Advertisement

Confocal Laser Scanning Microscopy

  • Akira Matsuno
  • Johbu Itoh
  • Tadashi Nagashima
  • R. Yoshiyuki Osamura
  • Keiichi Watanabe

Abstract

Immunoelectron microscopy and electron microscopic in situ hybridization are undoubtedly the best methods for following the dynamic changes of subcellular organelles; however, these techniques require specific tissue preparation and equipment. More recent developments include a more refined and sophisticated technique, confocal laser scanning microscopy (CLSM), which was originally described by Minsky in 1957 (1) and has since been applied to the field of medical biology. In early experiments, only fluorescent signals were detectable by CLSM (2–9); however, recent innovations have made possible the visualization of nonfluorescent signals such as horseradish peroxidase (HRP) and diaminobenzidine (DAB) signals by CLSM (10,11). Moreover, the combination of CLSM and the image analysis system (IAS) (12) has allowed us to visualize subcellular organelles three-dimensionally in routinely processed light microscopic specimens. We applied CLSM to specimens prepared for light microscopy (12) and demonstrated the intracellular identification of subcellular organelles and pituitary hormone mRNA, comparable to that of electron microscopy (13,14). We also applied CLSM to the study of tumor angiogenesis (15) and the microvessel environment of hormone-secreting cells (16). The visualization of subcellular organelles, mRNA and protein products, as well as three-dimensional images of microvessel environment of hormone-secreting cells is discussed in this chapter.

