Polarization Microscopy

Shinya Inoué1

1 Marine Biological Laboratory, Woods Hole, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.9
DOI:  10.1002/0471143030.cb0409s13
Online Posting Date:  February, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit provides an introduction to polarization microscopy, the optics involved, and practical considerations for observing submicroscopic structures. The unit also includes specific examples for visualization of microtubules in the mitotic spindle, chromatin within maturing spermatids, and the biocrystalline skeletal spicules in larval echinoderms.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Color in Polarized Light
  • Optical Rotation
  • Video Microscopy
  • Applications of Polarization Microscopy
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Allen, R.D., Travis, J.L., Allen, N.S., and Yilmaz, H. 1981. Video‐enhanced contrast polarization (AVEC‐POL) microscopy: A new method applied to the detection of birefringence in the motile reticulopodial network of Allogromia laticollaris. Cell Motil. 1:275‐289.
   Ambronn, H. and Frey, A. 1926. Das Polarisationsmikroskop, seine Anwedung in der Kolloidforschung in der Färbarei. Akademische Verlag, Leipzig, Germany.
   Belar, K. 1928. Die Technik der Deskriptiven Cytologie. In Methodik der Wissenschaftlichen Biologie, Vol.1 (T. Péterfi, ed.), pp. 638‐735. Julius Springer, Berlin.
   Belar, K. 1929. Beiträge zur Kausalanalyse der Mitose. II. Untersuchungen an den Spermatocyten von Chorthippus (Stenobothrus) lineatus Panz. Arch. Entwicklungsmech. Organismen 118:359‐484, plus 8 plates.
   Bennett, H.S. 1950. The microscopical investigation of biological materials with polarized light. In Handbook of Microscopical Technique (C.E. McClung, ed.) pp. 591‐677. Harper & Row (Hoeber), New York.
   Breton, J., Michel‐Villaz, M., and Paillotin, G. 1973. Orientation and structural proteins in the photosynthetic membrane of spinach chloroplasts: A linear dichroism study. Biochim. Biophys. Acta. 314:42‐56.
   Burgos, M.H., Goda, M., and Inoué, S. 2000. Fertilization‐induced changes in the fine structure of stratified Arbacia eggs. II. Observations with electron microscopy. Biol. Bull. 199:213‐214.
   Cachon, J., Sato, H., Cachon, M., and Sato, Y. 1989. Analysis by polarizing microscopy of chromosomal structure among dinoflagellates and its phylogenetic involvement. Biol. Cell 65:51‐60.
   Costello, D.P., Davidson, M.E., Eggers, A., Fox, M.H., and Henley, C. 1957. Methods for Obtaining and Handling Marine Eggs and Embryos. Lancaster Press, Lancaster, Pa.
   Deitz, R. 1969. Bau und Funktion des Spindelapparats. Naturwissenschaften 56:237‐248.
   Forer, A. 1965. Local reduction of spindle fiber birefringence in living Nephrotoma suturalis (Loew) spermatocytes induced by ultraviolet microbeam irradiation. J. Cell Biol. 25:95‐117.
   Frey‐Wyssling, A. 1953. Fine structure of protoplasmic derivatives. In Submicroscopic Morphology of Protoplasm, Chapter 3, pp. 279‐370. Elsevier/North‐Holland, Amsterdam.
   Green, P.B. 1963. On mechanisms of elongation. In Cytodifferential and Macromolecular Synthesis (M. Locke, ed.) pp. 203‐234. Academic Press, New York.
   Hárosi, F.I. 1981. Microspectrophotometry and optical phenomena: Birefringence, dichroism, and anomalous dispersion. In Springer Series in Optical Sciences, Vol. 23: Vertebrate Photoreceptor Optics (J.M. Enoch and F.L. Tobey, Jr., eds.) pp. 337‐399. Springer‐Verlag, Berlin.
   Hárosi, F.I. and MacNichol, Jr., E.F. 1974. Dichroic microspectrophotometer: A computer‐assisted, rapid, wavelength‐scanning photometer for measuring linear dichroism in single cells. J. Opt. Soc. Am. 64:903‐913.
