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.

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.

scholarly journals Fracture faces of frozen membranes: 50th anniversary

2016 ◽  
Vol 27 (3) ◽  
pp. 421-423
Author(s):  
Daniel Branton
Keyword(s):  
Electron Microscopy ◽  
Lipid Bilayers ◽  
Fracture Process ◽  
Relative Proportion ◽  
50Th Anniversary ◽  
Biological Membranes ◽  
Bilayer Membrane ◽  
Biological Cells ◽  
Membrane Surfaces ◽  
Freeze Etching

In 1961, the development of an improved freeze-etching (FE) procedure to prepare rapidly frozen biological cells or tissues for electron microscopy raised two important questions. How does a frozen cell membrane fracture? What do the extensive face views of the cell’s membranes exposed by the fracture process of FE tell us about the overall structure of biological membranes? I discovered that all frozen membranes tend to split along weakly bonded lipid bilayers. Consequently, the fracture process exposes internal membrane faces rather than either of the membrane’s two external surfaces. During etching, when ice is allowed to sublime after fracturing, limited regions of the actual membrane surfaces are revealed. Examination of the fractured faces and etched surfaces provided strong evidence that biological membranes are organized as lipid bilayers with some proteins on the surface and other proteins extending through the bilayer. Membrane splitting made it possible for electron microscopy to show the relative proportion of a membrane’s area that exists in either of these two organizational modes.

High Resolution FESEM and TEM Reveal Bacterial Spore Attachment

2007 ◽  
Vol 13 (4) ◽  
pp. 251-266 ◽  
Author(s):  
Barbara J. Panessa-Warren ◽  
George T. Tortora ◽  
John B. Warren
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Field Emission ◽  
Freeze Fracture ◽  
Bacterial Spore ◽  
Attachment Structures ◽  
Spore Attachment ◽  
Electron Microscopes ◽  
The 1960S ◽  
Field Emission Electron Microscopy

Transmission electron microscopy (TEM) studies in the 1960s and early 1970s using conventional thin section and freeze fracture methodologies revealed ultrastructural bacterial spore appendages. However, the limited technology at that time necessitated the time-consuming process of imaging serial sections and reconstructing each structure. Consequently, the distribution and function of these appendages and their possible role in colonization or pathogenesis remained unknown. By combining high resolution field emission electron microscopy with TEM images of identical bacterial spore preparations, we have been able to obtain images of intact and sectioned Bacillus and Clostridial spores to clearly visualize the appearance, distribution, resistance (to trypsin, chloramphenicol, and heat), and participation of these structures to facilitate attachment of the spores to glass, agar, and human cell substrates. Current user-friendly commercial field emission scanning electron microscopes (FESEMs), permit high resolution imaging, with high brightness guns at lower accelerating voltages for beam sensitive intact biological samples, providing surface images at TEM magnifications for making direct comparisons. For the first time, attachment structures used by pathogenic, environmental, and thermophile bacterial spores could be readily visualized on intact spores to reveal how specific appendages and outer spore coats participated in spore attachment, colonization, and invasion.

Scanning Electron Microscopy Study of Cerebellar Synaptic Junctions

1990 ◽  
Vol 48 (3) ◽  
pp. 148-149
Author(s):  
Orlando J. Castejón
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Cerebellar Cortex ◽  
Salmo Trutta ◽  
Freeze Fracture ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Specimen Preparation ◽  
Scanning Electron

Conventional and high resolution scanning electron microscopy have been applied to trace short cerebellar nerve circuits and to explore the outer and inner surfaces of spine, glomerular and axodendritic synapses. Samples of teleost fishes (Arius spixii and Salmo Trutta) cerebellar cortex were processed according to the slicing technique for conventional scanning electron microscopy (SEM), ethanol cryofracturing technique and freeze-fracture scanning electron microscopy (FFSEM) (Castejón, 1988). Primate (Rhesus monkey) cerebellar cortex was processed for high resolution scanning electron microscopy (Castejón and Apkarian, 1990), according to the protocol of delicate specimen preparation (Peters, 1980). Observations were made in JEOL 100B with (ASID), scanning attachment ISI DS-130 equiped with LaB6 emitter and Hitachi S-900 SEM with a cold cathode field emitter. Micrographs for HRSEM were soft focus printed to reduce instrumental noise (Peters, 1985). A comparison was made of gold-palladium and chromium coating cerebellar samples.The slicing technique and the cryofracture process exposed the neuronal outer surface revealing hidden surface ensheathed by neuroglial cells.

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:

scholarly journals More Than One Ever Wanted To Know About X-ray Detectors Part IV: Windows for Elements Heavy and Light

1995 ◽  
Vol 3 (3) ◽  
pp. 3-4
Author(s):  
Mark W. Lund
Keyword(s):  
Electron Microscopy ◽  
Chemical Element ◽  
Energy Dispersive Spectrometer ◽  
Silicon Crystal ◽  
Energy Dispersive ◽  
X Rays ◽  
Element Analysis ◽  
X Ray ◽  
Imaging Capability ◽  
The Road

I attend a local entrepreneur's luncheon once a month. Since the small town 1 live is the home of Word Perfect Corporation, and Novell, inc. is just down the road, you can imagine that mamy members are doing exciting things with software. When I tell them that MOXTEK makes windows (or X-ray detectors they really light up, until I tell them that! mean real windows.X-ray detectors are used in electron microscopy to add chemical element analysis to the imaging capability of the microscope. A typical energy dispersive spectrometer uses a silicon crystal about the si2e of a shirt button to detect and measure the energy of incoming X-rays.

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.

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