Connective Tissue Ultrastructure: A Direct Comparison between Conventional Specimen Preparation and High‐Pressure Freezing/Freeze‐Substitution

The investigation of connective tissue ultrastructure has historically relied on chemical fixation to stabilize micro architecture. These fixatives are not effective in retaining many matrix molecules, including proteoglycans, and allow precipitation of many other components in subsequent processing steps. The result is a preponderance of open space between matrix components. Cells shrink considerably and a precipitation of molecules in highly concentrated regions leads to artifacts including the lamina lucida and lamina densa of basement membranes. Cryo-fixation at high pressure followed by freeze substitution seeks to immobilize tissue components within vitreous (non-crystalline) ice, which is later substituted with acetone at temperatures below the recrystalization temperature of water. Formation of large ice crystals during any part of the preparation protocol significantly distorts structure and can be recognized in the compound microscope. Recognition of smaller freezing artifacts, including microcrystalline ice, is imperative for interpretation of resulting images.

Download Full-text

The ultrastructure of the connective tissue matrix of skin and cartilage after high-pressure freezing and freeze-substitution.

Journal of Histochemistry & Cytochemistry ◽

10.1177/41.8.8331280 ◽

1993 ◽

Vol 41 (8) ◽

pp. 1141-1153 ◽

Cited By ~ 33

Author(s):

D R Keene ◽

K McDonald

Keyword(s):

High Pressure ◽

Connective Tissue ◽

Freeze Substitution ◽

Collagen Fibrils ◽

High Pressure Freezing ◽

Dermal Matrix ◽

Dense Granules ◽

Dermal Collagen ◽

Connective Tissue Matrix ◽

Tissue Matrix

Studies designed to investigate the ultrastructure of the connective tissue matrix have historically relied on chemical fixatives to stabilize tissue microarchitecture. However, conventional fixatives are not completely effective in retaining many matrix constituents, including proteoglycans. Fixative recipes have been modified to include agents that retain proteoglycans, but they precipitate the glycosaminoglycan moiety into electron-dense granules and therefore do not preserve native microstructure. To avoid the structural artifacts introduced by aqueous fixatives, we prepared cartilage and skin by a cryostabilization procedure that included high-pressure freezing and freeze-substitution. Although similar approaches have been applied previously for study of connective tissue, our results and interpretation of matrix structure are significantly dissimilar. In optimally preserved areas of cartilage, collagen fibrils are continually surrounded by a densely staining sol. Empty fluid spaces are absent. In less optimally preserved areas, artifacts are noted and described, including a network that mimics the expected structure of proteoglycan. Similarly, the dermal matrix of human skin contains a preponderance of densely staining material that almost fills the voids commonly seen after aqueous fixation. Decorin, immunolocalized to the surface of dermal collagen fibrils, appears to be retained after this procedure.

Download Full-text

High-pressure freezing/freeze substitution of plant cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084600 ◽

1991 ◽

Vol 49 ◽

pp. 60-61

Author(s):

J.Z. Kiss ◽

L.A. Staehelin

Keyword(s):

High Pressure ◽

Biological Samples ◽

Cell Structure ◽

Plant Cells ◽

Freeze Substitution ◽

Specimen Preparation ◽

High Pressure Freezing ◽

New Information ◽

Cold Metal ◽

And Function

Electron microscopy of chemically fixed plant tissues has lead to important insights into the relationship between structure and function of plant cells. However, the slow rate of chemical fixation (seconds to minutes) potentially permits numerous artifacts to be induced. Most of these limitations ofs chemical fixatives can be overcome by the use of cryofixation techniques since cell structure is stabilized rapidly (milliseconds). Several types of cryofixation techniques have been developed such as cold metal block freezing and propane jet freezing. Although application of these techniques has yielded exciting new information, they are limiting in that specimens can be preserved only to a relatively shallow depth (approx. 40 μm). In contrast, under optimal conditions, high pressure freezing (HPF) at 2100 bar can produce excellent freezing of biological samples up to 600 μm in thickness. Since a commercial HPF apparatus has only recently become available, the number of systematic structural studies of biological samples utilizing HPF is still rather limited, and basic questions concerning specimen preparation and processing, HPF artifacts, and interpretation of images need to be addressed.

