A Langendorf preparation for quick-freezing small hearts

1992 ◽  
Vol 50 (1) ◽  
pp. 774-775
Author(s):  
Joachim Sommer ◽  
Teresa High ◽  
Peter Ingram ◽  
Rashid Nassar ◽  
Neal Shepherd
Keyword(s):  
Membrane Damage ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Tissue Preparation ◽  
Cell Structures ◽  
Freeze Dried ◽  
Quick Freezing ◽  
Cardiac Ultrastructure

The validity of studies of cell structures at high spatial and temporal resolution depends on the fidelity with which tissue preparation maintains in vivo conditions. Optimal preservation of structural substrates of precisely timed physiological intracellular events is offered by cryopreservation followed by freeze-fracture and freeze-substitution; we have established criteria for gauging that quality of cryopreservation in skeletal and cardiac muscle. Ciyopreservation is indispensable for electron probe x-ray microanalysis (EPXMA) of freeze-dried cryosections. We have developed a Langendorf preparation for small hearts (Figs. 1,2) suitable for use in our quick-freeze device (“Cryopress”; Med-Vac, Inc., St. Louis, MO 63117) to a) investigate the spatial distribution of physiologically important elements (e.g. calcium) during excitation-contraction coupling (ECC), especially in intact avian hearts and, b) assess damage to cardiac ultrastructure that is caused by pathological conditions (e.g. ischemia), rather than by artifacts due to chemical fixation (e.g. membrane damage by glutaraldehyde). In our Langendorf preparation, the tips of hearts can be quick-frozen at optimal freezing conditions, and comparative studies of the hearts of different animal species performed.

How to sample the entire length of a single muscle fiber quick-frozen after electrical point stimulation for high resolution EM

1992 ◽  
Vol 50 (1) ◽  
pp. 776-777
Author(s):  
Joachim R. Sommer ◽  
Teresa High ◽  
Betty Scherer ◽  
Isaiah Taylor ◽  
Rashid Nassar
Keyword(s):  
Skeletal Muscle ◽  
High Resolution ◽  
Action Potential ◽  
Muscle Fiber ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Electrical Field Stimulation ◽  
Skeletal Muscle Fiber ◽  
Freeze Dried ◽  
Quick Freezing

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.

Quick-Freezing to Catch the Membrane Changes that Occur During Exocytosis

1977 ◽  
Vol 35 ◽  
pp. 676-679
Author(s):  
J.E. Heuser
Keyword(s):  
Synaptic Vesicle ◽  
Structural Changes ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Frog Neuromuscular Junction ◽  
The Past ◽  
Synaptic Vesicle Exocytosis ◽  
Quick Freezing ◽  
Vesicle Exocytosis ◽  
Membrane Changes

The technique that we have used to capture synaptic vesicle exocytosis at the frog neuromuscular junction - that of quick-freezing muscles followed by freeze fracture (3) or freeze substitution (6) - works sufficiently well now that it may be useful in other sorts of membrane studies, or studies of fast structural changes with the electron microscope. This note reviews the quickfreezing technique we use, and describes its application to the problem of synaptic vesicle exocytosis and recycling at the synapse.Here, many of the membrane changes of interest occur during the brief delay in synaptic transmission, on a time scale of milliseconds or fractions of milliseconds, and leave only traces thereafter. In the past, we have studied these left-over traces in tissues fixed with the standard chemicals for electron microscopy (1), and have inferred from them that vesicles discharge the quanta of neurotransmitters, as the physiologists would predict.

