 19–24.

    PubMed  CAS  Google Scholar 

  6. Bustamante, C., Smith, S. B., Liphardt, J., and Smith, D. (2000) Single-molecule studies of DNA mechanics. Curr. Opin. Struct. Biol. 10, 279–285.

    Article  PubMed  CAS  Google Scholar 

  7. Capitanio, M., Vanzi, F., Broggio, C., et al. (2004) Exploring molecular motor and switches at the single-molecule level. Microsc. Res. Tech. 65, 194–204.

    Article  PubMed  CAS  Google Scholar 

  8. Ashkin, A., Dziedzic, J. M., and Yamane, T. (1987) Optical trapping and manipulation of single cells using infrared laser beams. Nature 330, 769–771.

    Article  PubMed  CAS  Google Scholar 

  9. Ashkin, A. and Dziedzic, J. M. (1987) Optical trapping and manipulation of viruses and bacteria. Science, 235, 1517–1520.

    Article  PubMed  CAS  Google Scholar 

  10. Zipfel, W. R., Williams, R. M., and Webb, W. W. (2003) Nonlinear magic: multiphoton microscopy in the bioscience. Nat. Biotech. 21, 1369–1377.

    Article  CAS  Google Scholar 

  11. Zipfel, W. R., Williams, R. M., Christie, R., Nikitin, A. Y., Hyman, B. T., and Webb, W. W. (2003) Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. USA 100, 7075–7780.

    Article  PubMed  CAS  Google Scholar 

  12. Miller, M. J., Wei, S. H., Parker, I., and Cahalan, M. D. (2002) Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873.

    Article  PubMed  CAS  Google Scholar 

  13. Maiti, S., Shear, J. B., Williams, R. M., Zipfel, W. R., and Webb, W. W. (1997) Measuring serotonin distribution in live cells with three-photon excitation. Science 275, 530–532.

    Article  PubMed  CAS  Google Scholar 

  14. Boyde, A. (1994) Bibliography on confocal microscopy and its applications. Scanning 16, 33–56.

    Google Scholar 

  15. Lichtman, J. W. (1994) Confocal microscopy. Scientific Am. 271, 40–45.

    Article  Google Scholar 

  16. Pawley, J. B., The Handbook of Biological Confocal Microscopy. Plenum, New York, 1990.

    Google Scholar 

  17. White, J. G., Amos, W. B., and Fordham, M. (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J. Cell. Biol. 105, 41–48.

    Article  PubMed  CAS  Google Scholar 

  18. Wilson, T. and Sheppard, C., Theory and Practice of Scanning Optical Microscopy. Academic Press, London 1994.

    Google Scholar 

  19. Centonze, V. E. and White, J. G. (1998) Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J. 75, 2015–2024.

    PubMed  CAS  Google Scholar 

  20. Soeller, A. and Cannell, M. B. (1999) Two-photon microscopy: imaging in scattering samples and three-dimensionally resolved flash photolysis. Microsc. Res. Tech. 47, 182–195.

    Article  PubMed  CAS  Google Scholar 

  21. Periasamy, A., Skoglund, P., Noakes, C., and Keller R. (1999) An evaluation of two-photon versus confocal and digital deconvolution fluorescence microscopy imaging in Xenopus morphogenesis. Microsc. Res. Tech. 47, 172–181.

    Article  PubMed  CAS  Google Scholar 

  22. Squirrel, J. M., Wokosin, D. L., White, J. G., and Bavister, B. D. (1999) Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nat. Biotechnol. 17, 763–767.

    Article  CAS  Google Scholar 

  23. Konig, K., So, P. T., Mantulin, W. W., Tromberg, B. J., and Gratton, E. (1996) Two-photon excited lifetime imaging of autofluorescence in cell during UVA and NIR photostress. J. Microsc. 183, 197–204.

    PubMed  CAS  Google Scholar 

  24. Koester, H. J., Baur, D., Uhl, R., and Hell, S. W. (1999) Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys. J. 77, 2226–2236.

    PubMed  CAS  Google Scholar 

  25. Hopt, A. and Neher, E. (2001) Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 2029–2036.

    PubMed  CAS  Google Scholar 

  26. Campagnola, P. J. and Loew, L. (2003) Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat. Biotechnol. 21, 1356–1360.

    Article  PubMed  CAS  Google Scholar 

  27. Dombeck, D. A., Kasischke, K. A., Vishwasrao, H. D., Ingelsson, M., Hyman, B. T., and Webb, W. W. (2003) Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy. Proc. Natl. Acad. Sci. USA 100, 7081–7086.

    Article  PubMed  CAS  Google Scholar 

  28. Dombeck, D. A., Blanchard-Desce, M., and Webb, W. W. (2004) Optical recording of action potentials with second-harmonic generation microscopy. J. Neurosci. 24, 999–1003.

