Excellent Structural Preservation Research Articles

Laser‐Assisted Direct Ink Writing for High‐Fidelity Fabrication of Elastomeric Complex Structures

AbstractPolydimethylsiloxane (PDMS) is extensively applied in the 3D printing field due to its excellent biocompatibility, flexibility, and gas permeability. However, as a thermal curing material, its low strength before curing inevitably causes structural deformation during the printing process and deteriorates the shape accuracy. In this paper, an innovative method, the infrared laser‐assisted direct ink writing (DIW) process, is developed to realize the in situ preheating and curing of silicone materials to improve the strength of PDMS filaments during processing. Therefore, the large‐span spatial structure with high‐fidelity morphology can be produced. First, the feasibility is validated that temperature ascension of the voxels arising from the irradiation energy of the infrared laser can accelerate the cross‐linking reaction and realize the precuring of PDMS; then, the relationship between the parameters of laser irradiation and the processing performance of 3D printing is systematically investigated to find the optimization. Finally, experiments are conducted, including the single silicone filament, silicone foams, and complex 3D features. Their excellent structural preservation proves the proposed method is practical to fabricate the high‐fidelity fabrication of complex structures such as large spans and thin wall features, which will help to broaden the 3D printing capabilities of silicone in the development of flexible electronics and soft robots.

Shale gas accumulation patterns in China

Although significant progress has been made in the geological theory and exploration and development of shale gas in China, the research on the geological particularity and enrichment regularity associated with shale gas accumulation remains weak. With this in mind, we analyzed the shale gas accumulation patterns in China. The findings show that: (1) A variety of organic-rich shales were deposited in the continental margin-type marine and transitional environments during the Pre-Mesozoic period and the basin-type terrestrial environment during the Mesozoic and Cenozoic eras. (2) The basins dominated by Mesozoic and Cenozoic terrestrial sediments superimpose on the Pre-Mesozoic continental margin-type marine–or transitional-facies sediments. The former developed the terrestrial shales with large thickness, high organic matter abundance, new age, and low thermal maturity; meanwhile, the deep parts of the basins are favorable for shale gas enrichment. However, the latter preserves the shale with old age, high thermal maturity, and undergoing multiphase tectonic movements; and the tectonically stable regions are favorable for shale gas enrichment. (3) The aulacogens mainly formed in the Meso-Neoproterozoic, Sinian-Ordovician, and Devon-Triassic periods are favorable for marine organic-rich shale deposition; moreover, this type of shale and other types including transitional facies shales with favorable reservoir-forming assemblages, coal-measure shales in small and medium-sized basins, and low-medium mature shales affected by magma intrusion, have jointly constituted the various shale gas accumulation patterns in China. (4) Based on the drilling discoveries in China, shale gas accumulation patterns can be categorized into two types: tectonic-controlled and stratigraphic-controlled. The former is mainly related to structural features, whereas the latter is mainly related to the variation of lithology and strata. (5) Shale development and gas content are mainly affected and controlled by geological factors including sedimentation, diagenesis, and later tectonic movements. Favorable sedimentary environments, appropriate thermal maturity, good sealing capacity of caprock, and excellent structural preservation are the necessary conditions for shale gas enrichment.

An improved procedure for low‐temperature embedding of high‐pressure frozen and freeze‐substituted plant tissues resulting in excellent structural preservation and contrast

Here we describe refinements in the processing of high-pressure frozen samples of delicate plant tissues for immuno-electron microscopy. These involve: shortened freeze-substitution schedules, lower temperatures during processing and polymerisation, the avoidance of temperature fluctuations and the optimisation of heat transfer from the specimens using small disposable aluminium containers. The application of these modifications leads to very good structural preservation and selective membrane contrast. As a result, the versatility of the method is increased since not only immuno-electron microscopical studies can be performed but often the quality is also quite suitable for structural investigations.

