Cryofixation of Dynamic Processes in Cells and Organelles

  • Gerd Knoll
  • Arie J. Verkleij
  • Helmut Plattner


Electron microscope analysis of cells depends on fixation; the goal is to trap the native state of dynamic structures and distribution patterns of components (Plattner and Bachmann 1982). This intention of fixation is, of course, not restricted to applications in electron microscopy. Attempts to literally “freeze” the living structure by cryofixation can be traced back to the last century, and were extended to electron microscopy in its very early days (compiled by Rash 1983). The main problem with simply freezing a biological specimen in order to fix it, is destruction caused by ice crystals. This freezing damage hampers further analysis, in particular at the high resolution level obtained with the electron microscope. For this reason, chemical fixation procedures have routinely been used, although their limitations and risks have been well recognized (Fernandez-Moran 1964; Rebhun 1965). On the other hand, the drawbacks of chemical treatments have stimulated the development of rapid freezing techniques which reduce the formation of ice crystals (Van Harreveld and Crowell 1964).


Membrane Fusion Chemical Fixation Freezing Time Lipidic Particle Rapid Freezing 
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  1. Allred DR, Staehelin LA (1986) Spatial organization of the cytochome b6-f complex within chloroplast thylakoid membranes. Biochim Biophys Acta 849:94–103.PubMedCrossRefGoogle Scholar
  2. Bald WB (1983) Optimizing the cooling block for the quick freeze method. J Microsc (Oxford) 131:11–23.CrossRefGoogle Scholar
  3. Bald WB (1986) On crystal size and cooling rate. J Microsc (Oxford) 143:89–102.CrossRefGoogle Scholar
  4. *.
    Bayer ME (1979) The fusion sites between outer membrane and cytoplasmic membrane of bacteria: their role in membrane assembly and virus infection. In: Inouye M (ed) Bacterial outer membranes. Biogenesis and functions. John Wiley & Sons, New York, pp 167–202.Google Scholar
  5. Bearer EL, Düzgünes N, Friend DS, Papahadjopoulos D (1982) Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion. Biochim Biophys Acta 693:93–98.PubMedCrossRefGoogle Scholar
  6. Brdiczka D, Riesinger I, Adams V, Bremm G (1986) Functions in metabolic regulation of contact sites between mitochondrial boundary membranes. In: 4th EBEC short reports (1986-200). Univ Press, Cambridge, pp 200.Google Scholar
  7. Bridgman PC, Reese TS (1984) The structure of cytoplasm in directly frozen cultured cells. I. Filamentous meshworks and the cytoplasmic ground substance. J Cell Biol 99:1655–1668.PubMedCrossRefGoogle Scholar
  8. Bridgman PC, Kachar B, Reese TS (1986) The structure of cytoplasm in directly frozen cultured cells. II. Cytoplasmic domains associated with organelle movements. J Cell Biol 102:1510–1521.PubMedCrossRefGoogle Scholar
  9. Buckley IK (1973) Studies in fixation for electron microscopy using cultured cells. Lab Invest 29:398–410.PubMedGoogle Scholar
  10. Caffrey M (1985) Kinetics and mechanism of the lamellar gel/lamellar liquid-crystal and lamellar/inverted hexagonal phase transition in phosphatidyl ethanolamine: a real-time X-ray diffraction study using synchrotron radiation. Biochemistry 24:4826–4844.PubMedCrossRefGoogle Scholar
  11. *.
    Carde J-P, Joyard J, Douce R (1982) Electron microscopic studies of envelope membranes from spinach plastids. Biol Cell 44:315–324.Google Scholar
  12. Chandler DE (1984) Comparison of quick-frozen and chemically fixed sea urchin eggs: Structural evidence that cortical granule exocytosis is preceded by a local increase in membrane mobility. J Cell Sci 72:23–36.PubMedGoogle Scholar
  13. Chandler DE, Heuser J (1979) Membrane fusion during secretion. Cortical granule exocytosis in sea urchin eggs as studied by quick-freezing and freeze-fracture. J Cell Biol 83:91–108.PubMedCrossRefGoogle Scholar
  14. Cheng TPO, Reese TS (1985) Polarized compartmentalization of organelles in growth cones from developing optic tectum. J Cell Biol 101:1473–1480.PubMedCrossRefGoogle Scholar
  15. Cline K, Keegstra K, Staehelin LA (1985) Freeze-fracture electron microscopic analysis of ultrarapidly frozen envelope membranes on intact chloroplasts and after purification. Protoplasma 125:111–123.CrossRefGoogle Scholar
  16. De Brabander M, Nuydens R, Geuens G, Moeremans M, De Mey Y (1986) The use of submicroscopic gold particles combined with video contrast enhancement as a simple molecular probe for the living cell. Cell Motil 6:105–113.CrossRefGoogle Scholar
  17. De Laat SW, Tertoolen LGJ, Van der Saag PT, Bluemink JG (1983) Quantitative analysis of modulations in numerical and lateral distribution of intramembrane particles during the cell cycle of neuroblastoma cells. J Cell Biol 96:1047–1055.PubMedCrossRefGoogle Scholar
  18. Denis-Pouxviel C, Riesinger I, Bühler C, Bremm G, Brdiczka D, Murat J-C (1987) Regulation of mitochondrial hexokinase in cultured HT29 human cancer cells. Biochim Biophys Acta (in press)Google Scholar
  19. *.