    Article  PubMed  CAS  Google Scholar 

  29. Goppert-Mayer, M. (1931) Uber Elementarekte mit zwei Quantensprunger. Ann. Phys. 9, 273.

    Article  CAS  Google Scholar 

  30. Kaiser, W. and Garret, C. G. B. (1961) Two-photon excitation in CaF2∶Eu2+. Phys. Rev. Lett. 7, 229–231.

    Article  CAS  Google Scholar 

  31. Denk, W., Strickler, H. J., and Webb, W. W. (1990) Two-photon laser scanning fluorescence microscopy. Science 248, 73–76.

    Article  PubMed  CAS  Google Scholar 

  32. Williams, R. M., Piston, D. W., and Webb, W. W. (1994) Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy microphotochemistry. FASEB J. 8, 804–813.

    PubMed  CAS  Google Scholar 

  33. Yuste, R. and Denk, W. (1995) Dendritic spine as basic function units of neuronal integration. Nature 375, 682–684.

    Article  PubMed  CAS  Google Scholar 

  34. Svodoba, K., Tank, D. W., and Denk, W. (1996) Direct measurement of coupling between dendritic spines and shafts. Science 272, 716–719.

    Article  Google Scholar 

  35. Denk, W., Piston, D. W., and Webb, W. W.. The Handbook of Confocal Microscopy, Plenum, New York, 1995.

    Google Scholar 

  36. Ashkin, A. (1970) Acceleration and trapping of particles by radiation pressure. Phys. Rev. Lett. 24, 156–159.

    Article  CAS  Google Scholar 

  37. Ashkin, A. (1971) Optical levitation by radiation pressure. Appl. Phys. Lett. 19, 283–285.

    Article  Google Scholar 

  38. Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., and Chu, S. (1986) Observation of a single-beam gradient force optical trap for dielectric particles. Opt. Lett. 11, 288–290.

    CAS  PubMed  Google Scholar 

  39. McCully, E. K. and Robinow, C. F. (1971) Mitosis in the fission yeast Schizosaccharomyces pombe: a comparative study with light and electron microscopy. J. Cell Sci. 9, 475–507.

    PubMed  CAS  Google Scholar 

  40. Svoboda, K., and Block, M. S. (1994) Biological applications of optical forces. Ann. Rev. Biophys. Biomol. Struct. 23, 247–85.

    Article  CAS  Google Scholar 

  41. Isenberg, G., Bielser, W., Meier-Ruge, W., and Remy, E. (1976) Cell surgery by laser micro-dissection: a preparative method. J. Microsc. 107, 19–24.

    PubMed  CAS  Google Scholar 

  42. Aist, J. R. and Berns, M. W. (1981) Mechanics of chromosome separation during mitosis in Fusarium (Fungi imperfecti): new evidence from ultrastructural and laser microbeam experiments. J. Cell Biol. 91, 446–458.

    Article  PubMed  CAS  Google Scholar 

  43. Leslie, R. J. and Pickett-Heaps, J. D. (1983) Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation. J. Cell. Biol. 96, 548–561.

    Article  PubMed  CAS  Google Scholar 

  44. Grill, S. W., Gonczy, P., Stelzer, E. H., and Hyman, A. A. (2001) Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo. Nature 409, 630–633.

    Article  PubMed  CAS  Google Scholar 

  45. Grill, S. W., Howard, J., Schaffer, E., Stelzer, E. H., and Hyman, A. A. (2003) The distribution of active force generators controls mitotic spindle position. Science 301, 518–521.

    Article  PubMed  CAS  Google Scholar 

  46. Khodjakov, A., Cole, R. W., and Rieder, C. L. (1997) A synergy of technologies: combining laser microsurgery with green fluorescent protein tagging. Cell Motil. Cytoskeleton 38, 311–317.

    Article  PubMed  CAS  Google Scholar 

  47. Konig, K., Riemann, I., and Fritzsche, W. (2001) Nanodissection of human chromosomes with near-infrared femtosecond laser pulses. Opt. Lett. 26, 819–821.

    Article  PubMed  CAS  Google Scholar 

  48. Tirlapur, U. K. and Konig, K. (2002) Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability. Plant J. 31, 365–374.

    Article  PubMed  Google Scholar 

  49. Tolic-Nørrelykke, I. M., Sacconi, L., Thon, G., and Pavone, F. S. (2004) Positioning and elongation of the fission yeast spindle by microtubule-based pushing. Curr. Biol. 14, 1181–1186.

