High-resolution, extended views of membrane surfaces revealed by fracture-flip

1989 ◽  
Vol 47 ◽  
pp. 738-739
Author(s):  
Pedro Pinto da Silva
Keyword(s):  
High Resolution ◽  
Colloidal Gold ◽  
Cell Membranes ◽  
Freeze Fracture ◽  
Surface Structures ◽  
New Method ◽  
Deep Etching ◽  
Membrane Surfaces ◽  
Routine Identification ◽  
Freeze Etching

I will describe a new method — fracture-flip — that uses commercially available equipment to produce extended views of cell and membrane surfaces. The resolution of this new method permits the routine identification of surface structures down to 5 nm diameter. Moreover, in contrast to freeze-etching/deep-etching, extended views are easily obtained.Conceptally, fracture-flip derives from label-fracture, another method developed in my laboratory. With label-fracture we showed that, after freeze-fracture, the exoplasmic (E) halves of cell membranes are stabilized by, and remain attached to, their platinum/carbon replicas. This allows the observation of co-incident views of the Pt/C replica of the E face, and of the distribution of colloidal gold labeled receptors or antigens. This is the sequence of steps in fracture-flip:

A Freeze Fracture Preparation Chamber Attached to the SEM

1977 ◽  
Vol 35 ◽  
pp. 588-589
Author(s):  
J. B. Pawley ◽  
T. L. Hayes
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Chemical Element ◽  
Fracture Process ◽  
Freeze Fracture ◽  
Element Analysis ◽  
Actual Sample ◽  
Membrane Surfaces ◽  
Subsequent Fracture ◽  
Standard Tool

The freeze fracture technique has developed into a standard tool of biological electron microscopy particularly for the study of membrane surfaces. While permitting high resolution (2.5 nm) study of replicated specimens, freeze fracture has always had certain technical limitations: 1) The carbon and platinum replicas are extremely fragile and much care and patience are required to prepare them for EM observation. This is particularly true when large areas must be replicated or when complementary replicas are required. 2) The carbon and platinum replica despite its very “real” appearance is not the actual sample. The sample itself has been completely destroyed in preparing the replica and can neither be subjected to chemical element analysis nor reprocessed for any sort of subsequent study. 3) The investigator can neither monitor the fracture process nor make a subsequent fracture of the same sample.

Identification of Intramembrane Particles by Pre- and Post-Fracture Labelling Techniques: A Progress Report

1980 ◽  
Vol 38 ◽  
pp. 692-695
Author(s):  
J. E. Rash ◽  
T. J. A. Johnson ◽  
C. S. Hudson ◽  
D. S. Copio ◽  
W. F. Graham ◽  
...  
Keyword(s):  
Neuromuscular Junctions ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Glycerol Solution ◽  
Protein Markers ◽  
Replica Technique ◽  
Deep Etching ◽  
Intramembrane Particles ◽  
Membrane Surfaces ◽  
Macromolecular Architecture

Conventional freeze-fracture replicas provide for high resolution, three-dimensional replication of the internal macromolecular architecture of cell membranes, but they have the distinct disadvantage that no biologically rele¬vant macromolecules remain for subsequent in situ labelling and positive identification. Recently, several ingenious freeze-fracture techniques have been devised to localize biochemical components before the tissues are diges-ted away. These methods include (a) freeze-fracture autoradiography of the frozen whole mount preparation (1, 2) and (b) deep etching (freeze-drying) to expose immunoglobins or other large protein markers attached to membrane surfaces (3). However, freeze-fracture autoradiography has insufficient resolution to permit localization of labels to individual intramembrane particles (IMPs) exposed by the cleaving process, while deep etching to reveal membrane surfaces does not reveal IMPs. To overcome some of these difficulties, we recently introduced the "sectioned replica technique" to provide a method for direct and unambiguous correlation of freeze-fracture and thin section images within a single electron micrograph (4). The fractured and replicated tissues are thawed, but instead of digesting the tissues, the replica-tissue sandwich is post-fixed in 30% buffered glycerol solution con-taining 1% OsO4, post-stained, embedded in plastic, and sectioned parallel to the replica-tissue interface. Using this sectioned-replica technique, we have been able to obtain (a) pre-fracture radioisotope-labelled mouse neuromuscular junctions and (b) pre- and post-fracture immunoferritin and colloidal gold-labelled membrane fragments and intact neuromuscular junctions.