Intercellular spaces in quick-frozen whole hearts of the frog and mouse

1986 ◽  
Vol 44 ◽  
pp. 262-263
Author(s):  
Sommer ◽  
N.R. Wallace ◽  
R. Nassar
Keyword(s):  
Electron Microscopy ◽  
Comparative Study ◽  
Freeze Fracture ◽  
High Fidelity ◽  
Ventricular Muscle ◽  
Intercellular Spaces ◽  
Cardiac Muscle Cells ◽  
Profound Influence ◽  
Quick Freezing

The geometry of cell apposition has a profound influence on certain electrophysiologic properties of aggregates of cardiac muscle cells, e.g. in bundles of frog versus mouse ventricular muscle. It should be assessed, ideally, in the absence of preparatory procedures that can be expected to change it. Since quick-freezing followed by freeze fracture eliminates all but freezing from the preparatory menue prior to electron microscopy, we have applied this technology to a comparative study of the geometry of intercellular spaces in frog and mouse hearts in an attempt to reproduce its in vivo state with high fidelity.

Quick-Freezing With High Time Resolution Of Fast Physiological Events

1985 ◽  
Vol 43 ◽  
pp. 6-9
Author(s):  
Joachim R. Sommer
Keyword(s):  
Time Scale ◽  
Biological Material ◽  
Morphological Changes ◽  
Freeze Fracture ◽  
Time Interval ◽  
High Time Resolution ◽  
Electron Microscopic ◽  
Freeze Dried ◽  
Quick Freezing ◽  
Spatial Displacements

Quick-freezing methodology has made three major contributions to our ability to relate structure to function: 1. Quick-freezing, especially when followed by freeze-fracture, is suited to study the ultrastructure of unfixed biological material as close to the native state as can possibly be obtained. 2. Physiological events associated with morphological changes at the level of electron microscopic resolution can be stopped at any desired time interval and, thus, analysed against a known time scale and, 3. Microchemical measurements, e.g. of elemental concentrations and their spatial displacements, can be obtained by electron dispersive X-ray microanalysis from freeze-dried frozen sections of quick-frozen biological material. Indeed, it is now possible to investigate, simultaneously, anatomical, physiological and microchemical parameters in a single cell, with a precise time scale thrown in for good measure.In the following I shall describe the techniques that we employ to study the morphology of single intact skeletal muscle fibers of the frog at known time intervals following electrical stimulation. The methodology is the judicious extension, including some modifications, of procedures whose efficacy is mostly uncontroversial and a matter of scientific record.

High-pressure freezing improves quantitative and qualitative structural data in the actinomycete microsymbiont frankia

1993 ◽  
Vol 51 ◽  
pp. 110-111
Author(s):  
R. Howard Berg
Keyword(s):  
High Pressure ◽  
Plant Root ◽  
Root Nodules ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
High Pressure Freezing ◽  
Void Space ◽  
En Bloc ◽  
Tem Analysis

Symbiotic plant root nodules containing the nitrogen-fixing bacterium Frankia occur on a variety of woody shrubs and trees. Ever since the first micrographs of freeze substituted cells of Frankia in culture were published there has been impetus to see if freeze substitued nodule tissue will improve imaging of Frankia in vivo. High pressure freezing/freeze substitution (HPFS) accomplishes this.Frankia is an actinomycete that fixes N2 in a specialized multicellular, spherical structure termed the “symbiotic vesicle” that is surrounded by a multilamellate envelope (MLE) comprised of lipids. Early work based on MLE birefringence suggested the MLE was a O2 diffusion barrier, thereby protecting nitrogenase from O2- inactivation. Recently this has been challenged by freeze fracture data. Traditionally it has been assumed that the MLE is electrontranslucent because the lamina of the MLE are extracted by dehydration solvents, producing the “void space”--an extraction artifact hindering TEM analysis of MLE structure.En bloc staining with chromic acid stains the MLE, showing that the MLE is present after exposure to dehydration solvents and that the void space results from tissue shrinkage in the symbiotic vesicle (Figure 1).

scholarly journals Immunoelectron microscopic localization of sarcoplasmic reticulum proteins in cryofixed, freeze-dried, and low temperature-embedded tissue.

