A new technique for removal of amorphous phase tissue water without ice crystal damage: a preparative method for ultrastructural analysis and immunoelectron microscopy

An apparatus has been produced that can remove amorphous phase tissue water via molecular distillation without devitrification or rehydration. This method represents a fundamental advance in tissue preparation, making possible for the first time ultrastructural localization of soluble molecular entities without the problems of alteration, re-distribution, and loss which have plagued conventional techniques. Fresh slices of rat brain, liver, or kidney, and monkey retinal tissue were cryofixed by bounce-free, metal mirror cooling on copper bars immersed in liquid nitrogen (LN2). Tissue transferred under LN2 was then placed in a precooled copper specimen block, which was subsequently lowered into a LN2-cooled stainless steel chamber. After rough pumping at 1 X 10(-3) mbar with a mechanical pump to remove LN2, the chamber was evacuated with a cryopump or turbomolecular pump to achieve a hydrocarbon-free, ultra-high vacuum of 1 X 10(-8) mbar. Equilibrium temperature in the chamber before the drying cycle was -192 degrees C. The copper specimen block was equipped with a thermocouple and a programmable feedback-controlled heating circuit. Tissue was dried by increasing the specimen block temperature 1 degree C/hr during the critical drying phase while monitoring the rate of water removal with a partial pressure analyzer. Results obtained indicate that drying is complete below the devitrification temperature of amorphous phase tissue water. Dried tissue was fixed with osmium tetroxide vapor, vacuum-embedded in a low-viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Processed tissue exhibits excellent morphological preservation without the use of pre-fixation or cryoprotective agents. Thin sections of this tissue are excellent for immunocytochemical staining and electron microprobe analysis.     

An efficient freeze-drying apparatus

Summary A freeze-drying apparatus with a practical temperature regulating system is described. Degassing, drying and embedding all take place in the drying chamber. The drying temperature can be set at a number of fixed temperature levels which may be chosen anywhere between -70° C and +60° C.     

香蕉果实冻干过程参数优化的研究

对香蕉果实冻干生产工艺中的物料厚度、冻结方法、加热板温度、干燥室真空度等参数进行比较试验,结果表明,各过程参数对香蕉冻干品质量和产量均有显著的影响。香蕉冻干过程中参数较佳的工艺条件建议为物料厚度选取5~7 mm,采用速冻方法冻结,加热板温度设定45℃,干燥室真空度控制于20~30 Pa。     

Effect of carbohydrates on the survival of Lactobacillus helveticus during vacuum drying

AIMS: To assess four carbohydrates for the protective effect against Lactobacillus helveticus cells inactivation during vacuum drying, and to study the effect of selected carbohydrate on changes of inactivation kinetics. METHODS AND RESULTS: Early stationary phase L. helveticus cells grown in MRS media were recovered from fermentation broth, washed with PBS buffer (pH 7.0), and then mixed with different concentrations of four carbohydrates, namely lactose, sorbitol, inulin, and xanthan gum. Cells were dried in a vacuum drier at 100 mbar, 43 degrees C for 12 h. Only cells with 1% sorbitol addition showed higher survival (18%) over cells without added carbohydrate (8%). Using in situ microbalance technique whereby cell weight during vacuum drying was continuously monitored via precision balances integrated into the vacuum chamber, drying and inactivation kinetics of cells and cells mixed with sorbitol were established. CONCLUSION: Survival of L. helveticus during the vacuum drying could be improved by the addition of optimal concentration of 1% sorbitol. Addition of sorbitol did not cause drastic changes in drying rate, water content and water activity of samples. The protection mechanisms of sorbitol seemed not to be due to a direct physical effect, which could be related to drying rate. SIGNIFICANCE AND IMPACT OF THE STUDY: The increase in survival of cells after vacuum drying by the addition of a protective carbohydrate may provide an alternative mean to preserve starter cultures at a higher level of activity.     

Critical Point Drying Method Using Dry Ice

A critical point drying method using dry ice (solid carbon dioxide) instead of liquid carbon dioxide is reported. After the specimens are placed in the chamber of the medical point drying apparatus, dry ice cut to the shape of the chamber is inserted. The chamber is closed and warmed to change the dry ice into liquid carbon dioxide. This method needs no gas cylinder and even minute or fragile specimens do not blow away because there is no flow of gas. This method can be used with any kind of critical point drying apparatus.     

Surface replicas of pulmonary endothelial cells in culture.