scholarly journals A COMPARISON OF CHLOROPLAST MEMBRANE SURFACES VISUALIZED BY FREEZE-ETCH AND NEGATIVE STAINING TECHNIQUES; AND ULTRASTRUCTURAL CHARACTERIZATION OF MEMBRANE FRACTIONS OBTAINED FROM DIGITONIN-TREATED SPINACH CHLOROPLASTS

1969 ◽  
Vol 43 (1) ◽  
pp. 16-31 ◽  
Author(s):  
C. J. Arntzen ◽  
R. A. Dilley ◽  
F. L. Crane
Keyword(s):  
Freeze Fracture ◽  
Internal Surface ◽  
Coupling Factor ◽  
Negative Staining ◽  
Fracture Plane ◽  
Chloroplast Membranes ◽  
Chloroplast Membrane ◽  
Membrane Surfaces ◽  
Large Particles ◽  
Freeze Etching

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.

scholarly journals Label-fracture: a method for high resolution labeling of cell surfaces.

1984 ◽  
Vol 99 (3) ◽  
pp. 1156-1161 ◽  
Author(s):  
P Pinto da Silva ◽  
F W Kan
Keyword(s):  
High Resolution ◽  
Cell Shape ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
Cell Surfaces ◽  
Surface Distribution ◽  
Membrane Surfaces ◽  
A Cell ◽  
Dense Marker

We introduce here a technique, "label-fracture," that allows the observation of the distribution of a cytochemical label on a cell surface. Cell surfaces labeled with an electron-dense marker (colloidal gold) are freeze-fractured and the fracture faces are replicated by plantinum/carbon evaporation. The exoplasmic halves of the membrane, apparently stabilized by the deposition of the Pt/C replica, are washed in distilled water. The new method reveals the surface distribution of the label coincident with the Pt/C replica of the exoplasmic fracture face. Initial applications indicate high resolution (less than or equal to 15 nm) and exceedingly low background. "Label-fracture" provides extensive views of the distribution of the label on membrane surfaces while preserving cell shape and relating to the freeze-fracture morphology of exoplasmic fracture faces. The regionalization of wheat germ agglutinin receptors on the plasma membranes of boar sperm cells is illustrated. The method and the interpretation of its results are straightforward. Label-fracture is appropriate for routine use as a surface labeling technique.

Photoreceptor disk membrane substructures by means of the complementary freeze-fracture-replica methods

1978 ◽  
Vol 36 (2) ◽  
pp. 156-157
Author(s):  
A. Tonosaki ◽  
M. Yamasaki ◽  
H. Washioka ◽  
J. Mizoguchi
Keyword(s):  
Cell Membranes ◽  
Freeze Fracture ◽  
Purple Membrane ◽  
Remarkable Case ◽  
Single Face ◽  
Disk Membrane ◽  
Associated Proteins ◽  
Photoreceptor Membranes

A vertebrate disk membrane is composed of 40 % lipids and 60 % proteins. Its fracture faces have been classed into the plasmic (PF) and exoplasmic faces (EF), complementary with each other, like those of most other types of cell membranes. The hypothesis assuming the PF particles as representing membrane-associated proteins has been challenged by serious questions if they in fact emerge from the crystalline formation or decoration effects during freezing and shadowing processes. This problem seems to be yet unanswered, despite the remarkable case of the purple membrane of Halobacterium, partly because most observations have been made on the replicas from a single face of specimen, and partly because, in the case of photoreceptor membranes, the conformation of a rhodopsin and its relatives remains yet uncertain. The former defect seems to be partially fulfilled with complementary replica methods.

