Chromosome Research

, 16:351 | Cite as

Three-dimensional structured illumination microscopy and its application to chromosome structure

  • Peter M. CarltonEmail author


This review discusses the exploration of chromosome structure with a recently developed high-resolution microscopy technique, three-dimensional structured illumination microscopy (3dSIM). 3dSIM surpasses the diffraction limit of conventional widefield optical microscopy, increasing the level of detail in images by a factor of 2, while retaining the sample preparation methods, ease of use and flexibility of conventional microscopy. Special attention will be given to the ways in which imaging beyond the diffraction limit can shed light on the structural organization of meiotic chromosomes.

Key words

chromosome imaging meiosis microscopy structured illumination synaptonemal complex 

Supplementary material (764 kb)
Supplementary Movie 1 is the raw data collected by 3dSIM. The stripe pattern created by the diffraction grating is easily distinguished. At each Z section, five phases of the stripe pattern are collected; the grating moves by 1/5th of a period at each shift, so the stripes appear to move smoothly throughout the Z stack. Three concatenated stacks are shown, each with the grating in a different rotational position. (MOV 763 kb) (4.8 mb)
Supplementary Movie 2 shows the cells after averaging the 5 different phases of the stripe pattern in one rotational position, removing the stripes (and associated high-frequency information), and resulting in an image that is equivalent to conventional widefield illumination. (MOV 4.81 mb) (1.8 mb)
Supplementary Movie 3 shows the results of deconvolving the image data of Movie 2. While out-of-focus blur is removed and features are more visible, resolution is not noticeably increased. (MOV 1.80 mb) (7.1 mb)
Supplementary Movie 4 shows the results of reconstructing the raw data into a 3dSIM image stack. For all movies, the Z step distance is 0.125 μm; also see scale bar in Figure 9. (MOV 7.12 mb)