High-pressure freezing combined with in vivo-DAB-cytochemistry: A novel approach for studies of endocytic compartments

Methods for fine structural and functional analyses of complex and dynamic cell compartments must ensure high temporal resolution together with an excellent fine structural preservation. High-pressure freezing followed by freeze-substitution, and resin embedding is state of the art but its use is limited in combination with preembedding cytochemical techniques. Here we show a new approach for the exploration of compartments of the endocytosis system, which combines high-pressure freezing with peroxidase-catalyzed cytochemistry, thus using the potencies of both synergistically. Uptake of horseradish peroxidase-labeled molecules is followed by in vivo-staining and immobilization of endocytic compartments by generation of diaminobenzidine precipitates. Subsequently, the specimens are high pressure frozen, freeze-substituted, and embedded in resin. The excellent fine structural preservation, together with the high temporal resolution, and differentiating visualization of endocytic compartments qualify the new approach for morpho-functional studies of the complex and dynamic components of the endocytosis system involved in physiologic and pathologic cellular traffic, and in routes utilized in drug targeting strategies. The distinct appearances of membranes and reactive compartments provide optimal conditions for 3D-analyses by electron tomography allowing to discern subtle details of the complex 3D-structures of endocytic compartments.

A balanced technique for preparation of specimens from pathogenicity studies for scanning electron microscopy.

This paper reports our experiences with preparing delicate biological specimens for scanning electron microscopy. Three different washing methods were evaluated: One method allowed the analysis of the location of the bacterium Mycoplasma mobile on piscine gill epithelium and the optimal evaluation of histopathologic changes caused by this microbe. These results were achieved when specimens were washed three times in a cacodylic acid buffer after completion of the in vitro infection experiment in gill explant cultures. We also found that of three different concentrations of glutaraldehyde, a fixation with a 1.5% solution was sufficient to achieve excellent structural preservation, even without using post fixation in osmium tetroxide. Furthermore, this study showed that the use of acetone-carbon dioxide in the critical point drying procedure resulted in well-preserved piscine gill epithelium and mycoplasmas. Finally, long-term storage of tissue specimens in 0.1 M cacodylic acid buffer is possible if the buffer is changed on a monthly basis to avoid growth of unwanted microorganisms, such as fungi.

Releasing the peri-neuronal net to patch-clamp neurons in adult CNS.

The extracellular matrix of adult neural tissue contains chondroitin sulphated proteogylcans that form a dense peri-neuronal net surrounding the cell body and proximal dendrites of many neuronal classes. Development of the peri-neuronal net beyond approximately postnatal day 17 obscures visualization and often access by patch electrodes to neuronal membranes with the result that patch clamp recordings are most readily obtained from early postnatal animals. We describe a technique in which the surface tension of a sucrose-based medium promotes partial dissociation of thin tissue slices from adult tissue. Surface tension spreads the tissue and loosens the peri-neuronal net from neuronal membranes within minutes and in the absence of proteolytic enzymes. Furthermore, the extent of dissociation can be controlled so as to maintain the overall slice structure and allow identification of specific cell classes. Excellent structural preservation of neurons and dendrites can be obtained and full access by patch electrodes made possible for current- or voltage-clamp recordings in tissue well beyond the development of peri-neuronal nets. We demonstrate the feasibility of using this approach through patch recordings from neurons in the brainstem and cerebellum of adult gymnotiform fish and in deep cerebellar nuclei of rats as old as 6 months.

Immunogold labelling of cryosectioned pea chloroplasts and initial localization of the proteins associated with the protein import machinery

The electron-microscopic technique for immunogold labelling of thawed cryosectioned material (K.T. Tokuyasu, 1989, Histochem J 21: 163–171) has been adapted for use with isolated chloroplasts. Percoll-purified pea (Pisum Sativum L. cv Feltham First) chloroplasts were fixed in a buffered glutaraldehyde solution and then infiltrated with a buffered solution of 10% polyvinylpyrrolidone in 2.07 M sucrose prior to freezing in liquid nitrogen and sectioning in an ultracryomicrotome. Sections were thawed, immunolabelled, and stained with ammonium molybdate in methyl cellulose on Formvar/carbon-coated Cu or Cu/Pd electron-microscope grids. Cryosectioning gave excellent structural preservation and retained antigenicity. The effectiveness of this technique in localizing proteins to their specific chloroplast compartment was assayed using antibodies raised against: (i) the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a stromal protein, (ii) the chloroplast ATP synthase (CF1), a peripheral thylakoid protein, and (iii) different envelope membrane proteins. Antibodies raised against three members of the chloroplasticouterenvelopeprotein (OEP) import machinery, a 34-kDa protein (OEP34 or IAP34), the channel-forming 75-kDa protein (OEP75 or IAP75), and the 86-kDa precursor protein receptor (OEP86 or IAP86) were tested for their localization. The previous localization of OEP86, OEP75 and OEP34 to the outer envelope by biochemical methods was confirmed by our immuno electronmicroscopic analysis. Additionally, a constituent of the chloroplastic inner envelope protein (IEP) import machinery IEP 110 (IAP 100) was clearly localized to this membrane. Therefore, cryosectioning and immunogold labelling of intact chloroplasts provides a method for studying the localization of chloroplast proteins, especially those residing in the inner and outer envelope membranes.

