Improved cryoprotection and freeze-substitution of embryonic quail retina: A tem study on ultrastructural preservation

Conditions for cryofixation and freeze-substitution crucial to the ultrastructural preservation of embryonic quail retina were improved. As freeze-substitution makes gentle dehydration and chemical fixation of tissue possible, the suitability of different cryoprotectants were tested in the preceding cryofixation. Additionally, different conditions for chemical prefixation were studied. In cryofixation, all of the “classic” cryoprotectants caused more or less severe tissue destruction. Only dimethylformamide (DMF) and–with certain reservations–dimethylsulfoxide (DMSO) yielded improved structure preservation. Perfusion fixation with a mixture of formaldehyde/glutaraldehyde (FA/GA) was superior to GA alone. In comparison to conventional fixation and dehydration methods, freeze-substitution yielded better ultrastructural preservation of the embryos with fewer artifacts.     

Design and operation of a simple environmental chamber for rapid freezing fixation

Rapid freezing is the most important step in sample preparation for freeze-fracture and other cryotechniques for electron microscopy. We present the design and operation of a simple environmental chamber coupled to a plunger-driven freezing device that has provided simple and reliable freezing from temperatures and humidities other than ambient. The chamber can be constructed and operated with equipment and techniques common to most electron microscopy labs. Temperature control of ±0.1°C and relative humidities of >90% were provided over the range ?5–60°C. Typical electron micrographs showing well preserved structures comparable to jet-freezing are presented.     

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 (Tl⁺) of Tl⁺-loaded muscle are localized at similar protein sites in freeze-substituted as in frozen-hydrated preparations. A comparison of X-ray micro-analytical 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 retention of diffusible cellular ions is dependent on their interactions with cellular macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze-substitution and low-temperature embedding.     

Advances in ultrarapid freezing for the preservation of cellular ultrastructure

Most of our current knowledge of cellular ultrastructure is derived from studies of chemically fixed and chemically cryoprotected preparations. In the first part of this review, we document the many artifacts associated with chemical techniques that render them unsuitable for further refinement of our understanding of cellular ultrastructure. The best method currently available for the preservation of cellular ultrastructure is ultrarapid freezing. The second part of this review is a consideration of the physics of ice crystal formation in biological systems, which suggests that ice crystals will be present in any frozen, uncryoprotected specimen. We define an ultrarapidly frozen preparation as one in which the ice crystals are so small as to be invisible at the electron microscopic level. Improvements in the ease of application and reliability of ultrarapid freezing techniques have reached the point that these techniques can be used by anyone requiring the best achievable preservation of cellular ultrastructure. In the third part of this review, we describe and critique the five methods of ultrarapid freezing in current use.     

Heptane and isooctane as embedding fluids for high-pressure freezing of Petunia ovules followed by freeze-substitution

In comparison with other fixation methods, high-pressure freezing and freeze-substitution of Petunia ovules lead to improved ultrastructural preservation of all tissues. Crucial for adequate high-pressure freezing is the absence of air in the specimen sandwich; air has to be replaced by an embedding fluid. Frequently, 1-hexadecene is used for this purpose. Using 1-hexadecene as an embedding fluid resulted in only 5–10% of Petunia ovules being preserved without disturbance of the ultrastructure due to ice-crystal damage. Since 1-hexadecene is not soluble in acetone at − 90 °C, freeze-substitution is hindered when ovules remained completely surrounded by it; this results in recrystallization when the temperature is raised. We tested and compared the suitability of heptane and isooctane as embedding fluids for high-pressure freezing and freeze-substitution, reasoning that because of their low melting points and low relative densities, phase separation during freeze-substitution would result in complete exposure of the ovules to the substitution medium, leading to adequate freeze-substitution. Using either heptane or isooctane as an embedding fluid yielded up to 90% ice-crystal-free ovules. Both compounds, however, have some damaging effects on the outer one or two cell layers of the ovule, but not on the inner tissues.     

Preservation of EDTA-expanded grid-mounted chromosomes and nuclei for electron microscopy using a specially designed freeze-dryer

We describe methods for freezing and drying EDTA-expanded, fixed metaphase chromosomes and nuclei, attached to grids as whole-mounts, for transmission electron microscopy. These methods use a special apparatus that is simple to construct. While separate freezers and dryers are commercially available, one for freezing blocks of tissue by slamming them against a cold metal surface, and the other for vacuum drying the frozen tissue, our apparatus is designed for gentler, cryogenic liquid plunge freezing and drying, sequentially, in the same apparatus, thus avoiding any compression or damage to the sepcimen. Use of a cryoprotectant is not essential; however, good results are obtained more often when 20% ethanol is used. Freezing is accomplished by rapid propulsion of the grid, with specimens attached, into slushy N₂ (-210°C) within the drying chamber; drying is automatic, by either sublimation under vacuum or by solvent substitution using absolute ethanol followed by acetone, which, in turn, is removed with a critical-point dryer. The apparatus offers a means of drying chromosomes and nuclei in an expanded state, and avoids the shrinkage of these structures that occurs during stepwise passage through increasing concentrations of ethanol or acetone.     