Pulmonary endothelial cells possess a variety of enzymes on their surface. However, the topographical distribution of these enzymes is not known. In this report we describe a simple technique for the preparation of surface replicas of pulmonary endothelial cells using an unmodified critical point drying apparatus and a high vacuum freeze-etch unit. The technique should be applicable for use with all cells in monolayer culture. Endothelial cells grown on glass slides or coverslips were washed thoroughly, fixed and dehydrated. The monolayers were then critical point dried. Surface replicas were prepared in a Balzer's freeze-etch unit. The advantages of surface replicas are that they can be examined with the resolving power of the transmission electron microscope and can be used to examine whole cells. The potential for the surface replica technique may lie in mapping specific enzymes, receptors and cell surface factors. Therefore, to provide a basis for such studies, we have described the appearance of pulmonary endothelial cells in surface replicas.     

Degassing‐assisted patterning of cell culture surfaces

We developed an alternative patterning technique which is capable of producing both topographic and biochemical features for cell culture studies. This technique is based on microaspiration induced with a degassed poly (dimethylsiloxane) (PDMS) mold. After degassing in a rough vacuum chamber and placed on a sample surface, liquid solution can be aspired through channels and cavities created in the PDMS mold. Depending on the composition of the solution and the associated drying or incubation processes, a variety of surface patterns can be produced without applying external pressure. For demonstration, we fabricated agarose gel microwells and biomolecule patterns either on a glass plate or in a cell culture Petri dish, both applicable for cell culture studies. Biotechnol. Bioeng. 2010. 105: 854–859. © 2009 Wiley Periodicals, Inc.     

A Modified Freezing-Drying Apparatus for Tissues

The preparation of tissues by the freezing-drying technic is preferred for many histochemical studies because of the rapid fixation, avoidance of deleterious action of chemical fixation and extraction by aqueous and lipid solvents in fixation, dehydration and clearing; to facilitate this procedure a freezing-drying apparatus has been constructed which permits cutting of sections within five hours after the fresh tissue is obtained. Liquid nitrogen provides a most efficient moisture trap and in conjunction with a heating element provides any desired temperature down to — 80°C. during tissue drying. Paraffin infiltration is started without disassembling the equipment and completed in a few minutes. Most stains and histochemical reactions for enzyme and other substances can be applied directly to sections of frozen-dried tissue cut from the same blocks.     

Combined influence of fermentation and drying conditions on survival and metabolic activity of starter and probiotic cultures after low-temperature vacuum drying

The influence of low temperature vacuum drying process parameters on the survival, metabolic activity and residual water content of three different bacterial strains (Lactobacillus paracasei ssp. paracasei, Lactobacillus delbrueckii ssp. bulgaricus and Bifidobacterium lactis) was investigated. Shelf temperature and chamber pressure were varied and optimized by response surface methodology with regard to survival and residual water content. It is shown that the survival rate after low temperature vacuum drying is comparable to that of freeze drying. Based on the optimization experiments the combined influence of fermentation pH and drying process parameters was studied for the most detrimental and the best process condition, respectively. The results show that interactions between process and fermentation conditions have to be taken in account and that these influences are highly strain specific.     

Testing of the method for water microleakage detection from OH hydroxyl spectral lines at the L-2M stellarator

Results are presented from L-2M stellarator experiments on testing a possible method for detection of water microleakages in the cooling system of the first wall and vacuum chamber of ITER. The method consists in the spectroscopic detection of spectral lines of the OH hydroxyl, which forms via the dissociation of water molecules in plasma. Emission in the spectral band of 305–310 nm can be detected even at water leakage rates less than 10^?4 Pa m³/s. Chemical reactions between water and boron compounds on the vacuum chamber wall delay the detection of leakages up to ～2000 s. A similar phenomenon can be expected when a leakage will occur in ITER, where the materials suggested for the first wall (Be, Li) can also chemically react with water.     

