A Versatile High-Vacuum Cryo-Transfer for Cryo-FESEM, Cryo-SPM and other Imaging Techniques

1999 ◽  
Vol 5 (S2) ◽  
pp. 424-425
Author(s):  
Martin Ritter ◽  
Didier Henry ◽  
Stefan Wiesner ◽  
Stephan Pfeiffer ◽  
Roger Wepf
Keyword(s):  
Structural Integrity ◽  
Imaging Techniques ◽  
High Vacuum ◽  
Specimen Preparation ◽  
Ice Crystal ◽  
Structural Water ◽  
Imaging Device ◽  
The Past ◽  
Cold Stage ◽  
Immobilization Techniques

A structure preservation of biological and organic samples, close to native state, can only be reached by cryo immobilization techniques. Cryo immobilization allows not only to preserve the high structural integrity but also to arrest dynamic processes in the μs- ms range.After freezing the sample and preparing the surface of interest, it is important to prevent the sample from ice crystal damage, removal of structural water, condensation of water or other contaminants until imaging. Therefore, ideally the samples are kept below the recrystallisation temperature of water (< 147K) during the transfer from the preparation environment into the imaging chamber.For the transfer of frozen samples several concepts have been followed in the,past: a) the specimen after manipulation/preparation were submersed in liquid nitrogen and transferred to the cold stage of the microscope or b) a preparation chamber was permanently attached to the microscope column allowing the direct transfers between the preparation chamber and the cold stage in the microscope. These concepts allow either a high grade of flexibility combined with a high risk of contamination or to prevent contamination but combined with inflexibility. In addition the later also does not allow using the microscope during the specimen preparation procedure, nor transferring the specimen to an other imaging device.

Specimen preparation, cryotransfer and microscope parameters for cold-stage work in SEM and application for the physical and biological sciences

1986 ◽  
Vol 44 ◽  
pp. 898-901
Author(s):  
J.A. Sargent
Keyword(s):  
Biological Sciences ◽  
Freeze Drying ◽  
Specimen Preparation ◽  
Ice Crystal ◽  
X Ray ◽  
Cold Stage ◽  
Critical Point Drying ◽  
Cryo Preservation ◽  
Precise Interpretation ◽  
Soluble Components

The availability of equipment which enables frozen-hydrated material to be viewed over prolonged periods in the SEM has greatly extended the versatility of the instrument and permitted more precise interpretation of the data obtained from it. Artifacts associated with chemical fixation are avoided and soluble components are retained which might otherwise be removed by solvents used for dehydration or critical point drying. In addition the shrinkage which frequently occurs during freeze drying is avoided. Two further advantages of cryo preservation can be invaluable: rapid cooling of motile specimens instantly arrests movement and the immobilization of diffusible compounds and ions enables X-ray microanalysis to be performed with confidence.The speed with which specimens are cooled is not critical if only surface features are to be studied. However, the examination of internal structure demands that ice crystal growth be minimised. Devices are available to maximise cooling rates using a variety of cryogens and specimen transfer to the SEM cold stage is achieved simply and effectively.

SEM evaluation of the TEM cold-stage double-film specimen

1984 ◽  
Vol 42 ◽  
pp. 272-273
Author(s):  
Jayesh Bellare
Keyword(s):  
Spatial Distribution ◽  
Sample Preparation ◽  
Sample Thickness ◽  
Specimen Preparation ◽  
Preparation Technique ◽  
Liquid Sample ◽  
Thin Specimen ◽  
Sample Preparation Technique ◽  
Cold Stage ◽  
Film Specimen

Seeing is believing, but only after the sample preparation technique has received a systematic study and a full record is made of the treatment the sample gets.For microstructured liquids and suspensions, fast-freeze thermal fixation and cold-stage microscopy is perhaps the least artifact-laden technique. In the double-film specimen preparation technique, a layer of liquid sample is trapped between 100- and 400-mesh polymer (polyimide, PI) coated grids. Blotting against filter paper drains excess liquid and provides a thin specimen, which is fast-frozen by plunging into liquid nitrogen. This frozen sandwich (Fig. 1) is mounted in a cooling holder and viewed in TEM.Though extremely promising for visualization of liquid microstructures, this double-film technique suffers from a) ireproducibility and nonuniformity of sample thickness, b) low yield of imageable grid squares and c) nonuniform spatial distribution of particulates, which results in fewer being imaged.

Electron Microscopic Observation of Biological Specimens in Their Native State by Employing Cryogenic Techniques

1972 ◽  
Vol 30 ◽  
pp. 410-411 ◽  
Author(s):  
Tokio Nei ◽  
Haruo Yotsumoto ◽  
Yoichi Hasegawa ◽  
Yuji Nagasawa
Keyword(s):  
Electron Microscopic Observation ◽  
Surface Structures ◽  
Specimen Preparation ◽  
Native State ◽  
Electron Microscopic ◽  
Biological Specimen ◽  
Specimen Holder ◽  
Cold Stage ◽  
Preparation Techniques ◽  
Scanning Electron

In order to observe biological specimens in their native state, that is, still containing their water content, various methods of specimen preparation have been used, the principal two of which are the chamber method and the freeze method.Using its recently developed cold stage for installation in the pre-evacuation chamber of a scanning electron microscope, we have succeeded in directly observing a biological specimen in its frozen state without the need for such conventional specimen preparation techniques as drying and metallic vacuum evaporation. (Echlin, too, has reported on the observation of surface structures using the same freeze method.)In the experiment referred to herein, a small sliced specimen was place in the specimen holder. After it was rapidly frozen by freon cooled with liquid nitrogen, it was inserted into the cold stage of the specimen chamber.