Stereo Electron Microscopy Applied to High Resolution Freeze-Etch Replicas

1970 ◽  
Vol 28 ◽  
pp. 306-307
Author(s):  
L. Andrew Staehelin
Keyword(s):  
Electron Microscopy ◽  
Plastic Deformation ◽  
High Resolution ◽  
Smooth Surfaces ◽  
Small Particles ◽  
Membrane Surfaces ◽  
Wide Acceptance ◽  
New Information ◽  
Number Of Particles ◽  
Small Holes

Freeze-etched membranes usually appear as relatively smooth surfaces covered with numerous small particles and a few small holes (Fig. 1). In 1966 Branton (1“) suggested that these surfaces represent split inner mem¬brane faces and not true external membrane surfaces. His theory has now gained wide acceptance partly due to new information obtained from double replicas of freeze-cleaved specimens (2,3) and from freeze-etch experi¬ments with surface labeled membranes (4). While theses studies have fur¬ther substantiated the basic idea of membrane splitting and have shown clearly which membrane faces are complementary to each other, they have left the question open, why the replicated membrane faces usually exhibit con¬siderably fewer holes than particles. According to Branton's theory the number of holes should on the average equal the number of particles. The absence of these holes can be explained in either of two ways: a) it is possible that no holes are formed during the cleaving process e.g. due to plastic deformation (5); b) holes may arise during the cleaving process but remain undetected because of inadequate replication and microscope techniques.

High Resolution Stereography of Complementary Freeze-Fracture Replicas Reveals the Presence of Depressions Opposite Membrane Associated Particles of Split Membranes

1971 ◽  
Vol 29 ◽  
pp. 442-443
Author(s):  
Russell L. Steere
Keyword(s):  
High Resolution ◽  
Cardiac Muscle ◽  
Electron Micrographs ◽  
Chromic Acid ◽  
Freeze Fracture ◽  
Distilled Water ◽  
Fine Scale ◽  
Acid Cleaning ◽  
Cleaning Solution

Complementary replicas have revealed the fact that the two common faces observed in electron micrographs of freeze-fracture and freeze-etch specimens are complementary to each other and are thus the new faces of a split membrane rather than the original inner and outer surfaces (1, 2 and personal observations). The big question raised by published electron micrographs is why do we not see depressions in the complementary face opposite membrane-associated particles? Reports have appeared indicating that some depressions do appear but complementarity on such a fine scale has yet to be shown.Dog cardiac muscle was perfused with glutaraldehyde, washed in distilled water, then transferred to 30% glycerol (material furnished by Dr. Joaquim Sommer, Duke Univ., and VA Hospital, Durham, N.C.). Small strips were freeze-fractured in a Denton Vacuum DFE-2 Freeze-Etch Unit with complementary replica tooling. Replicas were cleaned in chromic acid cleaning solution, then washed in 4 changes of distilled water and mounted on opposite sides of the center wire of a Formvar-coated grid.

Freeze-Fracture Replication of Frozen Outer Segments of Rat Retinal Rods

1969 ◽  
Vol 27 ◽  
pp. 392-393
Author(s):  
Thomas S. Leeson ◽  
C. Roland Leeson
Keyword(s):  
Electron Microscopy ◽  
Cross Section ◽  
Outer Segment ◽  
Freeze Fracture ◽  
X Ray Diffraction ◽  
X Ray ◽  
Outer Segments ◽  
Retinal Rods ◽  
Temperature Electron ◽  
Freeze Etching

Numerous previous studies of outer segments of retinal receptors have demonstrated a complex internal structure of a series of transversely orientated membranous lamellae, discs, or saccules. In cones, these lamellae probably are invaginations of the covering plasma membrane. In rods, however, they appear to be isolated and separate discs although some authors report interconnections and some continuities with the surface near the base of the outer segment, i.e. toward the inner segment. In some species, variations have been reported, such as longitudinally orientated lamellae and lamellar whorls. In cross section, the discs or saccules show one or more incisures. The saccules probably contain photolabile pigment, with resulting potentials after dipole formation during bleaching of pigment. Continuity between the lamina of rod saccules and extracellular space may be necessary for the detection of dipoles, although such continuity usually is not found by electron microscopy. Particles on the membranes have been found by low angle X-ray diffraction, by low temperature electron microscopy and by freeze-etching techniques.

How to sample the entire length of a single muscle fiber quick-frozen after electrical point stimulation for high resolution EM

1992 ◽  
Vol 50 (1) ◽  
pp. 776-777
Author(s):  
Joachim R. Sommer ◽  
Teresa High ◽  
Betty Scherer ◽  
Isaiah Taylor ◽  
Rashid Nassar
Keyword(s):  
Skeletal Muscle ◽  
High Resolution ◽  
Action Potential ◽  
Muscle Fiber ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Electrical Field Stimulation ◽  
Skeletal Muscle Fiber ◽  
Freeze Dried ◽  
Quick Freezing

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.

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