An inexpensive device for freeze drying and plastic embedding tissues at low temperatures

The preparation of biological tissues for electron microscopy by rapid freezing retains the original localization of ions and molecules. A reproducible freezing regime was established by quenching tissues in liquid propane according to the method of Elder et al. (1981). Tissue was thereafter freeze dried in a custom built freeze drying device with a liquid nitrogen cooled stage to prevent ice recrystallization during drying. The device was also designed to allow the vacuum embedding of tissue in low temperature resin such as Lowicryl® and polymerization in situ. This paper describes the design of the device and an example of its use in the freeze drying of cartilage. The results show that minimal ice damage occurs to the chondrocytes and that intracellular organelles are clearly visible. The regime described may prove a useful and pragmatic alternative to cutting tissue in the frozen state. Translocation of elements is unlikely except perhaps in the case of very labile elements such as Na and K, but this remains to be fully elucidated.     

The preparation of leaf surfaces for scanning electron microscopy: a comparative study

Cryopreservation is the superior technique for viewing leaf surfaces in the SEM. Epidermal cells become distorted when freeze dried and disrupt the orientation of epicuticular wax structures. The latter are largely lost during critical point drying. Nevertheless, the appearance of surface structures after subjecting them to each drying method is valuable in interpreting the features observed by cryopreservation.     

Cryofixation and ultra-low-temperature freeze-drying as a preparative technique for TEM

We have developed cryofixation and ultra-low-temperature molecular distillation drying as a method for preparing biological samples for electron microscopic analysis. To validate this approach, we have investigated the relationship between the drying characteristics and ice phases present within frozen samples. Two sample types were investigated. In the first, pure deuterium oxide (D₂O), or heavy water, was vapour condensed under vacuum conditions onto a gold-coated copper sample holder held at ?175 or ?110°C. Additionally, D₂O was slow-rate cooled from room temperature under an ultra-pure dry nitrogen gas atmosphere. The second sample type was rat liver biopsies from animals after 5 days of feeding with D₂O loaded water and ultra-rapid cooling by metal-mirror cryofixation. Ice forms present in the latter samples, determined by electron diffraction of frozen-hydrated cryosections, were amorphous, cubic, and hexagonal. Drying of samples was achieved using a molecular distillation configuration with continuous, microprocessor-controlled sample heating. The vacuum contents of the drying column were monitored by residual gas analysis (RGA) throughout the drying cycle. D₂O vapour in the vacuum chamber, as analysed by RGA, was found to increase in a phasic manner across a broad temperature range. These phases had characteristic onset temperatures and could be removed sequentially. For condensed D₂O samples, these onset temperatures were — 160, — 148, — 125 and — 90°C. Rat liver samples also demonstrated phasic drying patterns which were more complex than those detected with pure D₂O samples. Ultrastructural analysis of samples cryofixed and dried in this manner demonstrated a morphology consistent with the ice phases demonstrated in the frozen-hydrated cryosections. This, together with the RGA results, suggests the absence of devitrification or ice crystal growth during the drying procedure.     

生物制品冷冻干燥技术与策略

基于对现代冻干技术进展研究和多年生物制品实际冻干经验,探讨了影响冻干的重要因素、环节以及技术和对策。冷冻干燥技术研发和工业化是复杂的系统工程,仅靠传统方法已难于有效控制,应建立以科学为基础,通过质量设计、风险评估和过程控制,优化和控制冻干过程,获取优良冻干产品,提高生产效率,降低成本。通过冻干应用技术研究,为生物制品冻干研发和生产以及产品的质量控制提供参考。     

Visualization of biological specimens by cryoHVEM

This article describes the operation and the characteristics of cryoHVEM imaging of biological specimens using a top-entry cryostage. The procedure for inserting frozen specimens into the microscope column is also presented. Whole mounts were thus observed under optimal imaging conditions by combining: (i) fixation by fast freezing for structure preservation without exposure to chemicals, (ii) observation in the hydrated (frozen) state or in the dried state without exposure to the atmosphere after the initial fixation by freezing, and (iii) ultrastructural visualization with the key imaging factors of resolution, penetration and beam-induced damage at their best by high-voltage electron microscopy.     