Mitochondrial Reconstructions Based on Serial Thick Sections and HVEM

1975 ◽  
Vol 33 ◽  
pp. 286-287
Author(s):  
Jerome J. Paulin
Keyword(s):  
Imaging Techniques ◽  
Three Dimensional ◽  
Posterior Pole ◽  
Dimensional Structure ◽  
Stereo Imaging ◽  
Thin Sections ◽  
Anatomical Features ◽  
The Past ◽  
Cellular Components ◽  
Mitochondrial Reticulum

Within the past decade it has become apparent that HVEM offers the biologist a means to explore the three-dimensional structure of cells and/or organelles. Stereo-imaging of thick sections (e.g. 0.25-10 μm) not only reveals anatomical features of cellular components, but also reduces errors of interpretation associated with overlap of structures seen in thick sections. Concomitant with stereo-imaging techniques conventional serial Sectioning methods developed with thin sections have been adopted to serial thick sections (≥ 0.25 μm). Three-dimensional reconstructions of the chondriome of several species of trypanosomatid flagellates have been made from tracings of mitochondrial profiles on cellulose acetate sheets. The sheets are flooded with acetone, gluing them together, and the model sawed from the composite and redrawn.The extensive mitochondrial reticulum can be seen in consecutive thick sections of (0.25 μm thick) Crithidia fasciculata (Figs. 1-2). Profiles of the mitochondrion are distinguishable from the anterior apex of the cell (small arrow, Fig. 1) to the posterior pole (small arrow, Fig. 2).

Studies of metaphase chromosomes in the scanning transmission x-ray microscope: Image fidelity, radiation damage and specimen preparation

1992 ◽  
Vol 50 (2) ◽  
pp. 1038-1039
Author(s):  
Shawn Williams ◽  
Xiaodong Zhang ◽  
Susan Lamm ◽  
Jack Van’t Hof
Keyword(s):  
Visible Light ◽  
Radiation Damage ◽  
Cross Section ◽  
Imaging Techniques ◽  
Dose Range ◽  
Specimen Preparation ◽  
X Rays ◽  
Metaphase Chromosomes ◽  
X Ray ◽  
Scanning Transmission

The Scanning Transmission X-ray Microscope (STXM) is well suited for investigating metaphase chromosome structure. The absorption cross-section of soft x-rays having energies between the carbon and oxygen K edges (284 - 531 eV) is 6 - 9.5 times greater for organic specimens than for water, which permits one to examine unstained, wet biological specimens with resolution superior to that attainable using visible light. The attenuation length of the x-rays is suitable for imaging micron thick specimens without sectioning. This large difference in cross-section yields good specimen contrast, so that fewer soft x-rays than electrons are required to image wet biological specimens at a given resolution. But most imaging techniques delivering better resolution than visible light produce radiation damage. Soft x-rays are known to be very effective in damaging biological specimens. The STXM is constructed to minimize specimen dose, but it is important to measure the actual damage induced as a function of dose in order to determine the dose range within which radiation damage does not compromise image quality.

An x-ray microprobe based upon a close-coupled focal-spot / glass capillary arrangement

1992 ◽  
Vol 50 (2) ◽  
pp. 1758-1759
Author(s):  
D. A. Carpenter ◽  
M. A. Taylor
Keyword(s):  
Imaging Techniques ◽  
Fluorescence Analysis ◽  
High Sensitivity ◽  
Specimen Preparation ◽  
X Rays ◽  
Glass Capillary ◽  
Close Coupling ◽  
X Ray ◽  
Point To Point ◽  
Beam Damage

The development of intense sources of x rays has led to renewed interest in the use of microbeams of x rays in x-ray fluorescence analysis. Sparks pointed out that the use of x rays as a probe offered the advantages of high sensitivity, low detection limits, low beam damage, and large penetration depths with minimal specimen preparation or perturbation. In addition, the option of air operation provided special advantages for examination of hydrated systems or for nondestructive microanalysis of large specimens.The disadvantages of synchrotron sources prompted the development of laboratory-based instrumentation with various schemes to maximize the beam flux while maintaining small point-to-point resolution. Nichols and Ryon developed a microprobe using a rotating anode source and a modified microdiffractometer. Cross and Wherry showed that by close-coupling the x-ray source, specimen, and detector, good intensities could be obtained for beam sizes between 30 and 100μm. More importantly, both groups combined specimen scanning with modern imaging techniques for rapid element mapping.