  1. Abbe E (1873) Beiträge zur Theorie: des Mikroskops und der mikroskopischen Wahrnehmung. Archiv für mikroskopische Anatomie 9: 413–468.Google Scholar
  2. Bak AL, Zeuthen J, Crick FH (1977) Higher-order structure of human mitotic chromosomes. Proc Natl Acad Sci U S A 74: 1595–1599.PubMedCrossRefGoogle Scholar
  3. Betzig E, Patterson GH, Sougrat R et al. (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313: 1642–1645.PubMedCrossRefGoogle Scholar
  4. Blat Y, Protacio RU, Hunter N, Kleckner N (2002) Physical and functional interactions among basic chromosome organizational features govern early steps of meiotic chiasma formation. Cell 111: 791–802.PubMedCrossRefGoogle Scholar
  5. Bolcun-Filas E, Costa Y, Speed R et al. (2007) SYCE2 is required for synaptonemal complex assembly, double strand break repair, and homologous recombination. J Cell Biol 176: 741–747.PubMedCrossRefGoogle Scholar
  6. Carlton PM, Cande WZ (2002) Telomeres act autonomously in maize to organize the meiotic bouquet from a semipolarized chromosome orientation. J Cell Biol 157: 231–242.PubMedCrossRefGoogle Scholar
  7. Colaiacovo MP (2006) The many facets of SC function during C. elegans meiosis. Chromosoma 115: 195–211.PubMedCrossRefGoogle Scholar
  8. Costa Y, Cooke HJ (2007) Dissecting the mammalian synaptonemal complex using targeted mutations. Chromosome Res 15: 579–589.PubMedCrossRefGoogle Scholar
  9. Dawe RK, Sedat JW, Agard DA, Cande WZ (1994) Meiotic chromosome pairing in maize is associated with a novel chromatin organization. Cell 76: 901–912.PubMedCrossRefGoogle Scholar
  10. Dernburg AF, McDonald K, Moulder G, Barstead R, Dresser M, Villeneuve AM (1998) Meiotic recombination in C. elegans initiates by a conserved mechanism and is dispensable for homologous chromosome synapsis. Cell 94: 387–398.PubMedCrossRefGoogle Scholar
  11. Dong H, Roeder GS (2000) Organization of the yeast Zip1 protein within the central region of the synaptonemal complex. J Cell Biol 148: 417–426.PubMedCrossRefGoogle Scholar
  12. Egner A, Geisler C, von Middendorff C et al. (2007) Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys J 93: 3285–3290.PubMedCrossRefGoogle Scholar
  13. Foss E, Lande R, Stahl FW, Steinberg CM (1993) Chiasma interference as a function of genetic distance. Genetics 133: 681–691.PubMedGoogle Scholar
  14. Golubovskaya IN, Hamant O, Timofejeva L et al. (2006) Alleles of afd1 dissect REC8 functions during meiotic prophase I. J Cell Sci 119: 3306–3315.PubMedCrossRefGoogle Scholar
  15. Gugel H, Bewersdorf J, Jakobs S, Engelhardt J, Storz R, Hell SW (2004) Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy. Biophys J 87: 4146–4152.PubMedCrossRefGoogle Scholar
  16. Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198: 82–87.PubMedCrossRefGoogle Scholar
  17. Gustafsson MG (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A 102: 13081–13086.PubMedCrossRefGoogle Scholar
  18. Gustafsson MGL, Shao L, Carlton PM et al. (2007) Three-dimensional resolution doubling in widefield fluorescence microscopy by structured illumination. Biophys J doi: 10.1529/biophysj.107.120352 [Epub ahead of print].
  19. Hamant O, Golubovskaya I, Meeley R et al. (2005) A REC8-dependent plant Shugoshin is required for maintenance of centromeric cohesion during meiosis and has no mitotic functions. Curr Biol 15: 948–954.PubMedCrossRefGoogle Scholar
  20. Heintzmann R, Ficz G (2006) Breaking the resolution limit in light microscopy. Brief Funct Genomic Proteomic 5: 289–301.PubMedCrossRefGoogle Scholar
  21. Heintzmann R, Jovin TM, Cremer C (2002) Saturated patterned excitation microscopy–a concept for optical resolution improvement. J Opt Soc Am A Opt Image Sci Vis 19: 1599–1609.PubMedCrossRefGoogle Scholar
  22. Hell SW (2003) Toward fluorescence nanoscopy. Nat Biotechnol 21: 1347–1355.PubMedCrossRefGoogle Scholar
  23. Hell SW (2007) Far-field optical nanoscopy. Science 316: 1153–1158.PubMedCrossRefGoogle Scholar
  24. Hess ST, Girirajan TPK, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91: 4258–4272.PubMedCrossRefGoogle Scholar
  25. Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319: 810–813.Google Scholar
  26. Jaramillo-Lambert A, Ellefson M, Villeneuve AM, Engebrecht J (2007) Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line. Dev Biol 308: 206–221.PubMedCrossRefGoogle Scholar