   Hartshorne, N.H. and Stuart, A. 1960. Crystals and the Polarising Microscope: A Handbook for Chemists and Others, 3rd Ed. Arnold, London.
   Harvey, E.B. 1940. A comparison of the development of nucleate and non‐nucleate eggs of Arbacia punctulata. Biol. Bull. 79:166‐187.
   Hecht, E. 1998. Polarization. In Optics, 3rd ed., Chapter 8, pp. 319‐377. Addison‐Wesley, Reading, Mass.
   Holzwarth, G., Webb, S.C., Kubinski, D.J., and Allen, N.S. 1997. Improving DIC microscopy with polarization modulation. J. Microscopy 188:249‐254.
   Holzwarth, G.M., Hill, D.B., and McLaughlin, E.B. 2000. Polarization‐modulated differential‐interference contrast microscopy with a variable retarder. Applied Optics 39:6288‐6294.
   Hsu, B.‐D. and Lee, Y.‐Y. 1987. Orientation of pigments and pigment‐protein complexes in the diatom Cylindrotheca fusiformis. A linear dichroism study. Biochim. Biophys. Acta 893:572‐577.
   Huxley, A.F. 1960. British patent specification 856,621. Improvements in or relating to polarizing microscopes (applied July 20, 1956).
   Inoué, S. 1952a. The effect of colchicine on the microscopic and submicroscopic structure of the mitotic spindle. Exp. Cell Res. Suppl. 2:305‐318.
   Inoué, S. 1952b. Effect of temperature on the birefringence of the mitotic spindle. Biol. Bull. 103:316.
   Inoué, S. 1964. Organization and function of the mitotic spindle. In Primitive Motile Systems in Cell Biology (R.D. Allen and N. Kamiya, eds.) pp. 579‐598. Academic Press, New York.
   Inoué, S. 1981a. Cell division and the mitotic spindle. J. Cell Biol. 91:131s‐147s.
   Inoué, S. 1981b. Video image processing greatly enhances contrast, quality, and speed in polarization based microscopy. J. Cell Biol. 89:346‐356.
   Inoué, S. 1986. Video Microscopy. Plenum, New York.
   Inoué, S. 1993. Porter and the fine architecture of dividing cells. In The Biological Century: Friday Evening Talks at the Marine Biological Laboratory (R.B. Barlow, Jr., J.E. Dowling, and G. Weissmann, eds.) pp. 100‐115. Harvard University Press, Boston.
   Inoué, S. and Dan, K. 1951. Birefringence of the dividing cell. J. Morphol. 89:423‐456.
   Inoué, S. and Goda, M. 2001. Fluorescence polarization of GFP crystals. Biol. Bull. 201:231‐233.
   Inoué, S. and Hyde, W.L. 1957. Studies on depolarization of light at microscope lens surfaces. II. The simultaneous realization of high resolution and high sensitivity with the polarizing microscope. J. Biophys. and Biochem. Cytol. 3:831‐838.
   Inoué, S. and Kubota, H. 1958. Diffraction anomaly in polarizing microscopes. Nature 182:1725‐1726.
   Inoué, S. and Bajer, A. 1961. Birefringence in endosperm mitosis. Chromosoma (Berl.) 12:48‐63.
   Inoué, S. and Sato, H. 1966. Deoxyribonucleic acid arrangement in living sperm. In Molecular Architecture in Cell Physiology (T. Haysahi and A.G. Szent‐Györgyi, eds.) pp. 209‐248. Prentice Hall, New York.
   Inoué, S. and Sato, H. 1967. Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement. J. Gen. Physiol. 50:259‐292.
   Inoué, S. and Ritter, H. 1978. Mitosis in Barbulanympha. II. Dynamics of a two‐stage anaphase, nuclear morphogenesis, and cytokinesis. J. Cell Biol. 77:655‐684.
   Inoué, S. and Salmon, E.D. 1995. Force generation by microtubule assembly/disassembly in mitosis and related movements. Mol. Biol. Cell 6:1619‐1640.