Direct STEM ADF imaging of gold on silicon (111)

1989 ◽  
Vol 47 ◽  
pp. 472-473
Author(s):  
P. Xu ◽  
E. J. Kirkland ◽  
J. Silcox
Keyword(s):  
Film Growth ◽  
Heavy Atom ◽  
High Vacuum ◽  
Dark Field ◽  
Thin Metal Film ◽  
Base Pressure ◽  
Ultra High Vacuum ◽  
Specimen Preparation ◽  
Dark Field Imaging ◽  
Wide Range

Many studies of thin metal film growth and the formation of metal-semiconductor contacts have been performed using a wide range of experimental methods. STEM annular dark field imaging could be an important complement since it may allow direct imaging of a single heavy atom on a thin silicon substrate. This would enable studies of the local atomic arrangements and defects in the initial stage of metal silicide formation.Preliminary experiments were performed in an ultra-high vacuum VG HB501A STEM with a base pressure of 1 × 10-10 mbar. An antechamber directly attached to the microscope for specimen preparation has a base pressure of 2×l0-10 mbar. A thin single crystal membrane was fabricated by anodic etching and subsequent reactive etching. The specimen was cleaned by the Shiraki method and had a very thin oxide layer left on the surface. 5 Å of gold was deposited on the specimen at room temperature from a tungsten filament coil monitored by a quartz crystal monitor.

A Biological Cryosystem for the Scanning Electron Microscope

1990 ◽  
Vol 48 (1) ◽  
pp. 414-415
Author(s):  
Zhang zhaohua ◽  
Luo Dong ◽  
Guo Yisong
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Natural State ◽  
Specimen Preparation ◽  
Cold Stage ◽  
Specimen Temperature ◽  
Successful Design ◽  
Scanning Electron

Since early 1970's the use of cold stage on SEM for observation of hydrated samples in their natural state has become more and more popular despite its high cost. Experiences gained from earlier experiments indicate that a successful design should incorporate thefollowing features:1. The specimen temperature should be below −135°C (the recrystallization point of water), lower the temperature, better the results.2. The frozen specimen, the cold block in the specimen preparation chamber, as well as the cold stage should be kept under vacuum at all times to keep them frost free.3. Different specimen preparation processes such as fracturing, coating and sublimation should be possible in one compact preparation chamber .

scholarly journals Diagnosis and Management of Thyroid Cancer

1995 ◽  
Vol 2 (2) ◽  
pp. 107327489500200
Author(s):  
Christopher L. Alexander ◽  
Roberto E. Izquierdo ◽  
James Figge ◽  
John Horton
Keyword(s):  
Thyroid Carcinoma ◽  
Radioactive Iodine ◽  
Imaging Techniques ◽  
Needle Aspiration ◽  
Differentiated Thyroid Carcinoma ◽  
The Past ◽  
Locally Recurrent ◽  
Improvement In Survival ◽  
Radioactive Iodine Ablation ◽  
Radionuclide Scanning

Thyroid carcinoma, which comprises the majority of endocrine malignancies, has a substantial annual morbidity and mortality based on age and other predisposing factors. Diagnosis of a growing thyroid nodule can be difficult, but ultrasonography, radionuclide scanning, and fine needle aspiration allow the majority of nodules to be properly characterized. Treatment of differentiated thyroid carcinoma remains controversial. Surgical resection continues to be the most important modality with long survival if the tumor is resected early. Newer imaging techniques have improved the diagnosis of locally recurrent or metastatic disease. Radioactive iodine ablation is indicated for patients with “high-risk” tumors or advanced age. Few patients respond to cytotoxic chemotherapy. In the past decade, advances in the screening and diagnosis of medullary thyroid carcinoma have led to earlier detection with improvement in survival.

Prenatal Diagnosis

1992 ◽  
Vol 13 (9) ◽  
pp. 334-342
Author(s):  
John H. DiLiberti ◽  
Mark A. Greenstein ◽  
Sally Shulman Rosengren
Keyword(s):  
Prenatal Diagnosis ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Prenatal Testing ◽  
The Past ◽  
Computerized Image Processing ◽  
The Family ◽  
Family History Assessment ◽  
Additional Explanation ◽  
Pediatric Disorders

The enormous progress witnessed in the field of prenatal diagnosis during the past two decades is likely to continue into the future. Improved imaging techniques are likely to enhance the resolution of noninvasively obtained fetal images considerably over their current excellent quality. Although this undoubtedly will be true for ultrasonography, the increased speed of magnetic resonance equipment may offer a new realm of imaging possibilities. Computerized image processing, analysis, and three-dimensional reconstructions all should make interpretation of fetal images easier and more understandable to the nonspecialist. Advances in molecular genetics will continue to accelerate, greatly expanding the range and accuracy of prenatal diagnosis. The alert pediatrician who is sensitive to genetic issues may, by early detection of pediatric disorders and careful family history assessment, be in a position to identify families at risk for serious genetic conditions and provide the opportunity to make informed decisions on reproductive options that avert a major tragedy. The pediatrician, working with obstetric colleagues, should be part of a team effort to support families going through prenatal testing. Familiarity with these rapidly changing technologies will make it far easier to support the family needing additional explanation about prenatal diagnosis issues.

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