Ultrastructure of heat-deproteinated compact bone

Heat-deproteinated bone (calcined bone) is a natural candidate for an osteoreproductive biomaterial. Ultrastructural investigation has pointed out an important aspect of the preparation technique. Treatment of bone at temperatures exceeding 500°C causes complete disruption of the tissue architecture and the reorganization of the mineral phase into tightly packed, dense crystals. At 500°C or less the structure and distribution of the mineral phase remain unaffected, so that cross-banded ‘shadows’ of collagen fibrils are still readily observable, although collagen is no longer present in the samples. By its excellent structural preservation and natural porosity, low-temperature calcined bone seems to be a promising alternative for osteoreproduction.

Secretion of cell wall polysaccharides in Vicia root hairs

Root hairs of hairy winter vetch (Vicia villosa Roth) synthesize and secrete abundant cell wall matrix polysaccharides, making this an excellent system for the study of secretion during tip growth. Roots with newly formed hairs were preserved by cryofixation and freeze substitution. Cryofixed root hairs showed excellent structural and antigenic preservation. Ultrastructural analyses showed numerous vesicles near the tip and a concentration of Golgi bodies in the subapical region of the hair. The distribution of polygalacturonic acid and xyloglucan in the endomembrane system and cell wall were revealed by immunolabeling, using previously characterized monoclonal antibodies. De‐esterified polygalacturonic acid was present on the external surface of the cell wall, but was not detected within the cell, although chemical de‐esterification revealed abundant antigen in Golgi bodies and secretory vesicles. Methyl‐esterified polygalacturonic acid epitopes were detected within the medial and trans cisternae of Golgi bodies, in secretory vesicles, and throughout the wall, indicating that pectin is secreted in a neutral form and may then be de‐esterified in muro. Xyloglucan was also detected within the trans cisternae of Golgi bodies, secretory vesicles and throughout the cell wall. Double labeling experiments demonstrated that both polysaccharides occur simultaneously in the same Golgi bodies, and that secretory vesicles containing both polygalacturonic acid and xyloglucan deliver the polysaccharides to the cell wall at the growing tip.

Immunocytochemical localization of nicotine in developing leaves of two cultivars ofNicotiana tabacum

Improved techniques have been developed for thein situlocalization of nicotine in developing leaves ofNicotiana tabacum. Earlier studies had shown that aldehyde fixed and partially dehydrated material embedded in different hydrophillic resins at low temperatures, retained up to 50% of the nicotine but invariably at the expense of good structural preservation. Significant improvements have now been made to the methods and it is now possible to obtain excellent structural preservation with high retention of nicotine.Tobacco plants were grown in a glasshouse maintained at 23-26°C with 16H light and were well watered and fed with a high nitrogen fertilizer. The flower heads were removed from the plants as they appeared. Samples were taken from between the main veins at a distance 10mm from the edge of the mid part of the leaf at 10, 13, 16, 20, and 22 weeks from the time of seed germination which corresponded to the Juvenile, Mature, Ripe, Senescent and Old stages of leaf development.

Use of Semithin Sections Embedded in a Water-Miscible Methacrylate for Light Microscopy of Central Nerve Tissues

Semithin sections embedded in water-miscible methacrylates were used for the study of fine structures of cells and tissues in the central nervous system by light microscopy instead of the conventional paraffin sections. This method used a water-miscible methacrylate mixture consisting of 2-hydroxypropyl methacrylate (HPMA), Quetol 523 and methyl methacrylate (MMA) as an embedding medium. The mixture had a low viscosity, was easy to handle and penetrated readily and completely into the specimen, producing a homogenous block from which it was easy to make sections 1.5 microns thick. Staining could be localized far more precisely in these sections than in paraffin sections owing to the thickness of the semithin sections and to the excellent structural preservation of cellular components.

