Freeze-substitution Procedure Research Articles

Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding

In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.

Epoxy resin as fixative during freeze-substitution

An alternative protocol for freeze-substitution is described. Araldite/Epon embedding medium (20% in acetone) is first used as a stabilizer (as e.g., OsO 4) and then as embedding medium. The major components of the Araldite/Epon resin formulation react with proteins and lipids and provide for an excellent preservation and reasonable visualisation of the ultrastructure. The ultrastructural appearance can be deliberately influenced with the standard freeze-substitution procedure [Van Harreveld, A., Crowell, J., 1964. Electron microscopy after rapid freezing on a metal surface and substitution fixation. Anat. Rec. 149, 381–386.] using OsO 4 as stabilizing agent by protocols which degrade cytoplasmic and membrane proteins. Epoxy stabilized and embedded samples may become an important tool to get information about the effects of different reagents and protocols used in freeze-substitution. We believe that an in-depth understanding of the procedures is required to correctly interpret images and to complement studies of dynamic processes by light microscopy with reliable, highly detailed ultrastructural information. The block face of epoxy stabilized samples after ultrathin sectioning is highly suited for the analysis of the ultrastructure by AFM.

Human Cerebral Microvessel Endothelial Cell Culture as a Model System to Study the Blood–Brain Interface in Ischemic/Hypoxic Conditions

(1) Cerebral ischemia and reperfusion induce several changes on the endothelial cells at the microcirculatory level. (2) Vasogenic brain edema due to compromised blood-brain barrier, transformation of the endothelial cell surface from an anticoagulant to a procoagulant surface are important factors in the pathogenesis of ischemic stroke. (3) Release of prostaglandins, endothelin-1, complement proteins, and matrix metalloproteinase-9 by microvascular endothelial cells are other components in the complex mechanism of brain ischemia/hypoxia. (4) Ultrastructural studies documented the opened paracellular avenues in the course of vasogenic edema in different experimental models (5) Tight junctions of endothelial cells have been characterized with freeze fracture electron microscopy, and the process of transvesiculation was analyzed using rapid freeze and freeze substitution procedure before electron microscopy studies (6) In endothelial cell-culture experiments, we used rodent and later human brains. (7) Endothelial cells co-cultured with astroglia resulted in an elaborate tight junctional complex. (8) This co-culture technique becomes the basis of in vitro blood-brain barrier studies On endothelial cells of human brain origin, different regulatory factors found to be responsible for the complex mechanism of ischemic stroke.

Chloride distribution in the CA1 region of newborn and adult hippocampus by light microscopic histochemistry.

GABA, the main inhibitory neurotransmitter in the central nervous system, exerts its effect by rendering the postsynaptic GABAA receptors permeable to chloride ions. Thus, depolarizing or excitatory effects of GABA, experienced in early postnatal life or in certain regions and/or conditions of the adult brain, is thought to be associated with a reversed transmembrane chloride gradient. However, there is only limited direct information about the correlation of the actual excitatory versus inhibitory effects of GABA and the local chloride distribution. Precipitation of chloride with silver is a potential way to immobilize and visualize chloride ions in biological tissue. We examined the applicability of light microscopic histochemistry, based on trapping tissue chloride with silver ions during freeze-substitution or aldehyde fixation, to visualize the chloride distribution in hippocampal slices. The freeze-substitution procedure yielded better chloride retention while with aldehyde fixation tissue preservation was more appropriate. Both methods were qualitative only, had limited applicability to the superficial 20-30 microns of slices, but were able to demonstrate a reduced extracellular-to-intracellular chloride gradient in the CA1 pyramidal neurons of the newborn hippocampus as compared to adult animals. In the 4-aminopyridine model of epilepsy, redistribution of chloride from extracellular to intracellular space could also be demonstrated.