    Düzgünes N (1985) Membrane fusion. In: Roodyn DB (ed) Subcellular biochemistry, vol 11. Plenum Press, New York, pp 195–286.Google Scholar
  20. Düzgünes N, Straubinger RM, Baldwin PA, Friend DS, Papahadjopoulos D (1985) Proton-induced fusion of oleic acid-phosphatidyl ethanolamine liposomes. Biochemistry 24: 3091–3098.PubMedCrossRefGoogle Scholar
  21. Escaig J (1982) New instruments which facilitate rapid freezing at 83 K and 6 K. J Microsc (Oxford) 126:221–229.CrossRefGoogle Scholar
  22. Fernandez DE, Staehelin LA (1985) Structural organization of ultrarapidly frozen barley aleurone cells actively involved in protein secretion. Planta 165:455–468.CrossRefGoogle Scholar
  23. *.
    Fernandez-Moran H (1964) New approaches in correlative studies on biological ultrastructure by high-resolution electron microscopy. J R Microsc Soc 83:183–195.PubMedCrossRefGoogle Scholar
  24. *.
    Friend DS (1982) Plasma-membrane diversity in a highly polarized cell. J Cell Biol 93:243–249.PubMedCrossRefGoogle Scholar
  25. Gaub H, Sackmann E, Büschl R, Ringsdorf H (1984) Lateral diffusion and phase separation in two-dimensional solutions of polymerized butadiene lipids in dimyristoylphosphatidyl-choline bilayers. Biophys J 45:725–731.PubMedCrossRefGoogle Scholar
  26. *.
    Grant CWM, Peters MW (1984) Lectin-membrane interactions. Information from model systems. Biochim Biophys Acta 779:403–422.PubMedGoogle Scholar
  27. Green CR, Severs NJ (1984) Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks. J Cell Biol 99:453–463.PubMedCrossRefGoogle Scholar
  28. Gulik-Krzywicki T, Costello MJ (1978) The use of low temperature X-ray diffraction to evaluate freezing methods used in freeze-fracture electron microscopy. J. Microsc (Oxford) 112: 103–113.CrossRefGoogle Scholar
  29. Hackenbrock CR (1968) Chemical and physical fixation of isolated mitochondria in low-energy and high-energy states. Proc Natl Acad Sci USA 61:598–605.PubMedCrossRefGoogle Scholar
  30. Heuser JE, Reese TS, Dennis MJ, Jan Y, Jan L, Evans L (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol 81:275–300.PubMedCrossRefGoogle Scholar
  31. Hobot JA, Carlemalm E, Villinger W, Kellenberger E (1984) Periplasmic gel: New concept resulting from the reinvestigation of bacterial cell envelope ultrastructure by new methods. J Bacteriol 160:143–152.PubMedGoogle Scholar
  32. Jones GJ (1984) On estimating freezing times during tissue rapid freezing. J Microsc (Oxford) 136:349–360.CrossRefGoogle Scholar
  33. Kachar B, Reese TS (1982) Evidence for the lipidic nature of tight junction strands. Nature (London) 296:464–466.CrossRefGoogle Scholar
  34. Kleemann W, McConnell HM (1974) Lateral phase separations in Escherichia coli membranes. Biochim Biophys Acta 345:220–230.PubMedCrossRefGoogle Scholar
  35. Klug GA, Krause J, Östlund A-K, Knoll G, Brdiczka D (1984) Alterations in liver mitochondrial function as a result of fasting and exhaustive exercise. Biochim Biophys Acta 764:272–282.PubMedCrossRefGoogle Scholar
  36. Knoll G (1984) Dynamische Interaktionen der mitochondrialen Hüllmembranen. Eine ultrastrukturelle Analyse auf der Basis schneller Kryofixation. Diss, Univ KonstanzGoogle Scholar
  37. Knoll G, Brdiczka D (1983) Changes in freeze-fractured mitochondrial membranes correlated to their energetic state. Dynamic interactions of the boundary membranes. Biochim Biophys Acta 733:102–110.PubMedCrossRefGoogle Scholar