1987 ◽  
Vol 35 (7) ◽  
pp. 723-732 ◽  
Author(s):  
A O Jorgensen ◽  
L J McGuffee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Freeze Drying ◽  
Tissue Preparation ◽  
Variable Degree ◽  
Chemical Fixation ◽  
Thin Sections ◽  
Microscopic Localization ◽  
Freeze Dried ◽  
Quick Freezing ◽  
Lowicryl K4m

Immunoelectron microscopic labeling of calsequestrin on ultra-thin sections of rat ventricular muscle prepared by quick-freezing, freeze-drying, and direct embedding in Lowicryl K4M was compared to that observed on ultra-thin sections prepared by chemical fixation, dehydration in ethanol, and embedding in Lowicryl K4M. Brightfield electron microscopic imaging of cryofixed, freeze-dried, osmicated, and Spurr-embedded rat ventricular tissue showed that the sarcoplasmic reticulum was very well preserved by cryofixation and freeze-drying. Therefore, the four structurally distinct regions of the sarcoplasmic reticulum (i.e., the network SR, the junctional SR, the corbular SR, and the cisternal SR) were easily identified even when myofibrils were less than optimally preserved. As previously shown by immunoelectron microscopic labeling of ultra-thin frozen sections of chemically fixed tissue, calsequestrin was confined to the lumen of the junctional SR and of a specialized non-junctional (corbular) SR, and was absent from the lumen of network SR in cryofixed, freeze-dried, Lowicryl-embedded myocardial tissue. In addition, a considerable amount of calsequestrin was also present in the lumen of a different specialized region of the non-junctional SR, called the cisternal sarcoplasmic reticulum. By contrast, relocation of calsequestrin to the lumen of the network SR was observed to a variable degree in chemically fixed, ethanol-dehydrated, and Lowicryl-embedded tissue. We conclude that tissue preparation by cryofixation, freeze-drying, and direct embedding in Lowicryl K4M for immunoelectron microscopic localization of diffusible proteins, such as calsequestrin, is far superior to that obtained by chemical fixation, ethanol dehydration, and embedding in Lowicryl K4M.

Effect of osmium vapor on calcium loss during sectioning of freeze-dried, embedded tissue

1986 ◽  
Vol 44 ◽  
pp. 258-259
Author(s):  
L. J. McGuffee ◽  
S. A. Little
Keyword(s):  
Smooth Muscle ◽  
Freeze Drying ◽  
Osmium Tetroxide ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Freeze Dried ◽  
Quick Freezing ◽  
Calcium Loss ◽  
Cellular Organelles

Our laboratory has been using electron microscopic autoradiography to localize 45Ca in smooth muscle. We prepare the tissue for these studies by quick freezing against a copper mirror, freeze-drying at low temperature, exposing the dry tissue to osmium tetroxide vapors in vacuo, and infiltrating and embedding in Spurr resin.Two requirements must be met before one can examine the distribution of a soluble ion, such as calcium, using this, or any morphological technique. First, morphological perservation of the tissue must be sufficient to identify cellular organelles and membranes. This requirement can be met in smooth muscle by using freeze-dried Spurr embedded tissue. Second, the distribution of calcium must be representative of the in vivo distribution.

Side bridge geometry after quick-freezing of stimulated and unstimulated frog skeletal muscle fibers

1983 ◽  
Vol 41 ◽  
pp. 464-465
Author(s):  
J.R. Sommer ◽  
R. Nassar ◽  
S. Walker
Keyword(s):  
Skeletal Muscle ◽  
Time Course ◽  
Muscle Fibers ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Frog Skeletal Muscle ◽  
Specimen Holder ◽  
Skeletal Muscle Fibers ◽  
Quick Freezing ◽  
The Impact

Quick-freezing allows the structural analysis of timed perturbations of morphology. We are presenting preliminary results concerning the feasibility of studying directly the side bridge geometry of actin-myosin interactions within the time course of a twitch in single intact frog skeletal muscle fibers, both by freeze-substitution and freeze-fracture after quick-freezing, and following various time intervals between stimulation and impact of the fibers on a liquid He-cooled copper block.Materials and Methods. The quick-freezing device was a "Slammer"(Polaron) for which the electronics had been redesigned; they are capable, in combination with a Grass S48 stimulator, of any stimulation interval between 0 and 1 sec prior to freezing, including tetanus. The actual elapsed time between stimulation and freezing is recorded with a digital clock. Single intact tendonto- tendon frog skeletal muscle fibers (semitendinosus of r. temporaria) or toe muscle bundles (r.pipiens) were isolated by sharp dissection and placed between coextensive Pt stimulation wires on blackened 2% agarose, the height of which on the specimen holder was adjusted appropriately with respect to a spacer ring both, to calibrate the impact time and to prevent smashing of the fibers.

scholarly journals Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane.

