Freeze-fracture replication of organized tissue without cryoprotection.
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Fresh pieces of rat liver and pancreas were rapidly frozen without prior chemical fixation or cryoprotection, and replicated folloing freeze-fracture. Replicas revealed small peripheral areas free of ice crystals or damage and, within such areas, general ultrastructural morphology was essentially similar to that seen in conventionally processed material. On fracture faces of plasma and nuclear membranes a population of less prominent particles in addition to conventional membrane-associated particles was seen, and smooth areas devoid of particles of any type were seen on some nuclear membranes. These smooth areas did not appear to be similar to smooth areas allegedly arising as artifacts of conventional processing. Tight junctions and gap junctions appeared as they do in cryoprotected specimens. The results provide a base-line for assessing the possible effects of processing steps or agents on the ultrastructure of organized tissues as revealed in freeze-fracture replicas. (+info)
Rough surfaced smooth endoplasmic reticulum in rat and mouse cerebellar Purkinje cells visualized by quick-freezing techniques.
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The in vivo structure of the smooth endoplasmic reticulum (ER) was visualized in rat and mouse cerebellar Purkinje cells by using quick-freezing techniques followed by freeze-substitution for ultrathin-sectioning or freeze-fracturing and deep-etching for replicas. High magnification electron microscopy of the ultrathin sections revealed a surprising finding that all the smooth ER are apparently rough surfaced, and heavily studded with a large number of small dense projections. In the soma the smooth ER appears to be similar to its rough counterpart, except that the projections are slightly smaller, less electron dense and less protrusive on the ER membranes than the ribosomes. The projections were short rectangles, 20 x 20 x 6 nm3 in size, covering the cytoplasmic surface of the smooth ER in a checker-board manner where closely packed. After freeze-etching and replication, they appeared to be composed of four subparticles, surrounding a central channel. Thus the projections are very similar to the foot structure (ryanodine receptor) of the sarcoplasmic reticulum. Furthermore, they were distributed exclusively in the ER compartment and were highly concentrated especially in the smooth ER. This localization of the projections coindides with the intracellular distribution of the inositol 1,4,5-trisphosphate (IP3) receptor determined by quantitative immunogold electron microscopy. These findings would suggest that the projections are tetramers of IP3 receptor molecules and could be used as a morphological marker for the smooth ER in Purkinje cells, which spreads from the soma to the axon and dendrite, up to the tips including the spines. In Purkinje cells tubular smooth ER runs freely in a serpentine fashion or are intertwined to make large membraneous tangles without forming cisternal stacks. It is highly probable that the ER cisternal stacks do not exist naturally in Purkinje cells but are formed artificially during the various procedures for chemical fixation. (+info)
The development of M cells in Peyer's patches is restricted to specialized dome-associated crypts.
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It is controversial whether the membranous (M) cells of the Peyer's patches represent a separate cell line or develop from enterocytes under the influence of lymphocytes on the domes. To answer this question, the crypts that produce the dome epithelial cells were studied and the distribution of M cells over the domes was determined in mice. The Ulex europaeus agglutinin was used to detect M cells in mouse Peyer's patches. Confocal microscopy with lectin-gold labeling on ultrathin sections, scanning electron microscopy, and laminin immuno-histochemistry were combined to characterize the cellular composition and the structure of the dome-associated crypts and the dome epithelium. In addition, the sites of lymphocyte invasion into the dome epithelium were studied after removal of the epithelium using scanning electron microscopy. The domes of Peyer's patches were supplied with epithelial cells that derived from two types of crypt: specialized dome-associated crypts and ordinary crypts differing not only in shape, size, and cellular composition but also in the presence of M cell precursors. When epithelial cells derived from ordinary crypts entered the domes, they formed converging radial strips devoid of M cells. In contrast to the M cells, the sites where lymphocytes invaded the dome epithelium were not arranged in radial strips, but randomly distributed over the domes. M cell development is restricted to specialized dome-associated crypts. Only dome epithelial cells that derive from these specialized crypts differentiate into M cells. It is concluded that M cells represent a separate cell line that is induced in the dome-associated crypts by still unknown, probably diffusible lymphoid factors. (+info)
Senescence-associated beta-galactosidase histochemistry for the primate eye.