Download Full-text

High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084624 ◽

1991 ◽

Vol 49 ◽

pp. 64-65

Author(s):

Marek Malecki ◽

James Pawley ◽

Hans Ris

Keyword(s):

Electron Microscopy ◽

High Pressure ◽

Low Voltage ◽

Culture Media ◽

Cell Structure ◽

Freeze Substitution ◽

Ice Crystal ◽

High Pressure Freezing ◽

Cell Culture Media ◽

Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

Download Full-text

High-pressure freezing and freeze substitution of plant tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124148 ◽

1992 ◽

Vol 50 (1) ◽

pp. 748-749

Author(s):

William P. Sharp ◽

Robert W. Roberson

Keyword(s):

High Pressure ◽

Cell Structure ◽

Leaf Tissue ◽

Freeze Substitution ◽

Crystal Formation ◽

Ice Crystal ◽

High Pressure Freezing ◽

Cell Components ◽

Cold Metal ◽

Brome Grass

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.

Download Full-text

High-pressure freezing and freeze substitution of teliospores of the rust fungus Gymnosporangium clavipes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161011 ◽

1990 ◽

Vol 48 (3) ◽

pp. 692-693

Author(s):

Robert W. Roberson

Keyword(s):

High Pressure ◽

Room Temperature ◽

Pathogenic Fungus ◽

Rust Fungus ◽

Freeze Substitution ◽

Ice Crystals ◽

High Pressure Freezing ◽

Thin Walled Structures ◽

Cytological Changes ◽

Dextran Solution

The use of cryo-techniques for the preparation of biological specimens in electron microscopy has led to superior preservation of ultrastructural detail. Although these techniques have obvious advantages, a critical limitation is that only 10-40 μm thick cells and tissue layers can be frozen without the formation of distorting ice crystals. However, thicker samples (600 μm) may be frozen well by rapid freezing under high-pressure (2,100 bar). To date, most work using cryo-techniques on fungi have been confined to examining small, thin-walled structures. High-pressure freezing and freeze substitution are used here to analysis pre-germination stages of specialized, sexual spores (teliospores) of the plant pathogenic fungus Gymnosporangium clavipes C & P.Dormant teliospores were incubated in drops of water at room temperature (25°C) to break dormancy and stimulate germination. Spores were collected at approximately 30 min intervals after hydration so that early cytological changes associated with spore germination could be monitored. Prior to high-pressure freezing, the samples were incubated for 5-10 min in a 20% dextran solution for added cryoprotection during freezing. Forty to 50 spores were placed in specimen cups and holders and immediately frozen at high pressure using the Balzers HPM 010 apparatus.

Download Full-text

PRE-MEETING SHORT COURSES: JULY 31

Microscopy and Microanalysis ◽

10.1017/s1431927605150302 ◽

2005 ◽

Vol 11 (I1) ◽

pp. 23-26

Keyword(s):

Freeze Substitution ◽

Specimen Preparation ◽

Digital Microscopy ◽

High Pressure Freezing ◽

Physical Sciences ◽

Special Events ◽

Nanoscale Imaging ◽

Art Exhibit ◽

Short Courses ◽

Full Day

Organizers: John Mansfield and Louis KerrAdditional fees required.SC01: Towards Nanoscale Imaging of Anything in VPSEM (including ESEM): From Basics to Current Practices. Full Day: 9:00 AM–5:00 PM, Room 317A.SC02: Image Processing and Analysis. Full Day: 9:00 AM–5:00 PM, Room 317B.SC03: Photoshop for Microscopy and Microanalysis. Full Day: 9:00 AM–5:00 PM, Room 318A.SC04: High Pressure Freezing Cryosectioning of Vitrified Samples for Tomography, and Freeze Substitution. Full Day: 9:00 AM–5:00 PM, Room 318B.SC05: Specimen Preparation for the Physical Sciences. Full Day: 9:00 AM–5:00 PM, Room 319A.SC06: Digital Microscopy and Image Analysis for Materials Characterization. Half Day: 9:00 AM–1:00 PM, Room 319B.SC07: Interpretation of Microstructure. Half-Day: 9:00 AM–1:00 PM, Room 323A.Special Events: Presidential happenings; IMS Henry Clifton Sorby award and lecture; and Art Exhibit.Educational Venues: Microscopic explorations: A workshop; and It's a Family Affair!

Download Full-text