Raman microscopy of freeze-dried mouse eyeball-slice in conjunction with the "in vivo cryotechnique"

The wavelength of Raman-scattered light depends on the molecular composition of the substance. This is the first attempt to acquire Raman spectra of a mouse eyeball removed from a living mouse, in which the eyeball was preserved using the "in vivo cryotechnique" followed by freeze-drying. Eyeballs were cryofixed using a rapid freezing cryotechnique, and then sliced in the cryostat machine. The slices were sandwiched between glass slides, freeze-dried, and analyzed with confocal Raman microscopy. Important areas including various eyeball tissue layers were selected using bright-field microscopy, and then the Raman spectra were obtained at 240 locations. Four typical patterns of Raman spectra were electronically mapped on the specimen images obtained by the bright-field microscopy. Tissue organization was confirmed by embedding the same eyeball slice used for Raman spectra into epoxy resin and the thick sections were prepared with the inverted capsule method. Each Raman spectral pattern represents a different histological layer in the eyeball which was mapped by comparing the images of toluidine blue staining and Raman mapping with different colors. In the choroid and pigment cell layer, the Raman spectrum had two peaks, corresponding to melanin. Some of the peaks of the Raman spectra obtained from the blood vessels in sclera and the photoreceptor layer were similar to those obtained from the purified hemoglobin and rhodopsin proteins, respectively. Our experimental protocol can distinguish different tissue components with Raman microscopy; therefore, this method can be very useful for examining the distribution of a biological structures and/or chemical components in rapidly frozen freeze-dried tissue.     

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.     

SEM sample preparation for cells on 3D scaffolds by freeze-drying and HMDS

Common dehydration methods of cells on biomaterials for scanning electron microscopy (SEM) include air drying, hexamethyldisilazane (HMDS) or tetramethysilane (TMS) treatment and critical point drying (CPD). On the other side, freeze-drying has been widely employed in dehydrating biological samples and also in preparing porous biomaterial scaffolds but not in preparing cells on three-dimensional (3D) biomaterials for SEM examination. In this study, we compare cells on porous hydroxyapatite (HA) prepared by air drying, HMDS and freeze-drying. The effects of fixation and using phosphate buffered saline (PBS) in the fixation were also assessed on three porous calcium phosphate (CaP) materials, namely, HA, α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP) samples. There is no significant difference in samples prepared by HMDS treatment and freeze-drying viewed at low magnification. Besides, it is better not to use phosphate buffer in the fixation step for CaP materials to avoid undesirable spontaneous precipitation of CaPs. On the other hand, fewer exchanges of liquids are required for freeze-drying and hence chemical fixation may not be absolutely required for samples prepared by freeze-drying. Other technical details of the preparation were also investigated and discussed. This study suggests both HMDS and freeze-drying can be employed to dehydrate cells on 3D scaffolds for SEM examination.     

A section press and low elemental support for enhanced preparation of freeze-dried cryosections

A press- and specimen holder system is described, whereby thin, freeze-dried cryosections can be obtained more conveniently and with greater protection against contamination and harmful temperature fluctuations. The use of a grid and retainer assembly as the basic working unit, greatly facilitates low temperature work particularly when delicate grids are preferable from an analytical point of view. A new press has been constructed in order to keep the sections permanently pressed during freeze-drying and rewarming. The press also protects the sections during transfer to an external freeze-dryer.     