Keywords

ACTH Cell Subcellular Organelle Dichroic Beam Splitter Sodium Chloride Sodium Citrate Biotinylated Oligonucleotide Probe 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Minsky, M. (1957) Microscopy apparatus. U.S. Patent No. 3013467.Google Scholar
  2. 2.
    Arndt-Jovin, D. J., Robert-Nicoud, M., and Jovin, T. M. (1990) Probing DNA structure and function with a multi-wavelength fluorescence confocal laser microscope. J. Microsc. 157, 61–72.PubMedCrossRefGoogle Scholar
  3. 3.
    Arndt-Jovin, D. J., Robert-Nicoud, M., Kaufman, S. J., and Jovin, T. M. (1985) Fluorescence digital imaging microscopy in cell biology. Science 230, 247–256.PubMedCrossRefGoogle Scholar
  4. 4.
    Bauman, J. G., Bayer, J. A., and van Dekken, H. (1990) Fluorescent in-situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy. J. Microsc. 157, 73–81.PubMedCrossRefGoogle Scholar
  5. 5.
    Hozak, P., Novak, J. T., and Smetana, K. (1989) Three-dimensional reconstructions of nucleo-lus-organizing regions in PHA-stimulated human lymphocytes. Biol Cell 66, 225–233.PubMedGoogle Scholar
  6. 6.
    Michel, E. and Parsons, J. A. (1990) Histochemical and immunocytochemical localization of prolactin receptors on Nb2 lymphoma cells: applications of confocal microscopy. J. Histochem. Cytochem. 38, 965–973.PubMedCrossRefGoogle Scholar
  7. 7.
    Takamatsu, T. and Fujita, S. (1988) Microscopic tomography by laser scanning microscopy and its three-dimensional reconstruction. J. Microsc. 149, 167–174.PubMedCrossRefGoogle Scholar
  8. 8.
    Tao, W., Walter, R. J., and Berns, M. W. (1988) Laser-transected microtubules exhibit individuality of regrowth; however, most free new ends of the microtubules are stable. J. Cell Biol. 107, 1025–1035.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    Itoh, J., Utsunomiya, H., Komatsu, N., Takekoshi, S., Osamura, R. Y., and Watanabe, K. (1992) A new application of confocal laser scanning microscopy (C-LSM) to observe subcellular organelles utilizing non fluorescent probe (osmium black). Histochem. J. 24,550.Google Scholar
  11. 11.
    Robinson, J. M. and Batten, B. E. (1989) Detection of diaminobenzidine reactions using scanning laser confocal reflectance microscopy. J. Histochem. Cytochem. 37, 1761–1765.PubMedCrossRefGoogle Scholar
  12. 12.
    Itoh, J., Osamura, R. Y., and Watanabe, K. (1992) Subcellular visualization of light microscopic specimens by laser scanning microscopy and computer analysis: a new application of image analysis. J. Histochem. Cytochem. 40, 955–967.PubMedCrossRefGoogle Scholar
  13. 13.
    Itoh, J., Sanno, N., Matsuno, A., Itoh, Y., Watanabe, K., and Osamura, R. Y. (1997) Application of confocal laser scanning microscopy (CLSM) to visualize prolactin (PRL) and PRL mRNA in the normal and estrogen-treated rat pituitary glands using non-fluorescent probes. Microsc. Res. Tech. 39, 157–167.PubMedCrossRefGoogle Scholar
  14. 14.
    Matsuno, A., Itoh, J., Osamura, R. Y., Watanabe, K., and Nagashima, T. (1999) Electron microscopic and confocal laser scanning microscopic observation of subcellular organelles and pituitary hormone mRNA: application of ultrastructural in situ hybridization and immu-nohistochemistry to the pathophysiological studies of pituitary cells. Endocr. Pathol. 10, 199–211.PubMedCrossRefGoogle Scholar
  15. 15.
    Itoh, J., Yasumura, K., Takeshita, T., et al. (2000) Three-dimensional imaging of tumor angiogenesis. Anal. Quant. Cytol. Histol. 22, 85–90.PubMedGoogle Scholar
  16. 16.
    Itoh, J., Kawai, K., Serizawa, A., Yasumura, K., Ogawa, K., and Osamura, R. Y. (2000) A new approach to three-dimensional reconstructed imaging of hormone-secreting cells and their microvessel environments in rat pituitary glands by confocal laser scanning microscopy. J. Histochem. Cytochem. 48, 569–578.PubMedCrossRefGoogle Scholar
  17. 17.
    Matsuno, A., Ohsugi, Y., Utsunomiya, H., et al. (1994) Ultrastructural distribution of growth hormone, prolactin mRNA in normal rat pituitary cells: a comparison between preembedding and postembedding methods. Histochemistry 102, 265–270.PubMedCrossRefGoogle Scholar
  18. 18.
    Matsuno, A., Teramoto, A., Takekoshi, S., et al. (1994) Application of biotinylated oligonucleotide probes to the detection of pituitary hormone mRNA using Northern blot analysis, in situ hybridization at light and electron microscopic levels. Histochem. J. 26, 771–777.PubMedGoogle Scholar
  19. 19.
    Matsuno, A., Ohsugi, Y., Utsunomiya, H., et al. (1995) Changes in the ultrastructural distribution of prolactin and growth hormone mRNAs in pituitary cells of female rats after estrogen and bromocriptine treatment, studied using in situ hybridization with biotinylated oligonucleotide probes. Histochem. Cell Biol. 104, 37–45.PubMedCrossRefGoogle Scholar
  20. 20.
    Matsuno, A., Nagashima, T., Osamura, R. Y., and Watanabe, K. (1998) Application of ultrastructural in situ hybridization combined with immunohistochemistry to pathophysiological studies of pituitary cell: technical review. Acta Histochem. Cytochem. 31, 259–265.CrossRefGoogle Scholar
  21. 21.
    Matsuno, A., Nagashima, T., Ohsugi, Y., et al. (2000) Electron microscopic observation of intracellular expression of mRNA and its protein product: technical review on ultrastructural in situ hybridization and its combination with immunohistochemistry. Histol. Histopa-thol. 15, 261–268.Google Scholar
  22. 22.
    Matsuno, A., Nagashima, T., Osamura, R. Y., and Watanabe, K. (2000) Electron microscopic in situ hybridization and its combination with immunohistochemistry, in Molecular Histo-chemical Techniques (Springer Lab Manual) (Koji, T., ed.), Springer, New York, pp. 204–221.Google Scholar
