ReviewFreeze-fracture studies on lipids and membranesH.W. Meyer*, W. RichterInstitut fuÈr Ultrastrukturforschung, Klinikum der Friedrich-Schiller-UniversitaÈt Jena, D-07740 Jena, GermanyReceived 10 April 2000; revised 13 June 2000; accepted 14 June 2000AbstractFreeze-fracture electron microscopy is especially useful for investigation of lipid structures by the advantageous fracture course withinhydrophobic zones. Freezing is, on the other hand, a restriction because the structures of lamellar and non-lamellar phase states withdisordered acyl chains (La,HII,cubic) are dif®cult to preserve. An important aspect of this method is therefore the lipid structure ofphase states with ordered acyl chains (crystal, gel), and with a different degree of hydration. Freeze-fracture of pure lipid systems createsa valid representation of the structure of non-lamellar phases and of the general structure of the ªlamellarº lipid bilayer, and lamellar phaseswith characteristic deformations (ripples, curvatures, plane sectors) can be identi®ed. Fracture through the hydrophobic bilayer centre ofbiological membranes reveals characteristic protein components, the intramembraneous particles (IMPs). The lateral distribution of the IMPsis a helpful marker for ¯uid and rigid phase states, also without deformation of the lamella. The overall history and the present state ofknowledge concerning the different structures revealed by the freeze-fracture and freeze-etch techniques in lipid systems, and to a limitedextent in biological membranes, is reviewed, taking into account studies from our own laboratory. q 2001 Elsevier Science Ltd. All rightsreserved.Keywords: Freeze-fracture electron microscopy; Lipids; Biomembranes; Phase states; Cold hydration; Cold storage; Ripples; Curvatures; Non-lamellarstructures; Phase separationContents1. Some history of the methods ................................................................ 6162. Lipid structures .......................................................................... 6162.1. Fats and oils ........................................................................ 6162.2. Membrane lipids .................................................................... 6162.2.1. Non-lipid lamellae ............................................................. 6172.2.2. Dry lipids .................................................................... 6172.2.3. Thermotropic states ............................................................ 6182.2.4. Lyotropic states ............................................................... 6242.2.5. Lateral phase separation in lipid bilayers and biological membranes ......................... 6302.3. Non-lamellar lipid structures ............................................................ 6342.3.1. Single micelles ................................................................ 6342.3.2. Aggregates of micelles .......................................................... 6342.3.3. Bicontinuous cubic phases of type II ................................................ 6363. Some contributions to biological membrane research .............................................. 6363.1. Surface views or membrane splitting . ..................................................... 636Micron 32 (2001) 615±644PERGAMON0968±4328/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.PII: S0968-4328(00)00050-0www.elsevier.com/locate/micron* Corresponding author. Tel.: 149-3641-633-123; fax: 149-3641-633-102.E-mail address: [email protected] (H.W. Meyer).Abbreviations: D2-HSPC: 1,2-Di(2-hexadecyl-stearoyl)-sn-glycero-3-phosphocholine; D4-DPPC: 1,2-Di(4-dodecyl-palmitoyl)-sn-glycero-3-phosphocholine;DEPC: 1,2-Dielaidoyl-sn-glycero-3-phosphocholine; DEPE: 1,2-Dielaidoyl-sn-glycero-3-phosphoethanolamine; DMPC: 1,2-Dimyristoyl-sn-glycero-3-phos-phocholine; DMPG: 1,2-Dimyristoyl-sn-glycero-3-phospho-glycerol; DPPC: 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; EF: Exoplasmic fracture face;F6C10PC: 1,2-C6F13(CH2)10-sn-glycero-3-phosphocholine; HII: Hexagonal phase of type II (water in oil); IMB: Intramembraneous particle; La: Lamellarphase with disordered chains (liquid crystalline); Lb0: Lamellar gel phase with ordered and tilted chains; Lc: Lamellar crystalline gel phase; Li: Lamellarinterdigitated gel phase; Pb0: Lamellar gel phase with ripples; Pcc: Lamellar crystalline gel phase with convex±concave curvatures; PF: Protoplasmic fractureface3.2. Membrane splitting and surface views ..................................................... 6363.3. Etching holes, indications for restricted membrane splitting . .................................... 6373.4. ªPlastic deformationº of proteins and intramembranous particles ................................. 6383.5. Membrane models ................................................................... 638Acknowledgements .......................................................................... 639References ................................................................................ 6391. Some history of the methodsInvestigations using freeze-fracture and freeze-etchingtechniques for the preparation of samples for electronmicroscopy started in 1950s. At ®rst the combination offreezing and etching (sublimation of ice to reveal surfacestructures) was introduced by Hall (1950) and Meryman(1950). The additional fracturing of frozen specimen wasused by Meryman and Ka®g (1955), and the ®rst structuresof biological materials Ð crystalline arrays of viruses Ðwas presented by Steere (1957). More general attention wasgiven to this new technique in the 1960s, especially by theimpressively spectacular three-dimensional ultrastructuralviews of freeze-fractured yeast cells, presented by Moorand his associates (Moor and MuÈhlethaler, 1963; Moor,1967a).Moor et al. (1961) developed a freeze-fracture/etchingapparatus which was then (Moor, 1965) commercially avail-able from BALZERS AG (Liechtenstein), but the machinewas relatively expensive, e.g. because a cooled precisionmicrotome for fracturing was constructed. However, Haggis(1961) used simpler instrumentation, based on preparativeequipment already present in most electron microscopelaboratories. Fracturing was performed by cross-breakingthe frozen sample together with its 100 mm thick chamberthat was made from glass coverslips and a platinum foil. Animproved and successful device, using a brass specimenblock and again standard laboratory