The Application of Freeze-Dry Methods to Plant Material

The freeze-dry method of preparing tissue for histological observation has been applied to a large variety of plant material with good results. This method involves freezing the tissue rapidly and dehydrating at a temperature below -30°C. under vacuum with a desiccant. The tissue, when dry, is infiltrated with paraffin under vacuum. The apparatus uses a liquid nitrogen-cooled condenser as the desiccant and the tissue is infiltrated with paraffin while in the original vacuum. Using root, stem, leaf, and reproductive tissue of common experimental species such as Vicia faba, Zea mays, Allium cepa, Lillium longiflorium, Pisum sativum and Phaseolus vulgaris, the effect of thickness on drying time was studied. It was found that there was greater difference between types of tissue than between species so that regardless of species, leaf tissue was easiest and root tips the most difficult to freeze and dry. Leaf segments required 1-2 days to dry while root material required 6 days or more. In all tissue except leaf, 1-2 mm. segments were optimal for freezing and drying although 4 mm. segments could occasionally be used.     

Ultrastructural observations on tissues processed by a quick-freezing,rapid-drying method: Comparison with conventional specimen preparation

A quick-freeze, rapid-dry method for processing unfixed tissue for electron microscopy has been developed. The technique employs freezing on a cryogenchilled metal surface and drying in a cryosorption vacuum apparatus that allows osmium-vapor fixation and epoxy-resin embedment under high vacuum. Liver, kidney, bone marrow, and monolayer cultures of ventricular myocytes were selected as tissue specimens representing a wide range of physical properties, to demonstrate the practical aspects of achieving good ultrastructural morphology by freeze drying. A comparison was made between freeze drying and conventional processing using aldehyde fixation and alcohol dehydration. The preservation of cellular ultrastructure achieved by freeze drying allowed the identification of specific cell types within each specimen. Membranous organelles were well preserved, surrounded by cytoplasmic ground substance devoid of ice crystal damage. Electron-dense material was observed within the rough endoplasmic reticulum and Golgi cisternae and vesicles of frozen-dried, but not conventionally processed cells. This suggests the preservation by freeze drying of cytoplasmic components otherwise extracted from the cell by solvent exposure.     

Modifications of Lebowich's Soap-Wax Technic

Lebowich's technic is outlined for simultaneous dehydration and infiltration of tissues by a medium composed of stearic acid, 56° C. paraffin, diethylene glycol, and monoethanolamine. The prices and places where these materials may be purchased are given.

Tissue for sectioning is placed in acetone, C.P., for 1 hour, then put directly into the soap-wax medium at 60° C. under reduced pressure, and finally embedded in new soap-wax.

Modifications include a simplification of the apparatus used by Lebowich. A preserving jar fitted with a rubber stopper serves as a vacuum chamber, and use of an aspirator accomplishes the reduction of pressure. With invertebrate embryos up to 1000 μ diameter no reduction of pressure is needed. Embryos are fixed, washed, placed in acetone, infiltrated in soap-wax, and embedded.

By this soap-wax method the alcohols, xylene, and overnight drying of affixed ribbons are eliminated. Tissue may be fixed, sectioned, stained, and permanently mounted within 6 to 8 hours.     

Thin Sections from Unfixed Tissues for Histochemical Staining

Sections were cut from fresh unfixed tissues by means of a microtome provided with an apparatus for the simultaneous cooling of the knife and freezing stage. These sections were of uniform thickness and were found to be very suitable for histochemical staining. Such sections were immersed while still frozen in the fluid which contained the necessary chemicals for a specific technic. After remaining in the fluid for an appropriate time, the sections were put on slides and dried in warm air. The remaining steps were carried out on the slides. Several histochemical procedures (phosphatase, esterase, glycogen) were found to give good results when this technic was used.     

Mechanically facilitated cell-cell electrofusion.

Apparatus and methods were developed to enable mechanically facilitated cell-cell electrofusion to be performed. The apparatus and methods mechanically place cells in contact before fusion. The key component of this fusion system was a newly developed fusion chamber. The chamber was composed of two functionally identical electrodes that were housed in a multi-layer structure. The layers functioned as support for the electrodes. They also allowed adjustment of the distance between opposing electrode faces. The electrodes were constructed in a manner that allowed cells to be deposited, by vacuum, onto each face. Electrode faces were positioned at a predetermined distance from each other to mechanically force cell-cell contact between the deposited cells. Fusion was induced by delivering direct current pulses to the juxtaposed cells. Fusion products were detected and quantitated by flow cytometry. Details of the chamber design and a protocol for using the fusion chamber are given. Mechanically facilitated cell-cell electrofusion was demonstrated by using the chamber to produce fusion products from like fusion partners. The practical applicability of the chamber was demonstrated by fusing unlike cell types. Mechanically facilitated cell-cell electrofusion is not specific to the cells used in this study; the chamber can be adapted for use with other cell types.     