    Article  PubMed  CAS  Google Scholar 

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

    Article  Google Scholar 

  51. Hagan, I. M. (1998) The fission yeast microtubule cytoskeleton. J. Cell Sci. 111, 1603–1612.

    PubMed  CAS  Google Scholar 

  52. Vogel, A. and Venugopalan, V. (2003) Mechanisms of pulsed laser ablation of biological tissues. Chem. Rev. 103, 577–644.

    Article  PubMed  CAS  Google Scholar 

  53. Berns, M. W., Aist, J. R., Wright, W. H., and Liang, H. (1992) Optical trapping in animal and fungal cells using a tunable, near-infrared titanium-sapphire laser. Exp. Cell Res. 198, 375–378.

    Article  PubMed  CAS  Google Scholar 

  54. Ashkin, A. and Dziedzic, J. M. (1989) Internal cell manipulation using infrared laser traps. Proc. Natl. Acad. Sci. USA 86, 7914–7918.

    Article  PubMed  CAS  Google Scholar 

  55. Berns, M. W., Wright, W. H., Tromberg, B. J., Profeta, G. A., Andrews, J. J., and Walter, R. J. (1989) Use of a laser-induced optical force trap to study chromosome movement on the mitotic spindle. Proc. Natl. Acad. Sci. USA 86, 4539–4543.

    Article  PubMed  CAS  Google Scholar 

  56. Ketelaar, T., Faivre-Moskalenko, C., Esseling, J. J., et al. (2002) Positioning of nuclei in Arabidopsis root hairs: an actin-regulated process of tip growth. Plant Cell 14, 2941–2955.

    Article  PubMed  CAS  Google Scholar 

  57. Tran, P. T., Marsh, L., Doye, V., Inoue, S., and Chang, F. (2001) A mechanism for nuclear positioning in fission yeast based on microtubule pushing. J. Cell Biol. 153, 397–411.

    Article  PubMed  CAS  Google Scholar 

  58. Daga, R. R. and Chang, F. (2005) Dynamic positioning of the fission yeast cell division plane. Proc. Natl. Acad. Sci. USA 102, 8228–8232.

    Article  PubMed  CAS  Google Scholar 

  59. Neuman, I. C., Chadd, E. H., Liou, G. F., Bergman, K., and Block, S. M. (1999) Characterization of photodamage to Escherichia coli in optical traps. Biophys. J. 77, 2856–2863.

    PubMed  CAS  Google Scholar 

  60. Sacconi, L., Tolic-Nørrelykke, I. M., Stringari, C., Antolini, R., and Pavone, F. S. (2005) Optical micromanipulations inside yeast cells. Appl. Opt. 44, 2001–2007.

    Article  PubMed  Google Scholar 

  61. Visser, T. D. and Wiersma, S. H. (1992) Diffraction of converging electromagnetic waves. J. Opt. Soc. Am. A 9, 2034–2047.

    Google Scholar 

  62. Tran, P. T., Marsh, L., Doye, V., Inoue, S., and Chang, F. (2001) A mechanism for nuclear positioning in fission yeast based on microtubule pushing. J. Cell Biol. 153, 397–411.

    Article  PubMed  CAS  Google Scholar 

  63. Tolic-Norrelykke, I. M., Sacconi, L., Stringari, C., and Pavone, F. S. (2005) Nuclear and division plane positioning revealed by optical micromanipulation. Curr. Biol. 15, 1212–1218.

    Article  PubMed  CAS  Google Scholar 

  64. Bloembergen, N. Nonlinear Optic. World Scientific, 1965.

  65. Moreaux, L., Sandre, O., and Mertz, J. (2000) Membrane imaging by second-harmonic generation microscopy. J. Opt. Soc. Am. B 17, 1685–1694.

    Article  CAS  Google Scholar 

  66. Shen, Y. R. (1989) Surface properties probed by second-harmonic and sum-frequency generation. Nature 337, 519–525.

    Article  CAS  Google Scholar 

  67. Hellwarth, R. and Christensen, P. (1974) Nonlinear optical microscopic examination of structure in polycrystalline ZnSe. Opt. Com. 12, 318–322.

    Article  CAS  Google Scholar 

  68. Gennaway, J. and Sheppard, C. J. R. (1978) Second-harmonic imaging in the scanning optical microscope. Opt. Quant. Elect. 10, 435–439.

    Article  Google Scholar 

  69. Ben-Oren, I., Peleg, G., Lewis, A., Minke, B., and Loew, L. (1996) Infrared nonlinear optical measurements of membrane potential in photoreceptor cells. Biophys. J. 71, 1616–1620.

    PubMed  CAS  Google Scholar 

  70. Peleg, G., Lewis, A., Linial, M., and Loew, L. M. (1999) Non-linear optical measurement of membrane potential around single molecules at selected cellular sites. Proc. Natl. Acad. Sci. USA 96, 6700–6704.