Controlled microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography

High‐pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 μm, the tubing protects small and fragile samples within the thickness constraints of high‐pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.     

圆材矫直的理论与实践

介绍了影响圆材矫直过程的一系列因素 ,并结合实际生产过程对这些因素进行了具体分析 ,提出了应用矫直理论于实际生产中的一些方法     

The cryopuncher: A pneumatic cryofixation device for X-ray microanalysis of tissue specimens

A cryopunching device is described which allows cryofixation of tissue specimens by quick contact with a precooled copper surface during excision. The advantage of the cryopuncher for analytical electron microscopy of cells and tissues in defined functional states is illustrated by electron probe X-ray microanalysis of freeze-dried cryosections from rat liver and dogfish kidney. In comparison with results obtained from specimens plunged into liquid propane, cryopunching in situ results in similar preservation of morphology and remarkably improved intracellular K/Na ratio.     

A device for the rapid freezing of biological specimens under precisely controlled and reproducible conditions

The construction and preliminary testing of a device is described which can be used to freeze biological specimens in any cryogenic liquid at temperatures down to the nitrogen freezing point (63 K) and which can operate in the pressure range 1.3 kNm^?2 to 1 MNm^?2. Ultra-rapid freezing can be carried out in a subcooled cryogenic liquid either hyperbarically or at atmospheric pressure. Slow freezing rates can be achieved by cooling the specimens in a controlled manner in the vapour phase above the liquid bath.     

Optimal preservation of the shark retina for ultrastructural analysis: An assessment of chemical, microwave, and high-pressure freezing fixation techniques

Recent advances in microwave chemical fixation (MCF) and/or high pressure freezing (HPF) combined with transmission electron microscopy have resulted in superior ultrastructural detail in a variety of tissue types. To date, selachian tissue has been fixed and processed using only standard chemical fixation (CF) methods, and the resulting ultrastructure has been less than ideal. In this study, we compared the ultrastructure of the fragile retinal tissue from the brown banded bamboo shark, Chiloscyllium punctatum, obtained using CF, MCF, and HPF methods. For all fixation protocols, ultrastructural preservation was improved by keeping the tissue in oxygenated Ringer solution until the time of fixation. Both MCF and HPF produced superior retinal ultrastructure compared to conventional CF. Although HPF occasionally resulted in very high quality ultrastructure, microwave fixation was almost comparable, quicker and far more consistent. Microsc. Res. Tech. 75:1218–1228, 2012. © 2012 Wiley Periodicals, Inc.     

高压水射流数值模拟及混凝土路面切割试验研究

探讨了高压水射流的切割机理,并对圆形喷嘴高压水射流垂直冲击水泥混凝土建立了流体动力学模型,进行了数值模拟。通过对模拟结果的分析,得到射流喷嘴最大出口压力和量纲一冲击高度对水泥混凝土在滞止点及其附近压力值的影响。根据结果选取主要参数,设计制造出高压水切割设备并且完成水泥混凝土试块的切割试验。试验达到预期效果,证明此高压水切割设备工作的有效性。     

Microtubule structure studied by quick freezing: Cryo-electron microscopy and freeze fracture

Microtubules have been quickly frozen and examined by electron microscopy using several techniques: (1) freezing of a thin layer of solution by plunging into cryogen, followed by cryo-electron microscopy of the unstained vitrified samples; (2) freezing by the propane-jet method, followed by freeze fracturing and metal replication. The unstained frozen-hydrated microtubules show a structure in agreement with X-ray diffraction data; they differ from negatively stained particles mainly by the better preservation of cylindrical shape. Secondly, they reveal a supertwist of the profilaments that is not detected reliably by other methods. This allows a determination of the number of protofilaments and the polarity. The structural resolution of unstained microtubules is similar to that of stained ones (about 2–3 nm); it is limited by low contrast and lack of crystalline order. Propane-jet or cryo-block freezing followed by freeze fracturing reveals the structures of the inner and outer surfaces of the microtubule wall at a resolution of 4 nm or better. The outside is dominated by the longitudinal protofilaments whereas on the inside one observes tilted cross-striations. Although the freezing temperatures of the two methods are different (liquid nitrogen or helium) they yield similar results for the case of thin layers of protein solution.     