  38. Kopstad G, Elgsaeter A (1982a) Theoretical analysis of the ice crystal size distribution in frozen aqueous specimens. Biophys J 40:155–161.PubMedCrossRefGoogle Scholar
  39. Kopstad G, Elgsaeter A (1982b) Theoretical analysis of specimen cooling rate during impact freezing and liquid-jet freezing of freeze-etch specimens. Biophys J 40:163–170.PubMedCrossRefGoogle Scholar
  40. Kruskal BA, Shak S, Maxfield FR (1986) Spreading of human neutrophils is immediately preceded by a large increase in cytoplasmic free calcium. Proc Natl Acad Sci USA 83:2919–2923.PubMedCrossRefGoogle Scholar
  41. Landis DMD, Reese TS (1981) Astrocyte membrane structure: Changes after circulatory arrest. J Cell Biol 88:660–663.PubMedCrossRefGoogle Scholar
  42. Malhotra SK, Van Harreveld A (1965) Some structural features of mitochondria in tissues prepared by freeze-substitution. J Ultrastruct Res 12:473–487.PubMedCrossRefGoogle Scholar
  43. Melchior DL, Bruggemann EP, Steim JM (1982) The physical state of quick-frozen membranes and lipids. Biochim Biophys Acta 690:81–88.PubMedCrossRefGoogle Scholar
  44. Mersey B, McCully ME (1978) Monitoring the course of fixation of plant cells. J Microsc (Oxford) 114:49–76.CrossRefGoogle Scholar
  45. Metcalf III TN, Villanueva MA, Schindler M, Wang JL (1986a) Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS-1. J Cell Biol 102:1350–1357.PubMedCrossRefGoogle Scholar
  46. Metcalf III TN, Wang JL, Schindler M (1986b) Lateral diffusion of phospholipids in the plasma membrane of soybean protoplasts: Evidence for membrane lipid domains. Proc Natl Acad Sci USA 83:95–99.PubMedCrossRefGoogle Scholar
  47. Miller DC, Dahl GP (1982) Early events in calcium-induced liposome fusion. Biochim Biophys Acta 689:165–169.PubMedCrossRefGoogle Scholar
  48. Miller TM, Goodenough DA (1985) Gap junction structures after experimental alteration of junctional channel conductance. J Cell Biol 101:1741–1748.PubMedCrossRefGoogle Scholar
  49. Nicolay K, Timmers RJM, Spoelstra E, Van der Neut R, Fok JJ, Huigen YM, Verkleij AJ, De Kruijff B (1984) The interaction of adriamycin with cardiolipin in model and rat liver mitochondrial membranes. Biochim Biophys Acta 778:359–371.PubMedCrossRefGoogle Scholar
  50. *.
    Peracchia C (1980) Structural correlates of gap junction permeation. Int Rev Cytol 66:81–146.PubMedCrossRefGoogle Scholar
  51. *.
    Pinto da Silva P, Kachar B (1980) Quick freezing vs. chemical fixation: capture and identification of membrane fusion intermediates. Cell Biol Int Rep 4:625–640.CrossRefGoogle Scholar
  52. *.
    Plattner H (1981) Membrane behaviour during exocytosis. Cell Biol Int Rep 5:435–459.PubMedCrossRefGoogle Scholar
  53. *.
    Plattner H, Bachmann L (1982) Cryofixation: A tool in biological ultrastructural research. Int Rev Cytol 79:237–304.PubMedCrossRefGoogle Scholar
  54. *.
    Plattner H, Zingsheim HP (1983) Electron microscopic methods in cellular and molecular biology. In: Roodyn DB (ed) Subcellular biochemistry, vol 9. Plenum Press, New York, pp 1–236.CrossRefGoogle Scholar
  55. Rand RP, Kachar B, Reese TS (1985) Dynamic morphology of calcium-induced interactions between phosphatidylserine vesicles. Biophys J 47:483–489.PubMedCrossRefGoogle Scholar
  56. *.
    Rash JE (1983) The rapid-freeze technique in neurobiology. TINS 6:208–212.Google Scholar
  57. *.