  27. Jones GH (1984) The control of chiasma distribution. Symp Soc Exp Biol 38: 293–320.PubMedGoogle Scholar
  28. Jones GH, Armstrong SJ, Caryl AP, Franklin FCH (2003) Meiotic chromosome synapsis and recombination in Arabidopsis thaliana; an integration of cytological and molecular approaches. Chromosome Res 11: 205–215.PubMedCrossRefGoogle Scholar
  29. Klar TA, Jakobs S, Dyba M, Egner A, Hell SW (2000) Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci U S A 97: 8206–8210.PubMedCrossRefGoogle Scholar
  30. Kleckner N, Zickler D, Jones GH et al. (2004)A mechanical basis for chromosome function. Proc Natl Acad Sci U S A 101: 12592–12597.PubMedCrossRefGoogle Scholar
  31. König P, Braunfeld M, Sedat J, Agard D (2007) The three-dimensional structure of in vitro reconstituted Xenopus laevis chromosomes by EM tomography. Chromosoma 116: 349–372.PubMedCrossRefGoogle Scholar
  32. Loidl J (1994) Cytological aspects of meiotic recombination. Experientia 50: 285–294.PubMedCrossRefGoogle Scholar
  33. Marko JF, Siggia ED (1997) Polymer models of meiotic and mitotic chromosomes. Mol Biol Cell 8: 2217–2231.PubMedGoogle Scholar
  34. Mezard C, Vignard J, Drouaud J, Mercier R (2007) The road to crossovers: plants have their say. Trends Genet 23: 91–99.PubMedCrossRefGoogle Scholar
  35. Moens PB, Pearlman RE, Heng HH, Traut W (1998) Chromosome cores and chromatin at meiotic prophase. Curr Top Dev Biol 37: 241–262.PubMedGoogle Scholar
  36. Morgan TH, Sturtevant AH, Muller HJ, Bridges CB (1915) The Mechanism of Mendelian Heredity. New York: Henry Holt and Company.Google Scholar
  37. Moses MJ (1969) Structure and function of the synaptonemal complex. Genetics 61(Supplement): 41–51.PubMedGoogle Scholar
  38. Nagorni M, Hell SW (1998) 4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution. J Struct Biol 123: 236–247.PubMedCrossRefGoogle Scholar
  39. Parisi S, McKay MJ, Molnar M et al. (1999) Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fission yeast to humans. Mol Cell Biol 19: 3515–3528.PubMedGoogle Scholar
  40. Robinson PJJ, Fairall L, Huynh VAT, Rhodes D (2006) EM measurements define the dimensions of the “30-nm” chromatin fiber: Evidence for a compact, interdigitated structure. Proc Natl Acad Sci 103: 6506–6511.PubMedCrossRefGoogle Scholar
  41. Russ JC (2002) The Image Processing Handbook, 4th edn. Boca Raton: CRC Press.Google Scholar
  42. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3: 793–795.PubMedCrossRefGoogle Scholar
  43. Schalch T, Duda S, Sargent DF, Richmond TJ (2005) X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature 436: 138–141.PubMedCrossRefGoogle Scholar
  44. Schrader M, Bahlmann K, Giese G, Hell SW (1998) 4Pi-confocal imaging in fixed biological specimens. Biophys J 75: 1659–1668.PubMedCrossRefGoogle Scholar
  45. Schweizer D, Loidl J, Hamilton B (1987) Heterochromatin and the phenomenon of chromosome banding. Results Probl Cell Differ 14: 235–254.PubMedGoogle Scholar
  46. Strukov YG, Wang Y, Belmont AS (2003) Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes. J Cell Biol 162: 23–35.PubMedCrossRefGoogle Scholar
  47. Sutton WS (1903) The chromosomes in heredity. Biol Bull 4: 231–251.CrossRefGoogle Scholar
  48. Sym M, Engebrecht JA, Roeder GS (1993) ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis. Cell 72: 365–378.PubMedCrossRefGoogle Scholar
  49. Tremethick DJ (2007) Higher-order structures of chromatin: the elusive 30 nm fiber. Cell 128: 651–654.PubMedCrossRefGoogle Scholar
  50. Tung KS, Roeder GS (1998) Meiotic chromosome morphology and behavior in zip1 mutants of Saccharomyces cerevisiae. Genetics 149: 817–832.PubMedGoogle Scholar
  51. Willig KI, Rizzoli SO, Westphal V, Jahn R, Hell SW (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440: 935–939.PubMedCrossRefGoogle Scholar
  52. Wilson EB (1925) The Cell in Development and Inheritance, 3rd edn. London: Macmillan.Google Scholar
  53. Zickler D, Kleckner N (1999) Meiotic chromosomes: integrating structure and function. Annu Rev Genet 33: 603–754.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2008

Authors and Affiliations

  1. 1.Department of Biochemistry and BiophysicsUniversity of California, San FranciscoSan FranciscoUSA

Personalised recommendations