  23. 23.
    Lakkakorpi, T. J., Yang, M., and Rajaniemi, H. J. (1994) Processing of the LH/CG receptor and bound hormone in rat luteal cells after hCG-induced down-regulation as studied by a double immunofluorescence technique in conjunction with confocal laser scanning microscopy. J. Histochem. Cytochem. 42, 727–732.PubMedCrossRefGoogle Scholar
  24. 24.
    Rummelt, V., Gardner, L. M., Folberg, R., et al. (1994) Three-dimensional relationships between tumor cells and microcirculation with double cyanine immunolabeling, laser scanning confocal microscopy, and computer-assisted reconstruction: an alternative to cast corrosion preparations. J. Histochem. Cytochem. 42, 681–686.PubMedCrossRefGoogle Scholar
  25. 25.
    Strong, L. H. (1964) The early embryonic pattern internal vascularization of the mammalian cerebral cortex. J. Comp. Neurol. 123, 121–138.PubMedCrossRefGoogle Scholar
  26. 26.
    Conradi, N. G., Engvall, J., and Wolff, J. R. (1980) Angioarchitectonics of rat cerebral cortex during pre- and postnatal development. Acta Neuropathol. (Berl.) 50, 131–138.CrossRefGoogle Scholar
  27. 27.
    Elias, K. A. and Weiner, R. I. (1984) Direct arterial vascularization of estrogen-induced prolactin-secreting anterior pituitary tumors. Proc. Natl. Acad. Sei. USA 81, 4549–4553.CrossRefGoogle Scholar
  28. 28.
    Kimura, K., Tojo, A., Nanba, S., Matsuoka, H., and Sugimoto, T. (1990) Morphometric analysis of arteriolar diameters in experimental nephropathies: application of microvascular casts. Virchows Arch. 417, 319–323.CrossRefGoogle Scholar
  29. 29.
    Yoshida, Y. and Ikuta, F. (1984) Three-dimensional architecture of cerebral microvessels with a scanning electron microscope: a cerebrovascular casting method for fetal and adult rats. J. Cereb. Blood Flow Metab. 4, 290–296.PubMedCrossRefGoogle Scholar
  30. 30.
    Nakamura, K. and Masuda, T. (1981) Scanning electron microscopy of corrosion cast of rat adrenal vasculatures with emphasis on medullary artery under ACTH administration. Tohoku J. Exp. Med. 134, 203–213.PubMedCrossRefGoogle Scholar
  31. 31.
    Bonner-Weir, S. and Orci, L. (1982) New perspectives on the microvasculature of the islets of Langerhans in the rat. Diabetes 31, 883–889.PubMedCrossRefGoogle Scholar
  32. 32.
    Marin-Padilla, M. (1985) Early vascularization of the embryonic cerebral cortex: Golgi and electron microscopic studies. J. Comp. Neurol. 241, 237–249.PubMedCrossRefGoogle Scholar
  33. 33.
    Novikoff, A. B. and Goldfischer, S. (1961) Nucleosidediphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc. Natl. Acad. Sei. USA 47, 802–810.CrossRefGoogle Scholar
  34. 34.
    Bell, M. A. and Scarrow, W. G. (1984) Staining for microvascular alkaline phosphatase in thick celloidin sections of nervous tissue: morphometric and pathological applications. Microvasc. Res. 27, 189–203.PubMedCrossRefGoogle Scholar
  35. 35.
    Holthöfer, H., Virtanen, I., Kariniemi, A. L., Hormia, M., Linder, E., and Miettinen, A. (1982) Ulex europaeus I lectin as a marker for vascular endothelium in human tissues. Lab. Invest. 47, 60–66.PubMedGoogle Scholar
  36. 36.
    Minamikawa, T., Miyaké, T., Takamatsu, T., and Fujita, S. (1987) A new method of lectin histochemistry for the study of brain angiogenesis. Lectin angiography. Histochemistry 87, 317–320.PubMedCrossRefGoogle Scholar
  37. 37.
    Hoyer, L. W., de Santos, R., and Hoyer, J. R. (1973) Antithemophilic factor antigen. Localization in endothelial cells by immunofluorescence microscopy. J. Clin. Invest. 52, 2737–2744.PubMedCrossRefGoogle Scholar
  38. 38.
    Jaffe, E. A., Hoyer, L. W., and Nachman, R. L. (1973) Synthesis of antihemophilic factor antigen by cultured human endothelial cells. J. Clin. Invest. 52, 2757–2764.PubMedCrossRefGoogle Scholar
  39. 39.
    Stemerman, M. B., Pitlick, F. A., and Dembitzer, H. M. (1976) Electron microscopic immunohistochemical identification of endothelial cells in the rabbit. Circ. Res. 38,146–156.PubMedCrossRefGoogle Scholar
  40. 40.
    Ghandour, M. S., Langley, O. K., and Varga, V. (1980) Immunohistological localization of gamma-glutamyl transpeptidase in cerebellum in light and electron microscope levels. Neurosci. Lett. 20, 125–129.PubMedCrossRefGoogle Scholar
  41. 41.
    Auerbach, R., Alby, L., Grieves, J., et al. (1982) Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells. Proc. Natl. Acad. Sei. USA 79, 7891–7895.CrossRefGoogle Scholar
  42. 42.
    Barsky, S. H., Baker, A., Siegal, G. P., Togo, S., and Liotta, L. A. (1983) Use of anti-basement membrane antibodies to distinguish blood vessel capillaries from lymphatic capillaries. Am. J. Surg. Pathol. 7, 667–677.PubMedCrossRefGoogle Scholar
  43. 43.
    Strottmann, J. M., Robinson, J. B., Jr., and Stellwagen, E. (1983) Advantages of preelectro-phoretic conjugation of polypeptides with fluorescent dyes. Anal. Biochem. 132, 334–337.PubMedCrossRefGoogle Scholar
  44. 44.
    D’Amato, R., Wesołowski, E., and Smith, L. E. (1993) Microscopic visualization of the retina by angiography with high-molecular-weight fluorescein-labeled dextrans in the mouse. Microvasc. Res. 46, 135–142.PubMedCrossRefGoogle Scholar
  45. 45.
    Nakane, P. K. (1975) Recent progress in the peroxidase-labeled antibody method. Ann. NY Acad. Sei. 254, 203–211.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2001

Authors and Affiliations

  • Akira Matsuno
  • Johbu Itoh
  • Tadashi Nagashima
  • R. Yoshiyuki Osamura
  • Keiichi Watanabe

There are no affiliations available

Personalised recommendations