Imaging of subcellular structures by scanning ion microscopy and mass spectrometry. Advantage of cryofixation and freeze substitution procedure over chemical preparation

With the IMS 4F, a scanning ion microscope and mass spectrometer (SIMS), it is possible to map chemical elements with a lateral resolution of about 250 nm over a field of view of 50 x 50 microns2. Such conditions should enable the imaging of subcellular structures with constitutive ionic species such as CN-, P-, S-. The study was performed on heart and renal tissues prepared either by chemical procedure or cryofixation-freeze substitution (CF-FS) prior to embedding. Heart tissue was chosen because cardiocytes display a simple structural organization whereas the structural organization of kidney tubular cells is more complex. Whatever the preparation procedure, nuclei were easily identified due to their high P- content. The CN-, P- and S- ion images obtained on heart and renal tissues prepared by chemical procedure showed weak contrasts inside the cytoplasm so that it was difficult to recognize the organelles. After CF-FS, enhanced contrasted images allow organelle (mitochondria, myofibrils, lysosomes, vacuoles, basal lamina, etc) characterization. This work demonstrated that CF-FS is a more suitable preparation procedure than chemical method to reveal organelle structures by their chemical composition. The improvements in the imaging of these structures is an essential step to establish the correlation between the localization of a trace element (or a molecule tagged with isotopes or particular atoms) and its subcellular targets.

Imaging of subcellular structures by scanning ion microscopy and mass spectrometry. Advantage of cryofixation and freeze substitution procedure over chemical preparation

With the IMS 4F, a scanning ion microscope and mass spectrometer (SIMS), it is possible to map chemical elements with a lateral resolution of about 250 nm over a field of view of 50 × 50 μm 2. Such conditions should enable the imaging of subcellular structures with constitutive ionic species such as CN −, P −, S −. The study was performed on heart and renal tissues prepared either by chemical procedure or cryofixation-freeze substitution (CF-FS) prior to embedding. Heart tissue was chosen because cardiocytes display a simple structural organization whereas the structural organization of kidney tubular cells is more complex. Whatever the preparation procedure, nuclei were easily identified due to their high P − content. The CN −, P −, and S − ion images obtained on heart and renal tissues prepared by chemical procedure showed weak contrasts inside the cytoplasm so that it was difficult to recognize the organelles. After CF-FS, enhanced contrasted images allow organelle (mitochondria, myofibrils, lysosomes, vacuoles, basal lamina, etc) characterization. This work demonstrated that CF-FS is a more suitable preparation procedure than chemical method to reveal organelle structures by their chemical composition. The improvements in the imaging of these structures is an essential step to establish the correlation between the localization of a trace element (or a molecule tagged with isotopes or particular atoms) and its subcellular targets.

Freeze substitution after fast‐freeze fixation in preparation for immunocytochemistry

As compared to classical chemical fixation, the physical immobilization of ultrastructures by fast-freeze fixation (FFF) and the subsequent exchange of water in its solid state by freeze substitution (FS) improve the preparation procedure for immunogold labeling (IGL). FFF-FS results in a morphological preservation of unchallenged quality, as well as in a better preservation of antigenic reactivity, thus allowing remarkable precision of labeling on sections. However, FFF, particularly over a cooled metal plate, requires a heavy and expensive machine. It is not suitable for all biological specimens and in the best conditions, which remain difficult to standardize, the thickness of the well-preserved portion of the specimen does not exceed a few microns for compact tissues, and exceptionally 30-40 microns for isolated cells. The FS procedure is long and must be adjusted empirically for every new specimen and antigenic detection. The preservation of a given antigen's reactivity in the presence of fixative agents and embedding resins remains unpredictable. The action of fixative agents is different and milder in FS than when they are used classically in chemical fixation. By chance, one of the best FS procedures for the preservation of both ultrastructure and antigenicity appears to be by using acetone alone, together with a molecular sieve to improve the water exchange process. A large choice of embedding resins usually allows us to find a compromise between ultrastructural and antigenic preservation.

Ultrastructure and X-ray microanalysis of epiphyseal growth cartilage of femoral head processed by rapid-freezing and freeze-substitution.

