High-Vacuum Evaporation and a Cryostage to Observe Fractured Yeast

1988 ◽  
Vol 46 ◽  
pp. 256-257
Author(s):  
William P. Wergin ◽  
Eric F. Erbe ◽  
Eugene L. Vigil
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Standard Specimen ◽  
High Vacuum ◽  
Specimen Holder ◽  
Sputter Coating ◽  
Fresh Frozen ◽  
Gain Access ◽  
Scanning Electron ◽  
Transfer Device

Investigators have long realized the potential advantages of using a low temperature (LT) stage to examine fresh, frozen specimens in a scanning electron microscope (SEM). However, long working distances (W.D.), thick sputter coatings and surface contamination have prevented LTSEM from achieving results comparable to those from TEM freeze etch. To improve results, we recently modified techniques that involve a Hitachi S570 SEM, an Emscope SP2000 Sputter Cryo System and a Denton freeze etch unit. Because investigators have frequently utilized the fractured E face of the plasmalemma of yeast, this tissue was selected as a standard for comparison in the present study.In place of a standard specimen holder, a modified rivet was used to achieve a shorter W.D. (1 to -2 mm) and to gain access to the upper detector. However, the additional height afforded by the rivet, precluded use of the standard shroud on the Emscope specimen transfer device. Consequently, the sample became heavily contaminated (Fig. 1). A removable shroud was devised and used to reduce contamination (Fig. 2), but the specimen lacked clean fractured edges. This result suggested that low vacuum sputter coating was also limiting resolution.

Use of platinum shadowing and magnetron sputter coating in an Oxford Cryotrans system to increase low temperature resolution of biological samples in a Hitachi field-emission Scanning Electron Microscope

1993 ◽  
Vol 51 ◽  
pp. 122-123
Author(s):  
William P. Wergin ◽  
Eric F. Erbe ◽  
Alan Robins
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Scanning Electron Microscope ◽  
Field Emission ◽  
High Vacuum ◽  
Water Ice ◽  
Temperature Observation ◽  
Sputter Coating ◽  
Magnetron Sputter ◽  
Scanning Electron

Previous studies in this laboratory have shown that the resolution of biological specimens could be increased at least two fold in a conventional as well as a field emission SEM by substituting high vacuum evaporation of Pt for standard sputter coating. Because the EMscope SP2000A Sputter Cryo System and the Oxford CT 1500 Cryotrans System, which were used in these experiments, employed standard sputter coating, Pt shadowing and C evaporation were carried out in a modified Denton DFE-3 freeze-etch module on a DV-503 high vacuum evaporator and the coated specimens were transferred to the cryostage (EMscope) or the prechamber (Oxford) of the cryosystem. Not only did this procedure require a high vacuum evaporator but as a result of a through air transfer into LN2, considerable contamination condensed on the surface of the specimen. Most of this contamination consisted of water ice that could be easily sublimed; however, other unidentifiable contaminants remained. To increase the versatility of the cryosystem, reduce surface contamination of the specimen and evaluate alternative coating procedures, Oxford Cryotrans Systems were equipped and tested with a Pt evaporator and a high resolution magnetron sputter head. Low temperature observation and evaluation of the coated specimens were performed in a Hitachi S4100 field emission scanning electron microscope.

Versatile and Inexpensive Specimen Holder for High Angle Azimuth Rotation in a Low Temperature Scanning Electron Microscope

2001 ◽  
Vol 7 (S2) ◽  
pp. 718-719
Author(s):  
William P. Wergin ◽  
Eric F. Erbe ◽  
Ronald Ochoa
Keyword(s):  
Low Temperature ◽  
Chemical Fixation ◽  
High Angle ◽  
Limited Sample ◽  
Specimen Holder ◽  
Critical Point Drying ◽  
Temperature Scanning ◽  
The Cost ◽  
Scanning Electron ◽  
Transfer Device

Low temperature scanning electron microscopy (LTSEM) avoids many of the artifacts associated with chemical fixation, dehydration and critical point drying. As a result, this technique has had numerous applications ranging from biology to hydrology. Unfortunately, experiments using LTSEM are frequently compromised by the limited sample manipulations that are available on most cryo-stages and by the cost of specimen holders. Whereas the conventional SEM stage enables 360° rotation and tilts as great as 90°, the braids or hoses required for cooling a cryo-stage generally limit rotation to about 30° and tilt to less than 40° from normal. Furthermore, the specimen holders for LTSEM may cost several hundred dollars each, thereby prohibiting experiments that require numerous holders. This study describes an inexpensive specimen holder that solves these problems.Figure la illustrates a drawing of the specimen holder supplied with the standard transfer device from the Oxford CT 1500 Cryotrans System.