   Inoué, S. and Spring, K.R. 1997. Environmental considerations. In Video Microscopy: The Fundamentals, 2nd ed., Section 13.5, pp. 595‐603. Plenum, New York.
   Inoué, S. and Oldenbourg, R. 1998. Video essay: Microtubule dynamics in mitotic spindle displayed by polarized light microscopy. Mol. Biol. Cell 9:1603‐1607.
   Inoué, S., Fuseler, J., Salmon, E.D., and Ellis, G.W. 1975. Functional organization of mitotic microtubules: Physical chemistry of the in vivo equilibrium system. Biophys. J. 15:725‐744.
   Inoué, S., Knudson, R.A., Suzuki, K., Okada, N., Takahashi, H., Iida, M., and Yamanaka, K. 1998. Centrifuge polarizing microscope. Microsc. Microanal. 4 (Suppl. 2):36‐37.
   Inoué, S., Knudson, R.A., Goda, M., Suzuki, K., Nagano, C., Okada, N., Takahashi, H., Ichie, K., Iida, M., and Yamanaka, K. 2001a. Centrifuge polarizing microscope. I. Rationale, design, and instrument performance. J. Microscopy 201:341‐356.
   Inoué, S., Goda, M., and Knudson, R.A. 2001b. Centrifuge polarizing microscope. II. Sample biological applications. J. Microscopy 201:357‐367.
   Jenkins, F.A. and White, H.E. 1957. Fundamentals of Optics, 3rd ed., pp. 20‐29, 407‐606. McGraw‐Hill, New York.
   Katoh, K., Hammar, K., Smith, P.J.S., and Oldenbourg, R. 1999. Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones. Mol. Biol. Cell 10:197‐210.
   Kinosita, K. 1999. Real time imaging of rotating molecular machines. In A Half Century of Advances in Microscopy (R.B. Silver, ed.). FASEB J. 13:s201‐s208.
   Langford, G.M. and Inoué, S. 1979. Motility of the microtubular axostyle in Pyrsonympha. J. Cell Biol. 80:521‐538.
   Lutz, D.A. and Inoué, S. 1986. Techniques for observing living gametes and embryos. In Methods in Cell Biology, Vol. 27 (T. Schroeder, ed.) pp. 89‐110. Academic Press, New York.
   McIntosh, J.R., Cande, W.Z., and Snyder, J.A. 1975. Structure and physiology of the mammalian mitotic spindle. In Molecules and Cell Movement (S. Inoué and R.E. Stephens, eds.) pp. 31‐76. Raven Press, New York.
   Mitchison, T. and Kirschner, M. 1984. Dynamic instability of microtubule growth. Nature 312:237‐242.
   Murphy, D.B. 2001. Polarization microscopy. In Fundamentals of Light Microscopy and Electronic Imaging, pp. 135‐151. Wiley‐Liss, New York.
   Nicklas, R.B. 1971. Mitosis In Advances in Cell Biology, Vol. 2 (D.M. Prescott, L. Goldstein, and E. McConkey, eds.) pp. 225‐289. Appleton‐Century‐Crofts, New York.
   Nicklas, R.B. 1979. Chromosome movement and spindle birefringence in locally heated cells: Interaction versus local control. Chromosoma (Berl.) 74:1‐37.
   Nicklas, R.B. and Staehly, C.A. 1967. Chromosome micromanipulation. I. The mechanics of chromosome attachment to the spindle. Chromosoma 21:1‐16.
   Nicklas, R.B., Brinkley, B.R., Pepper, D.A., Kubai, D.F., and Rickards, G.K. 1979. Electron microscopy of spermatocytes previously studied in life: Methods and some observations on micromanipulated chromosomes. J. Cell Sci. 35:87‐104.
   Nicklas, R.B., Ward, S.C., and Gorbsky, G.J. 1995. Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint. J. Cell Biol. 130:929‐939.
   Okazaki, K. and Inoué, S. 1976. Crystal property of the larval sea urchin spicule. Development, Growth and Differentiation 18:413‐434.