    Article  PubMed  CAS  Google Scholar 

  71. Lewis, A., Khatchattouriants, A., Trenin, M. et al. (1999) Second harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. Elegans. Chem. Phys. 245, 133–144.

    Article  CAS  Google Scholar 

  72. Cohen, I. B. and Salzberg, B. M. (1978) Optical measurement of membrane potential. Rev. Physiol. Biochem. Pharmacol. 83, 35–88.

    PubMed  CAS  Google Scholar 

  73. Bouevitch, O., Lewis, A., Pinevsky, I., Wuskell, J. P., and Loew, L. M. (1993) Probing membrane potential with nonlinear optics. Biophys. J. 65, 672–679.

    PubMed  CAS  Google Scholar 

  74. Campagnola, P. J., Wei, M., Lewis, A., and Loew, L. M. (1999) High resolution nonlinear optical imaging of live cells by second harmonic generation. Biophys. J. 77, 3341–3349.

    PubMed  CAS  Google Scholar 

  75. Moreaux, L., Pons, T., Dambrin, V., Blanchard-Desce, M., and Mertz, J. (2003) Electro-optic response of second-harmonic generation membrane potential sensors. Opt. Lett. 28, 625–627.

    Article  PubMed  CAS  Google Scholar 

  76. Pons, T., Moreaux, L., Mongin, O., Blanchard-Desce, M., and Mertz, J. (2003) Mechanisms of membrane potential sensing with second-harmonic generation microscopy. J. Biomed. Opt. 8, 428–431.

    Article  PubMed  CAS  Google Scholar 

  77. Millard, A. C., Jin, L., Lewis, A., and Loew, L. M. (2003) Direct measurement of the voltage sensitivity of second harmonic generation from a membrane dye in patchclamped cells. Opt. Lett. 28, 1221–1223.

    Article  PubMed  CAS  Google Scholar 

  78. Millard, A. C., Jin, L., Wei, M. D., Wuskell, J. P., Lewis, A., and Loew, L. M. (2004) Sensitivity of second harmonic generation from styryl dyes to transmembrane potential. Biophys. J. 86, 1169–1176.

    Article  PubMed  CAS  Google Scholar 

  79. Sacconi, L., D'Amico, M., Vanzi, F. et al. (2005) Second harmonic generation sensitivity to transmembrane potential in normal and tumor cells. J. Biomed. Opt. 10, 024014.

    Article  PubMed  CAS  Google Scholar 

  80. Rajfur, Z., Roy, P., Otey, C., Romer, L., and Jacobson, K. (2002) Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins. Nat. Cell Biol. 2, 286–294.

    Article  CAS  Google Scholar 

  81. Sakurai, T., Wong, E., Drescher, U., Tanaka, H., and Jay, D. G. (2002), Ephrin-A5 restricts topographically specific arborization in the chick retinotectal projection in vivo. Proc. Natl. Acad. Sci. USA 99, 10,795–10,800.

    Article  CAS  Google Scholar 

  82. Horstkotte, A., Schroder, T., Niewohner, J., Thiel, E., Jay, D., and Henning, S. (2004) Towards understanding the mechanism of CALI—evidence for the primary photochemical steps. Photochem. Photobiol. Epub ahead of print.

  83. Sacconi, L., Froner, E., Antolini, R., Taghizadeh, M. R., Choudhury, A., and Pavone, F. S. (2003) Multiphoton multifocal microscopy exploiting a diffractive optical element. Opt. Lett. 28, 1918–1920.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. European Laboratory for Non-Linear Spectroscopy, via Nello Carrara 1, I-50019, Sesto Fiorentino (Florence), Italy

    L. Sacconi, I. M. Tolic-Nørrelykke, F. Vanzi & F. S. Pavone

  2. Department of Physics, University of Trento, via Sommarive 14, I-38050, Povo (Trento), Italy

    L. Sacconi & R. Antolini

  3. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    I. M. Tolic-Nørrelykke

  4. Department of Experimental Pathology and Oncology, University of Florence, viale Morgagni 50, I-50100, Florence, Italy

    M. D'Amico & M. Olivotto

  5. Department of Biology and Genetics, University of Florence, via Romana 14, I-50100, Florence, Italy

    F. Vanzi

  6. Department of Physics, University of Florence, via Sansone 1, 50019, Sesto Fiorentino (Florence), Italy

    F. S. Pavone

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  1. L. Sacconi
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Sacconi, L., Tolic-Nørrelykke, I.M., D'Amico, M. et al. Cell imaging and manipulation by nonlinear optical microscopy. Cell Biochem Biophys 45, 289–302 (2006). https://doi.org/10.1385/CBB:45:3:289

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