    Rebhun LI (1965) Freeze-substitution: fine structure as a function of water concentration in cells. Fed Proc 24(Suppl 15):217–233.Google Scholar
  58. Robinson JM, Okada T, Castellot JJ, Karnovsky MJ (1986) Unusual lysosomes in aortic smooth muscle cells: presence in living and rapidly frozen cells. J Cell Biol 102:1615–1622.PubMedCrossRefGoogle Scholar
  59. Saffman PG, Delbrück M (1975) Brownian motion in biological membranes. Proc Natl Acad Sci USA 72:3111–3113.PubMedCrossRefGoogle Scholar
  60. Schleyer M, Neupert W (1985) Transport of proteins into mitochondria: translocation intermediates spanning contact sites between outer and inner membranes. Cell 43:339–350.PubMedCrossRefGoogle Scholar
  61. Schmidt W, Patzak A, Lingg G, Winkler H, Plattner H (1983) Membrane events in adrenal chromaffin cells during exocytosis: a freeze-etching analysis after rapid cryofixation. Eur J Cell Biol 32:31–37.PubMedGoogle Scholar
  62. *.
    Shotton D (1980) Quick freezing — the new frontier in freeze-fracture. Nature (London) 283:12–14.CrossRefGoogle Scholar
  63. Siegel DP (1984) Inverted micellar structures in bilayer membranes. Formation rates and half-lifes. Biophys J 45:399–420.PubMedCrossRefGoogle Scholar
  64. Thompson TE, Allietta M, Brown RE, Johnson ML, Tillack TW (1985) Organization of ganglioside GM1 in phosphatidylcholine bilayers. Biochim Biophys Acta 817:229–237.PubMedCrossRefGoogle Scholar
  65. Torri-Tarelli F, Grohovaz F, Fesce R, Ceccarelli B (1985) Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine. J Cell Biol 101:1386–1399.PubMedCrossRefGoogle Scholar
  66. Van Harreveld A, Crowell J (1964) Electron microscopy after rapid freezing on a metal surface and substitution fixation. Anat Rec 149:381–386.CrossRefGoogle Scholar
  67. Van Harreveld A, Crowell J, Malhotra SK (1965) A study of extracellular space in central nervous tissue by freeze-substitution. J Cell Biol 25:117–137.CrossRefGoogle Scholar
  68. Van Harreveld A, Trubatch J (1979) Progression of fusion during rapid freezing for electron microscopy. J Microsc (Oxford) 115:243–256.CrossRefGoogle Scholar
  69. Van Meer G, Gumbiner B, Simons K (1986) The tight junction does not allow lipid molecules to diffuse from one epithelial cell to the next. Nature (London) 322:639–641.CrossRefGoogle Scholar
  70. Van Venetie R, Verkleij AJ (1981) Analysis of the hexagonal II phase and its relation to lipidic particles and the lamellar phase. A freeze fracture study. Biochim Biophys Acta 645:262–269.PubMedCrossRefGoogle Scholar
  71. Van Venetie R, Verkleij AJ (1982) Possible role of non-bilayer lipids in the structure of mitochondria. A freeze-fracture electron microscopic study. Biochim Biophys Acta 692:379–405.Google Scholar
  72. *.
    Verkleij AJ (1984) Lipidic intramembranous particles. Biochim Biophys Acta 779:43–63.PubMedGoogle Scholar
  73. *.
    Verkleij AJ, Ververgaert PHJT (1975) Architecture of biological and artificial membranes, as visualized by freeze-etching. Annu Rev Phys Chem 26:101–122.CrossRefGoogle Scholar
  74. *.
    Verkleij AJ, Ververgaert PHJT (1978) Freeze-fracture morphology of biological membranes. Biochim Biophys Acta 515:303–327.PubMedGoogle Scholar
  75. Verkleij AJ, Ververgaert PHJT, Van Deenen LLM, Elbers PF (1972) Phase transitions of phospholipid bilayers and membranes of Acholeplasma laidlawii B. visualized by freeze-fracturing electron microscopy. Biochim Biophys Acta 288:326–332.PubMedCrossRefGoogle Scholar
  76. Verkleij AJ, Mombers C, Gerritsen WJ, Leunissen-Bijvelt J, Cullis PR (1979) Fusion of phospholipid vesicles in association with the appearance of lipidic particles as visualized by freeze-fracturing. Biochim Biophys Acta 555:358–361.PubMedCrossRefGoogle Scholar
  77. Ververgaert PHJT, Verkleij AJ, Verhoeven JJ, Elbers PF (1973) Spray-freezing of liposomes. Biochim Biophys Acta 311:651–654.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1987

Authors and Affiliations

  • Gerd Knoll
    • 1
  • Arie J. Verkleij
    • 2
  • Helmut Plattner
    • 1
  1. 1.Fakultät für BiologieUniversität KonstanzKonstanzGermany
  2. 2.Department of Molecular Cell BiologyUniversity of UtrechtThe Netherlands

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