Recent progress in freeze-fracturing of high-pressure frozen samples

Pancreatic tissue, bacteria and lipid vesicles were high‐pressure frozen and freeze‐fractured. In addition to the normal holder, a new type of high‐pressure freezing holder was used that is particularly suitable for suspensions. This holder can take up an EM grid that has been dipped in the suspension and clamped in between two low‐mass copper platelets, as used for propane‐jet freezing. Both the standard and the new suspension holder allowed us to make cryo‐fractures without visible ice crystal damage. High‐pressure frozen rat pancreas tissue samples were cryo‐fractured and cryo‐sectioned with a new type diamond knife in the microtome of a freeze‐etching device. The bulk fracture faces and blockfaces were investigated in the frozen‐hydrated state by use of a cryo‐stage in an in‐lens SEM. Additional structures can be made visible by controlled sublimation of ice (‘etching’), leading to a better understanding of the three‐dimensional organization of organelles, such as the endoplasmic reticulum. With this approach, relevant biological structures can be investigated with a few nanometre resolution in a near life‐like state, preventing the artefacts associated with conventional fixation techniques.     

Freeze substitution in 3 hours or less

Freeze substitution is a process for low temperature dehydration and fixation of rapidly frozen cells that usually takes days to complete. New methods for freeze substitution have been developed that require only basic laboratory tools: a platform shaker, liquid nitrogen, a metal block with holes for cryotubes and an insulated container such as an ice bucket. With this equipment, excellent freeze substitution results can be obtained in as little as 90 min for cells of small volume such as bacteria and tissue culture cells. For cells of greater volume or that have significant diffusion barriers such as cuticles or thick cell walls, one can extend the time to 3 h or more with dry ice. The 3-h method works well for all manner of specimens, including plants and Caenorhabditis elegans as well as smaller samples. Here, we present the basics of the techniques and some results from Nicotiana leaves, C. elegans adult worms, Escherichia coli and baby hamster kidney tissue culture cells.     

透射电镜生物样品冷冻置换技术的方法

本文介绍了制备透射电镜生物样品冷冻置换技术的基本方法及其优点。     

Chloroplast ultrastructure in leaves of Urtica dioica L. analyzed after high-pressure freezing and freeze-substitution and compared with conventional fixation followed by room temperature dehydration

In this article, we report on the adaptation of high-pressure freezing and freeze-substitution (HPF-FS) for ultrastructural analysis of leaf tissue with special emphasis on chloroplasts. To replace the gas in the intercellular spaces, a mixture of water and methanol (MeOH) was employed. We compared three different supplements for FS--osmiumtetroxide, uranyl acetate, and safranin--with regard to the preservation of the ultrastructure of chloroplasts and other cellular compartments. The results show that (i) replacement of air within intercellular spaces by 8% (v/v) MeOH has no influence on the ultrastructure of the chloroplasts, (ii) undulation of membranes frequently observed after conventional preparation of specimens does not occur during chemical fixation but during room temperature dehydration, and (iii) uranyl acetate or osmium tetroxide employed during FS are not superior over safranin.     

An improved cryo-jet freezing method

A new cryo-jet freezing apparatus is described that is easy to use and gives good results using a propane-butene mixture (3: 1). Our use of the freezer in the study of mouse spinal cord explant cultures is discussed. At the tissue surface, the quality of tissue preservation from freezing, followed by freeze substitution, rivals that of conventional electron microscopic methods. Certain intracellular structures are better visualized using our methods. There is no evidence of the tissue being distorted by the cryogen jet when the freezer is operated correctly. A new freeze substitution device is also discussed.     

Ultrastructural preservation of nuclei and chromatin: improvement with low-temperature methods

The ultrastructure of chromatin has been examined in nuclei prepared by a variety of low-temperature methods. Embedding glutaraldehyde (GA)-fixed nuclei in Lowicryl K4M or K11M following dehydration by the progressive lowering of temperature (PLT) method, or in K11M following spray freezing and freeze substitution (FS), produces chromatin fibres that have, in situ, a diameter close to the in vivo state, and show internal structural details consistent with patterns of nucleosome packing previously observed only in preparations of isolated fibres. This is a temperature-dependent effect; fibres conventionally dehydrated and embedded in Lowicryl at 0°C or in conventional epoxy resin at 60°C have lower and less uniform diameters, and lack internal structural details. Of the techniques used, spray freezing followed by FS resulted in the most notable improvement over conventional methods. Inclusion of GA during FS of rapidly frozen, unfixed nuclei in methanol does not result in cross-linking of nuclear proteins. In acetone, however, cross-linking by GA occurs at — 45°C, or at lower temperatures if the water content of the acetone-based FS media is kept deliberately high. Substitution regimes employing GA alone or in combination with uranyl acetate and/or osmium tetroxide do not result in fibre morphologies comparable to either prefixed or unfixed nuclei substituted in additive-free substitution media. Whole fibroblasts show excellent preservation of nuclei and the nuclear/cytoplasmic interface after spray freezing followed by FS and low-temperature embedding.     

Preparation of the nematode-trapping fungus,Arthrobotrys oligospora,for scanning electron microscopy by freeze substitution

A freeze-substitution technique for preparing fungal specimens for scanning electron microscopy is described. This involves cryofixation in liquid nitrogen, freeze substitution in methanol at — 20°C and critical-point drying. The trapping complexes and conidiophores of the nematophagous fungus Arthrobotrys oligospora are well preserved and retain their normal three-dimensional arrangement.     

A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues

In this paper, we review some published studies using correlative light and electron microscopy methods. We further refined our criteria to include only those studies using live cells for light microscope and where high-pressure freezing was the method of specimen preparation for electron microscopy. High-pressure freezing is especially important for some difficult-to-fix samples, and for optimal preservation of ultrastructure in samples larger than a few micrometres. How the light microscope observations are done is completely sample dependent, but the choice of high-pressure freezer depends on the speed required to capture (freeze) the biological event of interest. For events requiring high time resolution (in the 4–5 s range) the Leica EM PACT2 with rapid transfer system works well. For correlative work on structures of interest that are either non-motile or moving slowly (minutes rather than seconds), any make of high-pressure freezer will work. We also report on some efforts to improve the capabilities of the Leica EM PACT2 rapid transfer system.     

Effects of extreme pressure additive chemistry on rolling element bearing surface durability

Lubricant additives have been known to affect rolling element bearing surface durability for many years. Tapered roller bearings were used in fatigue testing of lubricants formulated with gear oil type additive systems. These systems have sulfur- and phosphorus-containing compounds used for gear protection as well as bearing lubrication. Several variations of a commercially available base additive formulation were tested having modified sulfur components. The variations represent a range of “active” extreme pressure (EP) chemistries. The bearing fatigue test results were compared with respect to EP formulation and test conditions. Inner ring near-surface material in selected test bearings was evaluated on two scales: the micrometer scale using optical metallography and the nanometer scale using transmission electron microscopy (TEM). Focused-ion beam (FIB) techniques were used for TEM specimen preparation. Imaging and chemical analysis of the bearing samples revealed near-surface material and tribofilm characteristics. These results are discussed with respect to the relative fatigue lives.     

Freeze-substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around –60°C

High‐pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high‐pressure frozen, freeze‐substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around –60°C.