High-Resolution Scanning Electron Microscopy of Biological Specimens

1988 ◽  
Vol 46 ◽  
pp. 186-187
Author(s):  
M. Müller ◽  
R. Hermann
Keyword(s):  
High Resolution ◽  
Freeze Drying ◽  
Room Temperature ◽  
Structural Information ◽  
Freeze Substitution ◽  
Critical Point Drying ◽  
Sem Study ◽  
Major Factors ◽  
Structural Preservation ◽  
Preparation Techniques

Three major factors must be concomitantly assessed in order to extract relevant structural information from the surface of biological material at high resolution (2-3nm).Procedures based on chemical fixation and dehydration in graded solvent series seem inappropriate when aiming for TEM-like resolution. Cells inevitably shrink up to 30-70% of their initial volume during gehydration; important surface components e.g. glycoproteins may be lost. These problems may be circumvented by preparation techniques based on cryofixation. Freezedrying and freeze-substitution followed by critical point drying yields improved structural preservation in TEM. An appropriate preservation of dimensional integrity may be achieved by freeze-drying at - 85° C. The sample shrinks and may partially collapse as it is warmed to room temperature for subsequent SEM study. Observations at low temperatures are therefore a necessary prerequisite for high fidelity SEM. Compromises however have been unavoidable up until now. Aldehyde prefixation is frequently needed prior to freeze drying, rendering the sample resistant to treatment with distilled water.

Simple desiccation method for SEM using dimethoxypropane

1992 ◽  
Vol 50 (1) ◽  
pp. 760-761
Author(s):  
Frantíšek Weyda
Keyword(s):  
Electron Microscopy ◽  
Freeze Drying ◽  
Simple Procedure ◽  
Aqueous Suspension ◽  
Freeze Substitution ◽  
Biological Specimen ◽  
Air Drying ◽  
Biological Objects ◽  
Critical Point Drying ◽  
Scanning Electron

A number of techniques such as critical point drying (the most commonly used method of drying cells and tissues), freeze-drying or freeze-substitution are available to process various tissues preparations for scanning electron microscopy (SEM). Those techniques are more or less complicated (depending also on expensive equipments) and time consuming. While more simple air-drying from aqueous suspension or organic solvents can be used for some rigid biological specimen, it is generally not satisfactory for most tissues and biological objects. On the opposite, rapid and simple procedure using hexamethyldisilazane (HMDS) and air drying has been successfully applied to insect and mite tissues without observable artifacts. We have developed similar simple and rapid method using dimethoxypropane (DMP) and air drying. DMP has previously been used for chemical dehydration of tissues.

Cryo - Fesem of Subcellular Organisation Using an In-Lens Rod with A Vacuum Transfer System

2001 ◽  
Vol 7 (S2) ◽  
pp. 724-725
Author(s):  
Terry Allen ◽  
Sandra Rutherford ◽  
Steve Bagley ◽  
Bob Morrison ◽  
Martin Goldberg
Keyword(s):  
High Resolution ◽  
Structural Information ◽  
Membrane Dynamics ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Surface Imaging ◽  
Thin Sections ◽  
Critical Point Drying ◽  
Cryo Preservation ◽  
Pore Complexes

The recent increase in use of Field Emission Scanning Instruments in biology has shown the potential of these systems for sub-cellular imaging, as they essentially provide transmission EM levels of resolution for surface imaging. in structures lacking inherent contrast in thin sections, such as nuclear pore complexes,(NPC) high resolution surface imaging has provided significant complementary structural information to the macromolecular architecture of the NPC. (Goldberg and Allen, 1996). A series of intermediate structural forms of the NPC during its formation has also been described using FESEM, which would not have been resolvable in TEM.(Goldberg, Weise, Allen, Wilson, 1997). All this work was performed with chemical fixation, dehydration and critical point drying, followed by high resolution Chromium coating. We now are extending these studies to a cryo-preservation approach, mindful of the requirement of rapid freezing to preserve the more labile events of membrane dynamics, and also to circumvent the requirement to expose membranes to organic solvents during conventional dehydration.

scholarly journals Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams

2015 ◽  
Vol 71 (5) ◽  
pp. 1184-1196 ◽  
Author(s):  
Nicolas Coquelle ◽  
Aaron S. Brewster ◽  
Ulrike Kapp ◽  
Anastasya Shilova ◽  
Britta Weinhausen ◽  
...  
Keyword(s):  
Silicon Nitride ◽  
High Resolution ◽  
Room Temperature ◽  
Structural Information ◽  
Protein Crystallography ◽  
Structural Data ◽  
Analysis Software ◽  
Raster Scanning ◽  
Hit Rate ◽  
Alternative Approach

High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, namedNanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out usingCrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering.

scholarly journals Enzyme catalysis captured using multiple structures from one crystal at varying temperatures

IUCrJ ◽  
2018 ◽  
Vol 5 (3) ◽  
pp. 283-292 ◽  
Author(s):  
Sam Horrell ◽  
Demet Kekilli ◽  
Kakali Sen ◽  
Robin L. Owen ◽  
Florian S. N. Dworkowski ◽  
...  
Keyword(s):  
High Resolution ◽  
Enzyme Catalysis ◽  
Density Functional ◽  
Room Temperature ◽  
Structural Information ◽  
Density Functional Theory Calculations ◽  
State Density ◽  
X Rays ◽  
Copper Site ◽  
Multiple Structures

High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate `structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a `top-hat' geometry, which was rapidly transformed to a `side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

The Use of Gach Technique in Stabilizing Biological Specimens For X-Ray Microanalysis