Study of the conditions necessary for propane-jet freezing of fresh biological tissues without detectable ice formation

The performance of a commercial double-propane-jet freezer (Balzers QFD 101) has been assessed, for rapid freezing of fresh tissues in freeze-etch work. Samples of diaphragm muscle and intestinal villi were frozen between copper sheets, with a spacer to give 20–30μm thickness of tissue. Fracture cuts were made with the Balzers BAF 400 freeze-etch microtome within 5–10μm of a freezing face (i.e. a tissue face in contact with the copper sheets of the frozen sandwich). After some modifications to the QFD 101, replicas showing no evidence of ice were obtained of muscle cells, although for intestinal epithelial cells some evidence of ice formation was found. Infiltration with 5% glycerol or dimethylsulphoxide improves the depth of good freezing. Results and problems arising from such infiltration are briefly discussed.     

Coefficients for solution of the analytical freezing equation in the range of states for rapid solidification of biological systems

Solution of the classical solidification heat transfer problem for many biological applications involves states for which roots of the transcendental equation associated with the governing differential equation are not available. These roots are calculated and presented for conditions that characterize a large ratio of solid to liquid phase thermal conductivities and diffusivities and large differences between the initial and final temperatures for the cooling process.     

Countercurrent plunge cooling: A new approach to increase reproducibility in the quick freezing of biological tissue

Conditions fulfilling all the accepted criteria for optimum cryoquenching are realized for the first time in a plunge freezing device. Samples are plunged down through a smoothly rising cryogen stream that is maintained within ± 0.25 K of a preselected temperature. Dry nitrogen gas at ambient temperature removes evaporated cryogen molecules from the plunging path immediately above the liquid coolant to prevent pre-cooling of the sample. Symmetrical instrument design and conditions of flow ensure symmetrical freezing under optimum conditions in a totally repeatable fashion. The device has been used with halocarbons, propane and ethane. It provides consistent cooling rates comparable with the best so far obtained by any other plunging method.     

Freeze-substitution and isothermal freeze-fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (-21°C)

Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethyl-sulphoxide, at cooling rates known to reduce viability by significantly different amounts. The reduction in viability was known to be related to ice formation. Freeze-substitution and isothermal freeze-fixation studies were carried out to determine the distribution of ice within the muscle at this temperature. Freeze-substitution using ethylene glycol was unsuccessful but a new method, using high concentrations of the cryoprotectant as the substituting solvent, was able to maintain ice configurations at this relatively high substitution temperature. The results of freeze-substitution in dimethylsulphoxide were confirmed by isothermal freeze-fixation when both techniques were conducted under identical cooling conditions. The results indicated that the functional differences produced by cooling muscle at either 0·3 K min^?1 or 2 K min^?1 were related to the distribution of the ice phase within the tissue.     

Comparison of slam-freezing and high-pressure freezing effects on the DNA cholesteric liquid crystalline structure

Using in parallel electron microscopy of ultrathin frozen-hydrated sections and freeze-fracture replicas, we compare the ultrastructural consequences of two freezing techniques: slam-freezing at liquid helium temperature and high-pressure freezing, on a model system, the DNA cholesteric liquid crystalline phase. Both freezing techniques are able to vitrify DNA liquid crystalline solutions containing up to 85% water, but induce structural rearrangements of the molecular organization. The cholesteric structure is preserved by the slam-freezing method despite the formation of periodic distortions induced by the mechanical compressive stress. In contrast, high-pressure freezing does not preserve the structure of the liquid crystal: the long-range cholesteric stratification disappears, and the local continuous twist between molecules is modified. These results show that vitrification, though necessary, may not be a sufficient token of preservation of the native state of hydrated materials. We discuss the possible origins of the molecular rearrangements that have time to occur in the specimens as a result of the low freezing rate permitted by the high-pressure freezing process.     

Methylamine tungstate and tannic acid in fixation for high contrast and preservation of fine structure: application to myxomycete plasmodia

The effects of several fixation methods for electron microscopy were compared, using macro-plasmodia of Badhamia utricularis (myxomycete) as test objects. A brief osmium tetroxide (Os) exposure followed by a mixture of glutaraldehyde (GA) and tannic acid (TA) with subsequent treatment with methylamine tungstate (MAT) overcame most commonly encountered difficulties in fixing these structures. Lead staining of the sections was necessary to bring out the range of densities inherent in the delicate detail of ground cytoplasm, membranes, and organelle structure retained by Os/GA-TA/MAT. Microfilaments were well preserved with good contrast and little evidence of breakage. Although GA as the initial fixative gave good organelle preservation, Os pretreatment was required to prevent artefactual changes in plasmodial strand morphology. The several fixation procedures tested gave pronounced differences in mitochondrial appearance, in some cases giving a negative stained appearance to the cristae. Some advantages in interpretation may result from such reversed contrast. The high contrast and range of densities achieved following the Os/GA-TA/MAT schedule permitted routine use of thin (silver-grey) sections, thereby potentially increasing the resolution.