A method for quick-freezing live muscles at known instants during contraction with simultaneous recording of mechanical tension.

We have developed a quick-freezing method, using a copper block cooled with liquid helium or nitrogen, which permits us to freeze muscles without any cryoprotectant at predetermined, precisely measured points in the recorded tension time-course of a single twitch or tetanus. Our aim is to arrest structural intermediates of the cross-bridge cycle for observation in the electron microscope. Chemically stimulated, demembranated muscles as well as electrically stimulated, live muscles can be frozen on the same apparatus. Good freezing of relaxed and contracting muscles has been obtained to a depth of 10-20 microns, with excellent structural preservation after freeze-substitution.

Whole head sectioning in [ 3H] deoxyglucose mapping of auditory responses in gerbils

Stimulus-specific neuronal responses in the central auditory pathway of Psammomys obesus were studied with deoxyglucose autoradiography. Responses were revealed mainly in the inferior colliculus. With whole head sectioning and subsequent gentle freeze-drying, excellent structural preservation was achieved. With the use of doubletritiated deoxyglucose, Ultrofilm 3H and vacuum-contact exposure, a mean spatial resolution of 43 μm Full Width Half Maximum was achieved.

Method for Large-Scale Isolation of Pancreatic Islets by Tissue Culture of Fetal Rat Pancreas

Detailed studies of the maturation of stimulus-secretion coupling of the pancreatic B-cell requires a supply of isolated fetal islets, which has so far been difficult to obtain. To overcome this problem we have maintained minced and mildly collagenase-digested fetal rat pancreatic glands (21.5 days gestational age) in tissue culture to enable degeneration of the acinar part, leaving the endocrine cells in an isolated and surviving state. Indeed, after 1 wk in culture there was a complete separation between acinar and endocrine cells with the appearance of numerous discrete islets and the disappearance or dedifferentiation of the exocrine cells. Isolated islets were either free floating or attached on top of a monolayer of fibroblast-like cells. Their number after 1 wk in culture was estimated as about 90 per explanted fetal pancreas and a total yield of about 5000 isolated islets was easily achieved. Both light arid electron microscopic examinations showed an excellent structural preservation with a marked predominance of well-granulated B-cells. Numerous islets of the same weight as that measured in cultured islets of adult rats were regularly found after 1 wk in culture. The insulin concentration of the cultured fetal islets was related to the glucose concentration of the growth medium. A similar relationship was found with respect to the insulin release in response to glucose. Thus, fetal islets cultured for 8 days in growth media containing 11.1 or 22.2 mM glucose showed a marked and significant insulin response to glucose in batch-type incubations at the end of the culture period. By contrast, the glucose stimulation of insulin release was insignificant in islets cultured at 5.5 or 2.8 mM glucose. When the culture period was confined to 1 day, there were no effects of glucose on the insulin release irrespective of the glucose concentration of the growth medium. It is concluded that the present technique for tissue culture of fetal rat pancreas makes it possible to isolate substantial amounts of fetal islets predominantly composed of B-cells. The transition in vitro from a poor glucose sensitivity to an adult-type insulin response indicates that the technique can be used in further detailed studies of the molecular mechanisms involved in the growth and development of the pancreatic B-cell.

A new STEM capable of observing single heavy atoms in frozen specimens

A new Scanning Transmission Electron Microscope (STEM) has been constructed in the Biology Department of Brookhaven National Laboratory. This instrument is designed specifically for imaging single heavy atoms in frozen-hydrated biological specimens. Observation of frozen-hydrated specimens has been shown to provide excellent structural preservation and reduced damage on biological specimens. The required components for this work are: a high resolution instrument with a suitable cold stage, a specimen changer capable of introducing frozen specimens into the microscope, and a suitable preparation chamber for fast freezing and transfer to the microscope.A STEM instrument was chosen for this work because of its superiority for low dose dark field operation and single heavy atom detection. The Brookhaven STEM is similar to instruments built at the University of Chicago and at Johns Hopkins University, except for the added capability for woking with frozen specimens. A field emission gun operates at a vacuum of < 10-10 Torr to produce a beam of 10-11 Amp at 50 kV.