   Okazaki, K., McDonald, K., and Inoué, S. 1980. Sea urchin larval spicule observed with the scanning electron microscope. In The Mechanisms of Biomineralization in Animals and Plants (M. Omori and N. Watabe, eds.) pp. 159‐168. Tokai University Press, Tokyo.
   Oldenbourg, R. 1991. Analysis of edge birefringence. Biophys. J. 60:629‐641.
   Oldenbourg, R. 1996. A new view on polarization microscopy. Nature 381:811‐812.
   Oldenbourg, R. 1999. Polarized light microscopy of spindles. In Methods in Cell Biology, Vol. 61 (C.L. Rieder, ed.) pp. 175‐208. Academic Press, New York.
   Oldenbourg, R. and Mei, G. 1995. New polarized light microscope with precision universal compensator. J. Microsc. 180:140‐147.
   Perutz, M.F. and Mitchison, J.M. 1950. State of hemoglobin in sickle‐cell anemia. Nature 166:677.
   Rieder, C. and Hard, R. 1990. Newt lung epithelial cells: Cultivation, use, and advantages for biomedical research. Int. Rev. Cytol. 122:153‐220.
   Ritter, H., Inoué, S., and Kubai, D. 1978. Mitosis in Barbulanympha. I. Spindle structure, formation and kinetochore engagement. J. Cell Biol. 77:638‐654.
   Salmon, E.D. and Segal, R.R. 1980. Calcium labile mitotic spindles isolated from sea urchin eggs (Lytechinus variegatus). J. Cell Biol. 89:355‐365.
   Sato, H., Ellis, G.W., and Inoué, S. 1975. Microtubular origin of mitotic spindle form birefringence: Demonstration of the applicability of Wiener's equation. J. Cell Biol. 67:501‐517.
   Schmidt, W.J. 1924. Die Bausteine des Tierkörpers in Polarisiertem Lichte. Cohen, Bonn.
   Schmidt, W.J. 1932. Der submikroskopische Bau des Chromatins. III. Mitteilung: Über die Doppelbrechung der Isosporenkerne von Thalassicolla. Arch. Protkde 78:613‐627.
   Schmidt, W.J. 1935. Doppelbrechung, Dichroismus und Feinbau des Auβengliedes der Sehzellen vom Frosch. Z. Zellforschg. 22:485‐522.
   Schmidt, W.J. 1937. Die Doppelbrechung von Karyoplasma, Zytoplasma und Metaplasma. In Protoplasma‐Monographien, Vol. 11. Borntraeger, Berlin.
   Schmidt, W.J. 1941. Einiges über optische Anisotropie und Feinbau von Chromatin und Chromosomen. Chromosoma 2:86‐110.
  Schroeder, T.E., ed. 1986. Echinoderm Gametes and Embryos. Methods in Cell Biology, Vol. 27. Academic Press, New York.
  Shotten, D.M. (ed.) 1993. Electronic Light Microscopy. Wiley‐Liss, New York.
   Swann, M.M. and Mitchison, J.M. 1950. Refinements in polarized light microscopy. J. Exp. Biol. 27:226‐237.
   Szent‐Györgyi, A., Cohen, C. and Philpott, D.E. 1960. Light meromyosin fraction I: A helical molecule from myosin. J. Mol. Biol. 2:133‐142.
   Walker, R.A., O'Brien, E.T., Pryer, N.K., Soboeiro, M.F., Voter, W.A., Erickson, H.P., and Salmon, E.D. 1988. Dynamic instability of individual microtubules analyzed by video light microscopy: Rate constants and transition frequencies. J. Cell Biol. 107:1437‐1448.
   Wilkins, M.H.F. 1951. Ultraviolet dichroism and molecular structure in living cells. II. Electron microscopy of nuclear membrane. Publ. Staz. Zool. Napoli 23 (Suppl.):104‐114.
   Zalokar, M. 1960. Cytochemistry of centrifuged hyphae of Neurospora. Exp. Cell Res. 19:114‐132.
   Zhang, D. and Nicklas, R.B. 1996. “Anaphase” and cytokinesis in the absence of chromosomes. Nature 382:466‐468.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library