Epiphyseal growth cartilage of the femoral head obtained from Wistar rats was investigated after fixation by a rapid-freezing and freeze-substitution. Liquid helium was used in order to achieve a fast cooling rate without ice-crystal damage during the rapid freezing. Use of the rapid-freezing and freeze-substitution procedure provided better ultrastructural preservation of the chondrocyte than conventional chemical fixation methods. This procedure allowed a more reliable approach to electron probe analysis. X-ray microanalysis of the specimens confirmed that calcium is not detected in the initial matrix vesicles as a result of the freezing process. The results suggest that calcium release from precipitates occurs in the free state without any detectable formation of hydroxyapatite at the initial stage of calcification and that calcium is not tightly bound to the matrix vesicles.

Immunogold Localization of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Developing Proplastids of Dark-Grown Wax-Rich Cells of Euglena gracilis

Wax-rich cells of Euglena gracilis Z grown in the dark without agitation were subjected to freeze-substitution procedures in electron microscopy, which gave intact images of wax accumulated as globules in the cytoplasm. The proplastids in these wax-rich cells were shown to contain no extensive internal structures. When these cells were transferred to an inorganic medium containing ammonium salt and aerated in the dark the specific activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) increased concurrently with the development of a prolamellar body and a rudimentary pyrenoid. When cell sections were labeled with antisera prepared against RuBisCO subunits followed by protein A-gold, gold particles (RuBisCO) were localized in a narrow peripheral region between the plastid envelope and the prolamellar body during an earlier phase of the dark incubation of cells. Subsequently, immunogold particles were concentrated over the rudimentary pyrenoid formed at a site adjacent to the prolamellar body, while the stroma was only slightly labeled with gold particles. It was postulated that the small subunits of RuBisCO made in the cytoplasm combined with the plastid-made large subunits to form the holoenzyme at the peripheral region near the prolamellar body before transfer to the rudimentary pyrenoid.

Freeze‐substitution and the preservation of diffusible ions

Freeze-substitution of biological material in pure acetone followed by low-temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry-cut sections shows that diffusible cellular thallium ions (T1+) of T1(+)-loaded muscle are localized at similar protein sites in freeze-substituted as in frozen-hydrated preparations. A comparison of X-ray microanalytical data obtained from freeze-dried cryosections and sections of freeze-substituted normal (potassium-containing) muscle shows that K+ ion retention in the freeze-substituted sample is highly dependent on the freeze-substitution procedure used so far, in the best case, about 67% of the cellular K+ is retained after freeze-substitution in pure acetone and low-temperature embedding. It is concluded that the macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze-substitution and low-temperature embedding.

Lowicryl resins

In the recent past the use of low temperature methods for specimen preparation have become more widespread as the limitations of conventional fixation and embedding procedures have become evident. Since conventional fixatives such as glutaraldehyde do not cause instantaneous cessation of cytoplasmic movement, there can result considerable post-fixation redistribution of cytoplasm and membrane distortions, forming such artifactual structures as lomasomes in fungi and mesosomes in bacteria. This in turn translates into a loss of fidelity between the ultrastructural image and the organization we can observe in a living cell. The deleterious effects of conventional fixation have been well documented in the literature of fungal ultrastructure and have fostered the use of freeze-substitution procedures. The advantage of employing freeze-substitution techniques to study the ultrastructure of bacteria is illustrated in Figure 1. E. coli were rapidly frozen and substituted at -80°C. These results correspond to the images seen in frozen hydrated sections (1). DNA is believed to lie in the ribosome-free region outlined in Fig. 1b.