1979 ◽  
Vol 82 (1) ◽  
pp. 150-173 ◽  
Author(s):  
J E Heuser ◽  
S R Salpeter
Keyword(s):  
Acetylcholine Receptors ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Deep Etching ◽  
Geometrical Pattern ◽  
Quick Freezing ◽  
Surface Patterns

The receptor-rich postsynaptic membrane of the elasmobranch electric organ was fixed by quick-freezing and then viewed by freeze-fracture, deep-etching and rotary-replication. Traditional freeze-fracture revealed a distinct, geometrical pattern of shallow 8.5-nm bumps on the E fracture-face, similar to the lattice which has been seen before in chemically fixed material, but seen less clearly than after quick-freezing. Fracture plus deep-etching brought into view on the true outside of this membrane a similar geometrical pattern of 8.5-nm projections rising out of the membrane surface. The individual projections looked like structures that have been seen in negatively stained or deep-etched membrane fragments and have been identified as individual acetylcholine receptor molecules. The surface protrusions were twice as abundant as the large intramembrane particles that characterize the fracture faces of this membrane, which have also been considered to be receptor molecules. Particle counts have always been too low to match the estimates of postsynaptic receptor density derived from physiological and biochemical studies; counts of surface projections, however, more closely matched these estimates. Rotary-replication of quick-frozen, etched postsynaptic membranes enhanced the visibility of these surface protuberances and illustrated that they often occur in dimers, tetramers, and ordered rows. The variations in these surface patterns suggested that in vivo, receptors in the postsynaptic membrane may tend to pack into "liquid crystals" which constantly appear, flow, and disappear in the fluid environment of the membrane. Additionally, deep-etching revealed a distinct web of cytoplasmic filaments beneath the postsynaptic membrane, and revealed the basal lamina above it; and delineated possible points of contact between these structures and the membrane proper.

X-Ray Microanalysis of Human Erythrocytes Artificially Jetted Through Tubes At Different Pressures by “in vitro Cryotechnique”

2001 ◽  
Vol 7 (S2) ◽  
pp. 732-733
Author(s):  
Y. Fujii ◽  
N. Terada ◽  
T. Baba ◽  
H. Ueda ◽  
S. Ohno
Keyword(s):  
Human Erythrocyte ◽  
Morphological Changes ◽  
Freeze Substitution ◽  
Substitution Method ◽  
In Vivo Cryotechnique ◽  
Blood Capillaries ◽  
X Ray ◽  
Freeze Dried

It is well known that flowing erythrocytes in blood capillaries were morphologically changing in vivo, as observed by light microscopy. Recently, dynamic morphological changes of flowing mouse erythrocytes in large blood vessels and hepatic sinusoids were demonstrated by scanning (SEM) or transmission (TEM) electron microscopy with our “in vivo cryotechnique”. Moreover, human erythrocyte deformability was already studied under artificially jetting conditions at different pressures by using “in vivo cryotechnique”, followed by freeze-substitution method for SEM. in this study, we have analyzed elemental changes of each human erythrocyte at different jetting pressures by “in vivo cryotechnique” combined with SEM for X-ray microanalysis.Human blood was collected with heparin-coated syringes, divided into two groups and kept at 4°C and 36°C. They were directly jetted into isopentane-propane cryogen (-193°C) through tubes (21 gauge) at different pressures (0-220mmHg) (Fig.la). The frozen blood samples were freeze-dried (4-6×10-7torr,-95°C,24h) in Eiko FD-3AS apparatus (Eiko Engineering, Japan) (Fig. lb).

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