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PURPOSE: To develop a senescence-associated beta-galactosidase histochemistry and bleaching protocol for the primate posterior pole. METHODS: Rhesus monkey eyes of different ages were enucleated after death, fixed in 4% paraformaldehyde for up to 16 hours, and cryoprotected using a graded sucrose infiltration technique. Ten-micrometer tissue sections were treated with beta-galactosidase, pH 4 (lysosomal) or pH 6 (senescence-associated) activity, for various times. Bleaching of retinal pigment epithelial (RPE) cell and choroidal melanocyte pigment was performed after beta-galactosidase histochemistry using 0.1% to 1% potassium permanganate incubation for 1 minute to 2 hours followed by 0.5% oxalic acid immersion. RESULTS: A 6-hour incubation with beta-galactosidase, pH 4 or 6, demonstrated optimal staining of the RPE. Uniform staining of the RPE for pH 4 beta-galactosidase was seen in both young and old eyes. In contrast, senescence-associated beta-galactosidase (pH 6) staining was seen in the RPE of 16 and 29-year-old, but not 1- and 2-year-old eyes. Senescence-associated beta-galactosidase staining was evident in RPE cells adjacent to cuticular drusen. Optimal bleaching without loss of beta-galactosidase staining was obtained using a 25-minute incubation with 0.05% permanganate. CONCLUSIONS: The senescence-associated beta-galactosidase histochemistry assay, adapted for use in the primate posterior pole, showed staining of RPE cells in older eyes. Visualization of beta-galactosidase activity in the RPE was enhanced by permanganate bleaching of melanin pigment. This technique could be valuable for identifying senescent RPE cells in human eyes. (+info)
Confocal microscopy in biomedical research.
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Confocal microscopy has allowed a major advance in biological imaging, since it represents a rapid, cost effective means of ecamining thick tissue specimens. In most cases, this involves fluorescence imaging and it is increasingly being used as a basic tool in biomedical research. Confocal microscopy allows the collection of thin optical sections, without the need for physical sectioning of the tissue. Additionally, confocal microscopes can usually produce images with greater sensitivity, contrast and resolution than those produced with normal light microscopes. We attempt to explain how this technology might be better used as a routine research tool. Since high quality, in-focus optical sections of thick tissue preparations can be generated quickly, confocal microscopy, in combination with immunofluorescence histochemistry, can now be used to examine complex three-dimensional distributions of distinct structures within tissues such as nerves within airways. Additionally, ultraviolet confocal microscopy allows the assessment of both dynamic and static phenomena in living cells and tissues. Thus, in addition to the imaging of fluorescence associated with structural elements, confocal microscopes can be used to quantitatively evaluate the distribution and fluxes of intracellular ions like calcium. Rapid, line-scanning confocal microscopes can be used in the assessment of dynamic events. For example, the in vivo imaging of microvascular permeability in airways becomes possible for the first time. By providing examples of some of our uses for confocal microscopy, we might encourage others to explore this relatively new and important texhnology for examining events and structures in single cells, tissue samples and in intact animals. (+info)
Application of the fractionator and vertical slices to estimate total capillary length in skeletal muscle.
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A new stereological method is proposed which combines vertical slice projections with the fractionator to estimate the total capillary length in a skeletal muscle. The method was demonstrated on the soleus muscle of a Wistar rat. The implementation required capillary highlighting, tissue sampling, and data acquisition in the form of intersection counts between capillary projections and cycloid test lines. The capillaries were demonstrated using vascular perfusion (with gelatine) of the hind leg of the rat. The sampling procedure followed the fractionator design, namely a multistage systematic sampling design with a known sampling fraction at each stage. To make the design unbiased, vertical slices were used; for efficiency, the vertical axis was chosen parallel to the main axis of the muscle. As prescribed to avoid bias, the cycloid test lines were superimposed on the slice projections, viewed under the light microscope, with their minor axes normal to the vertical axis. The estimation precision was compared for different sampling and subsampling fractions. The proposed method was globally highly efficient, unbiased, and easy to implement. (+info)
Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane.
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Transport intermediates (TIs) have a central role in intracellular traffic, and much effort has been directed towards defining their molecular organization. Unfortunately, major uncertainties remain regarding their true structure in living cells. To address this question, we have developed an approach based on the combination of the green fluorescent protein technology and correlative light-electron microscopy, by which it is possible to monitor an individual carrier in vivo and then take a picture of its ultrastructure at any moment of its life-cycle. We have applied this technique to define the structure of TIs operating from the Golgi apparatus to the plasma membrane, whose in vivo dynamics have been characterized recently by light microscopy. We find that these carriers are large (ranging from 0.3-1.7 microm in maximum diameter, nearly half the size of a Golgi cisterna), comprise almost exclusively tubular-saccular structures, and fuse directly with the plasma membrane, sometimes minutes after docking to the fusion site. (+info)
The morphology of heart development in Xenopus laevis.
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We have used serial histological sections to document heart formation in Xenopus laevis, from the formation of a linear heart tube to the appearance of morphologically distinct atrial and ventricular chambers. 3D reconstruction techniques have been used to derive accurate models from digital images, revealing the morphological changes that accompany heart differentiation. To demonstrate the utility of this approach in analysing cardiac gene expression, we have reexamined the distribution of Hand1 transcripts in the linear and looped heart tube. Our results demonstrate that prior to looping, an initial asymmetric, left-sided pattern is replaced by more symmetrical localisation of transcripts to the ventral portion of the myocardium. After the onset of looping, Hand1 expression is restricted to the ventral ventricular myocardium and extends along the entire length of the single ventricle. (+info)