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.

Using high-vacuum evaporation to obtain high-resolution low-temperature images of freeze-fractured membranes from yeast

1991 ◽  
Vol 49 ◽  
pp. 514-515 ◽  
Author(s):  
William P. Wergin ◽  
Eric F. Erbe
Keyword(s):  
Low Temperature ◽  
Plasma Membranes ◽  
High Vacuum ◽  
Vacuum Evaporation ◽  
Macromolecular Structure ◽  
Membrane Structures ◽  
Membrane Particles ◽  
Cold Stage ◽  
Sputter Coating ◽  
Coating Procedure

In previous low temperature studies of fractured yeast membranes that were examined in a conventional SEM, Wergin and Erbe demonstrated that resolution could be enhanced by substituting conventional sputter coating of gold with high vacuum evaporation of platinum/carbon. Although this procedure did not clearly resolve membrane particles, the frozen sample could be removed from the SEM and the Pt/C “coat” or replica could be recovered for further examination. Using instruments with greater resolution such as a TEM, as well as a field emission (FE) SEM, to examine the replica did reveal the membrane particles on the replica; therefore the authors concluded that failure to resolve these membrane structures in a conventional SEM probably resulted from limitations of the instrument rather than the coating procedure. Recently, we had the opportunity to interface a FESEM with a cryosystem; this combination was used to determine if samples shadowed in a high vacuum evaporator could be transferred to a cold stage in a FESEM to resolve the macromolecular structure of yeast plasma membranes.

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.

Multiple-fracturing and viewing of the same frozen sample at different depths using a low temperature FESEM

1996 ◽  
Vol 54 ◽  
pp. 820-821
Author(s):  
M.V. Parthasarathy ◽  
C. Daugherty
Keyword(s):  
Temperature Field ◽  
Low Temperature ◽  
Field Emission ◽  
Multiple Fracture ◽  
Precise Control ◽  
Cold Stage ◽  
Different Depths ◽  
Transfer Device

The versatility of Low Temperature Field Emission SEM (LTFESEM) for viewing frozen-hydrated biological specimens, and the high resolutions that can be obtained with such instruments have been well documented. Studies done with LTFESEM have been usually limited to the viewing of small organisms, organs, cells, and organelles, or viewing such specimens after fracturing them.We use a Hitachi 4500 FESEM equipped with a recently developed BAL-TEC SCE 020 cryopreparation/transfer device for our LTFESEM studies. The SCE 020 is similar in design to the older SCU 020 except that instead of having a dedicated stage, the SCE 020 has a detachable cold stage that mounts on to the FESEM stage when needed. Since the SCE 020 has a precisely controlled lock manipulator for transferring the specimen table from the cryopreparation chamber to the cold stage in the FESEM, and also has a motor driven microtome for precise control of specimen fracture, we have explored the feasibility of using the LTFESEM for multiple-fracture studies of the same sample.

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.

Advantages of Low Temperature SEM for Bacterial Studies

1995 ◽  
Vol 53 ◽  
pp. 1070-1071
Author(s):  
Stéphane Roy ◽  
Isabelle Babic ◽  
Alley E. Watada ◽  
William P. Wergin
Keyword(s):  
Electron Microscopy ◽  
Low Temperature ◽  
Field Emission ◽  
Taxonomic Identification ◽  
Chemical Fixation ◽  
Cold Stage ◽  
Transmission Electron ◽  
Critical Point Drying ◽  
Scanning Electron ◽  
Spinach Leaves

The application of transmission electron microscopy (TEM) has greatly increased our understanding of structure-function relationships in bacteriology. However, to achieve further advancements investigators are seeking preparation procedures that would avoid the artifacts associated with conventional chemical fixation, dehydration and critical point drying or embedding. In our laboratory a field emission scanning electron microscope (SEM) was recently equipped with a cold stage. This combination of techniques, referred to as low temperature (LT) SEM, allowed us to examine frozen, fully hydrated biological specimens. The present investigation images bacteria that were cryofixed for LTSEM observations and then freeze-substituted for TEM observations. In addition an attempt was made to culture the samples that had been cryofixed and observed with LTSEM so that taxonomic identification and further microscopic observations could be made.Bacteria used in this study were isolated from spinach leaves (variety New Jersey). LTSEM observations of cryofixed samples were performed on a Hitachi S-4100 field emission SEM equipped with an Oxford CT-1500HF Cryotrans System.

scholarly journals The distribution of autelectronic emission from single crystal metal points. I. Tungsten, molybdenum, nickel in the clean state

1940 ◽  
Vol 176 (965) ◽  
pp. 262-279 ◽  
Keyword(s):  
Crystal Structure ◽  
Angular Distribution ◽  
Low Temperature ◽  
External Field ◽  
Single Crystal ◽  
Field Emission ◽  
High Vacuum ◽  
Geometrical Shape ◽  
Point Temperature ◽  
Surface Mobility

E. Müller has shown that the angular distribution of field emission from fine metal points can be related to the crystal structure of the metal. He also suggested that the modifications in the pattern from tungsten which took place when the point temperature was raised could be attributed to a movement of the surface atoms which caused local changes in the work function. Further experiments are described in this paper using tungsten, molybdenum and nickel. It is shown that this surface mobility occurs above 1170° K for tungsten, 770° K for molybdenum, and 370° K for nickel. The changes in emission distribution, which are very much more marked for molybdenum and nickel than for tungsten, can be completely explained in terms of a change in the geometrical shape of the point. This is brought about by the action of the high external field upon the mobile surface atoms. The flash-over phenomenon in high vacuum is discussed and an explanation offered in terms of these observations. The effect of small traces of gas is illustrated, and it is shown that the gas film is removed at the relatively low temperature of 620-670° K. Several possible suggestions are put forward to explain the dependence of the emission distribution on the crystal structure of the metal, but no definite conclusion can be reached with the data at present available.

A Field Emission System For Transmission Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 298-299
Author(s):  
Michel Troyonal ◽  
Huei Pei Kuoal ◽  
Benjamin M. Siegelal
Keyword(s):  
Electron Microscope ◽  
Field Emission ◽  
Optical System ◽  
High Vacuum ◽  
Ultra High Vacuum ◽  
Objective Lens ◽  
Electron Optical System ◽  
Cross Sectional ◽  
Transmission Electron ◽  
Emission System

A field emission system for our experimental ultra high vacuum electron microscope has been designed, constructed and tested. The electron optical system is based on the prototype whose performance has already been reported. A cross-sectional schematic illustrating the field emission source, preaccelerator lens and accelerator is given in Fig. 1. This field emission system is designed to be used with an electron microscope operated at 100-150kV in the conventional transmission mode. The electron optical system used to control the imaging of the field emission beam on the specimen consists of a weak condenser lens and the pre-field of a strong objective lens. The pre-accelerator lens is an einzel lens and is operated together with the accelerator in the constant angular magnification mode (CAM).

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