HYPOTHERMIC PERFUSION OF RABBIT KIDNEYS WITH SOLUTIONS CONTAINING GELATIN POLYPEPTIDES

Rabbit kidneys were perfused with a solution of extracellular electrolyte composition, made hypertonic with glucose and containing the gelatin polypeptide preparation Haemaccel (Hoechst) as the only colloid. Perfusions were carried out at 5 and 10 C for 19 hr, and function was tested by autografting. All of the kidneys perfused at the higher temperature showed immediate life-sustaining function after transplantation and contralateral nephrectomy, whereas only one graft of five perfused at the lower temperature showed any function. The suitability of the Haemaccel solution as a vehicle for introducing the cryoprotective agent glycerol was tested by perfusing kidneys for 4 hr with a solutiont containing 2% glycerol; the function of these organs was similar to that of kidneys transplanted without perfusion. Ultrastructural examination of kidneys perfused for 24 hr at 10 C showed excellent structural preservation, but measurements of water and ion contents and the penetration of marker molecules in nonmetabolizing kidneys showed 2.8% Haemaccel to be somewhat less effective than 6% bovine serum albumin in stabilizing these values. The Haemaccel perfusate is considered to be highly suitable for the introduction and removal of cryoprotective agents, and the results of hypothermic preservation by continuous perfusion are encouraging.

Improved procedures for immunoferritin labeling of ultrathin frozen sections.

In employing fixed frozen ultrathin sections as substrates for immunoferritin labeling of intracellular antigens, we have found that conventional glutaraldehyde fixation sometimes permits very little specific staining of the sections, either because it inactivates certain protein antigens, or because it renders them inaccessible to the antibody stains. We have developed several fixation procedures that are chemically milder and allow a uniform but less extensive cross-linking of the specimen. With these procedures and precautions in the handling of the more fragile frozen sections, excellent structural preservation and specific immunoferritin labeling has been achieved with several systems.

Rapid Chemical Dehydration of Mammalian Tissues for Scanning Electron Microscopy using 2 ,2-Dimethoxypropane

Muller and Jacks used 2,2-dimethoxypropane (DMP) as a dehydrating agent for biological specimens prior to embedment, ultrathin sectioning, and examination by transmission electron microscopy. Acidified DMP reacts endothermically with water to produce methanol and acetone. Muller and Jacks demonstrated excellent fine structural preservation in a variety of specimens dehydrated with DMP.We have used the same method, prior to critical point drying, to prepare specimens for scanning electron microscopy (SEM) . Portions of the trachea, small intestine, and kidney from a laboratory mouse killed by cervical dislocation were fixed for 16 hours in cold 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2.

The use of glycol methacrylate embedding for studies of chromatin distribution and fine structure in salmon spermatid nuclei.

Glycol methacrylate has been used as an embedding medium for studies of spermiogenesis in the salmon. DNA and basic proteins are shown to stain with the same specificity in thick (0.5-1.0 mu) sections of GMA-embedded salmon testes as in sections of comparably fixed, paraffin-embedded testes. Stain can be localized far more precisely in GMA sections than in paraffin sections due to the thinness of the sections and to the excellent structural preservation of nuclei. In addition, ultra-thin sections of GMA-embedded salmon testes can be observed with the electron microscope, and this permits exact correlation between nuclear fine structure and chemical composition in consecutive sections of the same nuclei.

Preparation of Frozen Hydrated Tissue Sections for X-ray Microanalysis in the Scannning Electron Microscope

THE technique of X-ray microanalysis, using a finely focused beam of electrons to excite characteristic X rays from the elements present in a specimen, is proving to be a powerful tool in the investigation of biological systems. Its main limitation has been that, in common with all electron-optical methods, the specimen must first be stabilised by removal or immobilisation of cell and interstitial water. Conventional chemical fixation techniques give excellent structural preservation, but also cause gross permeability changes in cell membranes and, together with subsequent dehydration, lead to substantial loss or redistribution of free electrolytes. Freeze drying suffers from a similar limitation, in that removal of water must inevitably redistribute electrolytes which move to the remaining structures such as cellular membranes.