Morphometrics of rapidly frozen goose lungs

To understand the structural basis of avian gas exchange better, we made a morphometric study of domestic and Canada goose lungs. The volume of glutaraldehyde-fixed domestic goose lungs (30 cm 3/kg body weight) was similar to that determined from silicone casts of Canada goose lungs by Duncker (33 cm 3/kg). To examine finer structures, we rapidly froze goose lungs under physiologic conditions, fixed tissue samples by a freeze substitution procedure and analyszed samples with stereological methods. From light micrographs we determined that about 55% of the lung is parabronchi in both species. Volume densities of air capillaries, blood capillaries and tissue and surface : volume ratios of these same structures were determined from electron micrographs. Our measurements agree with those from glutaraldehyde-fixed Canada goose lungs from other laboratories. Gas exchange surface area was largest in the good flier (Canada goose) but both birds had larger surface areas than comparably sized mammals. The harmonic mean blood-gas barrier thickness is smaller in both species of birds (0.3 μm) than in mammals. Thus, membrane diffusing capacities for gases should be larger in birds than in mammals. Pulmonary blood capillary transit time, as calculated from blood capillary volume and normal levels of cardiac output, are longer in birds than in mammals and should allow more time for blood-gas equilibrium. Pleats and folds were frequently observed in air and blood capillaries, suggesting that the avian lung may not be as rigid as was previously thought and that capillary volumes and surface areas may change under physiologic conditions.

Identification of additional histaminergic neurons in Aplysia: Improvement of single cell isolation techniques for in tandem physiological and chemical studies

Identifiable neurons in the marine mollusc, Aplysia californica, were used to improve isolation techniques for in tandem physiological and chemical studies of the same neuron. A previous study demonstrated that the histaminergic neurons, RC-2 and LC-2, which had been subjected to intracellular recording and staining, were particularly sensitive to a freeze-substitution procedure used in isolating neurons for subsequent chemical assay. The present report describes an improvement of the freeze-substitution procedure which permits excellent recovery of histamine in physiologically manipulated C-2 neurons. The in tandem physiological and chemical procedures developed for identified neurons were used to characterize additional histamine-containing neurons which cannot be identified by visual inspection alone. These new histaminergic neurons, designated RC-3 and LC-3, are located on the ventral surface of the cerebral ganglion and are distinguishable from other adjacent histamine-containing neurons by their monosynaptic connections with some of the same follower neurons previously described for RC-2 and LC-2. The results of this study further support the role of histamine as a multiaction neurotransmitter and demonstrate the feasibility of measuring endogenous levels of various neurotransmitters in neuronal somata subsequent to intracellular recording.

Hyphal tip cell ultrastructure of the fungus Fusarium: Improved preservation by freeze-substitution

Ultrastructure of freeze-substituted hyphal tip cells of Fusarium acuminatum was compared to that after conventional chemical fixation. As a result of improved preservation by a modified freeze-substitution procedure, we document here: (i) an extensive system of smooth membrane cisternae with electron-dense contents, including tubules and single, fenestrated cisternae; (ii) two types of apical vesicles with a homogeneously electron-dense interior; (iii) an extensive system of microtubules which extend well into the apical dome; (iv) four distinct layers in the apical “primary” cell wall, with basipetal thickening due almost exclusively to enlargement of the outer cell wall layer; (v) hexagonal microvesicles; and (vi) a smooth profile of all cellular membranes and most organelles.

PARAMETERS OF TEXTURE CHANGE IN PROCESSED FISH: PROTEIN CRYSTALLIZATION.

Processed muscle of the Sacramento blackfish (Orthodon microlepidotus) was examined for changes in crystallinity by X-ray diffraction and relative water vapor uptake, the specimens having been prepared by the freeze-substitution procedure. Although only a slight increase in crystallinity occurred on processing, both methods agreed in showing a small increase in crystallinity on freezing, a somewhat greater increase on cooking, and a still greater increase on dehydrating. Further increases in crystallinity occurred during storage of frozen and dehydrated muscles.

Fluorescent-antibody staining of exo-erythrocytic stages of Plasmodium gallinaceum

The exo-erythrocytic stages of P. gallinaceum were stained in tissues by the direct fluorescent antibody method following a freeze-substitution procedure.