Obtaining complementary images with sputter coating and high-vacuum evaporation in an field emission SEM equipped with a cryostage

1991 ◽  
Vol 49 ◽  
pp. 74-75
Author(s):  
William P. Wergin ◽  
Eric F. Erbe ◽  
Deryck J. Mills
Keyword(s):  
Low Temperature ◽  
Field Emission ◽  
High Vacuum ◽  
Vacuum Evaporation ◽  
Yeast Suspension ◽  
Cold Stage ◽  
Sputter Coating ◽  
Transfer Device

An Oxford CT 1500 Cryotrans System was mounted on a Hitachi S-4000 FESEM to perform low temperature manipulations and observations. The sample, consisting of a yeast suspension, was frozen in each of six hinged gold specimen holders, which were clamped onto a complementary freeze-etch specimen cap. The cap was mounted on a precooled modified Oxford holder (Fig. 1), and transferred to the dedicated cryochamber and cryostage where the yeast was fractured, etched and sputter coated with Pt. A second sample, mounted in the same type of gold holders, was frozen, fractured, etched, shadowed with platinum and coated with carbon in a Denton DV-503 high vacuum evaporator equipped with a modified DFE-2 freeze-etch module.1 The standard Oxford specimen transfer device was used to insert the holder through the cryochamber and onto the cold stage in the microscope.

A Base Specific Single Heavy Atom Stain for Electron Microscopy

1972 ◽  
Vol 30 ◽  
pp. 184-185
Author(s):  
J. P. Langmore ◽  
N. R. Cozzarelli ◽  
A. V. Crewe
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Elastic Scattering ◽  
Heavy Atom ◽  
High Vacuum ◽  
Heavy Atoms ◽  
Large Movement ◽  
Base Sequencing ◽  
Scanning Electron

A system has been developed to allow highly specific derivatization of the thymine bases of DNA with mercurial compounds wich should be visible in the high resolution scanning electron microscope. Three problems must be completely solved before this staining system will be useful for base sequencing by electron microscopy: 1) the staining must be shown to be highly specific for one base, 2) the stained DNA must remain intact in a high vacuum on a thin support film suitable for microscopy, 3) the arrangement of heavy atoms on the DNA must be determined by the elastic scattering of electrons in the microscope without loss or large movement of heavy atoms.

Universal ESEM

1993 ◽  
Vol 51 ◽  
pp. 786-787
Author(s):  
G.D. Danilatos
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
High Vacuum ◽  
Natural Extension ◽  
Necessary Condition ◽  
Environmental Scanning ◽  
Detection Systems ◽  
Small Gain ◽  
Detection Medium ◽  
Scanning Electron

The environmental scanning electron microscope (ESEM) has evolved as the natural extension of the scanning electron microscope (SEM), both historically and technologically. ESEM allows the introduction of a gaseous environment in the specimen chamber, whereas SEM operates in vacuum. One of the detection systems in ESEM, namely, the gaseous detection device (GDD) is based on the presence of gas as a detection medium. This might be interpreted as a necessary condition for the ESEM to remain operational and, hence, one might have to change instruments for operation at low or high vacuum. Initially, we may maintain the presence of a conventional secondary electron (E-T) detector in a "stand-by" position to switch on when the vacuum becomes satisfactory for its operation. However, the "rough" or "low vacuum" range of pressure may still be considered as inaccessible by both the GDD and the E-T detector, because the former has presumably very small gain and the latter still breaks down.

Mounting an individual submicrometer sized single crystal

1996 ◽  
Vol 211 (6) ◽  
Author(s):  
R. B. Neder ◽  
M. Burghammer ◽  
Th. Grasl ◽  
H. Schulz
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Single Crystal ◽  
Single Crystals ◽  
High Vacuum ◽  
Nanometer Resolution ◽  
Micro Manipulator ◽  
Scanning Electron

AbstractWe developed a new micro manipulator for mounting individual sub-micrometer sized single crystals within a scanning electron microscope. The translations are realized via a commercially available piezomicroscope, adapted for high vacuum usage and realize nanometer resolution. With this novel instrument it is routinely possible to mount individual single crystals with sizes down to 0.1

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