1985 ◽  
Vol 43 ◽  
pp. 452-453
Author(s):  
W.T. Gunning ◽  
R.E. Crang
Keyword(s):  
Surface Morphology ◽  
Critical Point ◽  
Freeze Drying ◽  
Freeze Substitution ◽  
Specimen Preparation ◽  
Preparation Methods ◽  
Bulk Specimen ◽  
X Ray ◽  
Critical Point Drying ◽  
Better Than

An alternative SEM preparative technique is proposed for biological specimens in which cellular diffusible constituents may be retained that are otherwise lost during conventional specimen preparation involving critical point drying. The technique utilizes the copolymerization of glutaraldehyde with carbohy-drazide, and is designated “GACH”. Surface morphology with GACH preparations is as good as, or better than, that preserved using critical point drying. Bulk specimen preparation for elemental analysis has been limited heretofore to frozen-hydrated, freeze drying, freeze substitution, unfixed air dried, and critical point dried specimens. All such techniques offer particular advantages, but with certain limitations. The GACH technique is not being proposed as a superior routine replacement for present biological bulk specimen preparation methods, but rather as a viable alternative technique that allows both the retention of bound ions and acceptable surface morphology without elaborate instrumentation.

Ca-Histochemistry in slime mold plasmodia

1978 ◽  
Vol 36 (2) ◽  
pp. 130-131
Author(s):  
Ulrich Dierkes
Keyword(s):  
Physarum Polycephalum ◽  
Carbon Coating ◽  
Freeze Drying ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Slime Mold ◽  
Staining Procedure ◽  
Protoplasmic Streaming ◽  
Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

The Critical Point Drying Method Applied to Scanning Electron Microscopy of L-929 Cells

1970 ◽  
Vol 28 ◽  
pp. 278-279
Author(s):  
Charles TurnbiLL ◽  
Delbert E. Philpott
Keyword(s):  
Electron Microscopy ◽  
Carbon Dioxide ◽  
Surface Tension ◽  
Critical Point ◽  
Freeze Drying ◽  
Attractive Alternative ◽  
Crystal Formation ◽  
Critical Point Drying ◽  
Time Required ◽  
Scanning Electron

The advent of the scanning electron microscope (SCEM) has renewed interest in preparing specimens by avoiding the forces of surface tension. The present method of freeze drying by Boyde and Barger (1969) and Small and Marszalek (1969) does prevent surface tension but ice crystal formation and time required for pumping out the specimen to dryness has discouraged us. We believe an attractive alternative to freeze drying is the critical point method originated by Anderson (1951; for electron microscopy. He avoided surface tension effects during drying by first exchanging the specimen water with alcohol, amy L acetate and then with carbon dioxide. He then selected a specific temperature (36.5°C) and pressure (72 Atm.) at which carbon dioxide would pass from the liquid to the gaseous phase without the effect of surface tension This combination of temperature and, pressure is known as the "critical point" of the Liquid.

A Liquid Nitrogen Cooled Stage for STEM

1974 ◽  
Vol 32 ◽  
pp. 444-445
Author(s):  
Louis T. Germinario
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Liquid Nitrogen ◽  
Room Temperature ◽  
Objective Lens ◽  
Thermal Drift ◽  
Specimen Holder ◽  
Resolution Capability ◽  
Focal Position

A liquid nitrogen stage has been developed for the JEOL JEM-100B electron microscope equipped with a scanning attachment. The design is a modification of the standard JEM-100B SEM specimen holder with specimen cooling to any temperatures In the range ~ 55°K to room temperature. Since the specimen plane is maintained at the ‘high resolution’ focal position of the objective lens and ‘bumping’ and thermal drift la minimized by supercooling the liquid nitrogen, the high resolution capability of the microscope is maintained (Fig.4).

Preservation of Plant Cell Surfaces For Scanning Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 472-473
Author(s):  
Linda M. Sicko ◽  
Thomas E. Jensen
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Critical Point ◽  
Filter Paper ◽  
Freeze Drying ◽  
Cell Surfaces ◽  
Air Drying ◽  
Popular Method ◽  
Critical Point Drying ◽  
Scanning Electron

The use of critical point drying is rapidly becoming a popular method of preparing biological samples for scanning electron microscopy. The procedure is rapid, and produces consistent results with a variety of samples. The preservation of surface details is much greater than that of air drying, and the procedure is less complicated than that of freeze drying. This paper will present results comparing conventional air-drying of plant specimens to critical point drying, both of fixed and unfixed material. The preservation of delicate structures which are easily damaged in processing and the use of filter paper as a vehicle for drying will be discussed.

Scanning Electron Microscopy-cuticular Lesions on the Roundworm Ascaris Suum

1970 ◽  
Vol 28 ◽  
pp. 114-115
Author(s):  
P. A. Madden ◽  
W. R. Anderson
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Freeze Drying ◽  
Room Temperature ◽  
Secondary Electron ◽  
Distilled Water ◽  
Freeze Dried ◽  
Silver Paint ◽  
To Come ◽  
Scanning Electron

The intestinal roundworm of swine is pinkish in color and about the diameter of a lead pencil. Adult worms, taken from parasitized swine, frequently were observed with macroscopic lesions on their cuticule. Those possessing such lesions were rinsed in distilled water, and cylindrical segments of the affected areas were removed. Some of the segments were fixed in buffered formalin before freeze-drying; others were freeze-dried immediately. Initially, specimens were quenched in liquid freon followed by immersion in liquid nitrogen. They were then placed in ampuoles in a freezer at −45C and sublimated by vacuum until dry. After the specimens appeared dry, the freezer was allowed to come to room temperature slowly while the vacuum was maintained. The dried specimens were attached to metal pegs with conductive silver paint and placed in a vacuum evaporator on a rotating tilting stage. They were then coated by evaporating an alloy of 20% palladium and 80% gold to a thickness of approximately 300 A°. The specimens were examined by secondary electron emmission in a scanning electron microscope.

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