植物材料快速石蜡制片方法

真空干燥箱已越来越广泛地应用于现代生物学研究领域。该文利用真空干燥箱温度和负压的可控制性能,将固定、脱水、透明和石蜡渗透等过程在真空干燥箱中进行,建立起一套可行的植物组织快速石蜡制片方法。结果显示,真空干燥箱的应用加速了多种试剂的渗透速率,提高了切片质量,达到了优化实验步骤、节省实验时间和减少室内有毒化学气体污染的目的。     

1H NMR investigation on the role of sorbitol for the survival of Lactobacillus paracasei ssp. paracasei in vacuum‐dried preparations

Aims: The aim of this work was to study the effect of sorbitol as protective agent on the survival of the probiotic strain Lactobacillus paracasei ssp. paracasei (F19) after vacuum drying. Methods and Results: The survival was studied after different drying times and for various concentrations of sorbitol by plate count method. Furthermore, time domain ¹H NMR studies on dehydrated suspensions of Lact. paracasei ssp. paracasei were performed to study the proton mobility in the dried samples. From the obtained signal, T2 relaxation times of single components and fractions with different proton mobility were determined. It was found out that the survival is increased by the presence of a minimum amount of sorbitol that is dependent on drying time. Furthermore, it is shown that the protective effect can only be observed below a critical water content of c. 20%. Nuclear magnetic resonance (NMR) results indicate a transition of sorbitol from liquid to solid like behaviour during drying. The onset of the transition coincides with the critical water content found for a protective effect. Conclusions: The data suggest that sorbitol protons are incorporated into the dried cells of Lact. paracasei ssp. paracasei (F19) below the critical water content and therefore leading to an enhanced survival. Significance and Impact of the Study: The results help to better understand the underlying mechanism of protection of Lact. paracasei ssp. paracasei using sorbitol and to establish vacuum drying as potential alternative drying technique to standard freeze drying.     

ELECTRON MICROSCOPIC OBSERVATION OF SPECIMENS UNDER CONTROLLED GAS PRESSURE

A technique for encasing specimens in a thin gas layer during their observation in the Siemens Elmiskop I is described. All gases can be employed at pressures up to one atmosphere. Destruction of specimens can occur in the beam; all organic specimens are particularly liable to decompose. The conditions under which this can be avoided are given. A useful application of the technique allows one to prevent specimens from drying out, as they normally do in vacuum. A further application uses the controlled removal of carbon for thinning organic layers and for selective etching of organic materials.     

A new controlled-rate cooling apparatus for freezing hematopoietic cells for storage at -196 degrees C

An apparatus has been constructed to cool biological material at a controlled rate. The material to be frozen is placed in glass ampuls which are immersed in an aluminum bath containing ethyl alcohol and the bath is placed inside a freezer cabinet. Liquid nitrogen is pumped intermittently into the cabinet by means of a single-speed electric pump. The rate of cooling is controlled by a device that varies the interval between successive pumping cycles. The temperature fall is monitored by thermocouples placed inside selected glass ampuls and recorded as a plot on moving graph paper.This simple instrument is capable of cooling at an accurately controlled rate over the range of 0 to 7 °C/min. We chose for our studies a cooling rate of 1 °C/min which we could maintain with an accuracy of ±0.1 °C. Temperature fluctuations were, however, observed at the freezing plateau and varied considerably in magnitude and temperature at onset even for the same material cooled under the same conditions. Mouse bone marrow cells frozen by our technique and stored for various periods of time may, on reconstitution, form colonies in vivo and in vitro identical in morphology and number to those from unfrozen control cells. Our results suggest that expensive and intricate devices may not be necessary to obtain optimal recovery of viable cells after storage in liquid nitrogen. The apparatus is now in regular use for the storage of human bone marrow cells intended for use in treatment of patients with leukemia refractory to conventional measures.     

Is thiamine pyrophosphatase a reliable marker of the neuronal Golgi apparatus? A critical analysis

The thiamine pyrophosphatase histochemical technique is believed to be a suitable approach to the selective staining of the Golgi vesicles of all animal cells, icluding the neuron. However, during the past decade a considerable number of data have been published, suggesting that the TPPase is in general a membrane-associated enzyme in the brain, which can be found in subcellular fractions other than Golgi lamellae. It has therefore become necessary to reconsider the view that thiamine pyrophosphatase is an exclusive marker enzyme of the Golgi apparatus.