A texture-analysis-based design method for self-adaptive focus criterion function

Autofocusing (AF) criterion functions are critical to the performance of a passive autofocusing system in automatic video microscopy. Most of the autofocusing criterion functions proposed are dependent on the imaging system and image captured by the objective being focused or ranged. This dependence destabilizes the performance of the system when the criterion functions are applied to objectives with different characteristics. In this paper, a new design method for autofocusing criterion functions is introduced. This method enables the system to have the ability to tell the texture directional information of the objective. Based on this information, the optimal focus criterion function specific to one texture direction is designed, voiding blindly using autofocusing functions which cannot perform well when applied to the certain surface and can even lead to failure of the whole process. In this way, we improved the self-adaptability, robustness, reliability and focusing accuracy of the algorithm. First, the grey-level co-occurrence matrices of real-time images are calculated in four directions. Next, the contrast values of the four matrices are computed and then compared. The result reflects the directional information of the measured objective surfaces. Finally, with the directional information, an adaptive criterion function is constructed. To demonstrate the effectiveness of the new focus algorithm, we conducted experiments on different texture surfaces and compared the results with those obtained by existing algorithms. The proposed algorithm excellently performs with different measured objectives.     

Sublimation rates of ice in a cryo-ultramicrotome

Sublimation rates of ice in the nitrogen-filled cryochamber of an ultramicrotome have been measured. The values are four to five orders of magnitude smaller than vacuum sublimation rates at the same temperatures. This is a consequence of the much shorter mean free path length of water molecules in nitrogen at atmospheric pressure, as compared to freeze drying under vacuum. The partial pressure at the phase boundary and gas diffusion and convection determine the rate of freeze drying. This has implications for section handling and the design of transfer equipment. Generally, it means that the danger of even partial dehydration of semi-thick (0·5–1 μm) sections used mainly for X-ray microanalysis is less serious than commonly assumed.     

烘丝机清洗的新方法

为了解决烘丝机管板积淀了大量烟垢无法清洗的现象,现采用新的清洗方法快速的清除烘丝机管板上的烟垢,有效消除了烟垢混入烟丝中所造成的质量隐患。     

Low-temperature scanning electron microscopy in fungus-nematode interaction

A comparative study has been made of different preparation techniques used for the scanning electron microscope (SEM), with regard to their application to fungus-nematode interaction. The preparation of frozen-hydrated specimens of both healthy and Arthrobotrys-oli-gospora-infected second-stage larvae of the root-knot nematode (Meloidogyne sp.) is described, and the results are compared with those obtained by critical point-drying and freeze-drying. In all cases the frozen-hydrated specimens consistently showed the best preservation.     

Quantitative tomography of early mouse embryos: laser scanning microscopy and 3D reconstruction

The cell volume alteration participates in a wide variety of cellular functions that may interfere with intra‐cellular homeostasis. The most adequate approach of estimation of the volume changes induced by osmotic misbalance, alteration in shape and size due to the action shape forming substances, etc., is the direct measurement of volumetric parameters of embryos. In the given research, the volume magnitude and kinetics of changes in volume and surface area of blastomere and polar bodies of early mouse embryos were determined using three‐dimensional reconstruction of the optical section stack obtained with laser scanning microscope (LSM). The size and surface area were determined for isotonic and anisosmotic conditions. The physiological significance of the findings is discussed.     

The observation of intact hepatic endothelial cells by cryo-electron microscopy

Liver sinusoidal endothelial cells (LSECs) can optimally be imaged by whole mount transmission electron microscopy (TEM). However, TEM allows only investigation of vacuum‐resistant specimens and this usually implies the study of chemically fixed and dried specimens. Cryo‐electron microscopy (cryo‐EM) can be used as a good alternative for imaging samples as whole mounts. Cryo‐EM offers the opportunity to study intact, living cells while avoiding fixation, dehydration and drying, at the same time preserving all solubles and water as vitrified ice. Therefore, we compared the different results obtained when LSECs were vitrified using different vitrification conditions. We collected evidence that manual blotting at ambient conditions and vitrification by the guided drop method results in the production of artefacts in LSECs, such as the loss of fenestrae, formation of gaps and lack of structural details in the cytoplasm. We attribute these artefacts to temperature and osmotic effects during sample preparation just prior to vitrification. By contrast, by using an environmentally controlled glove box and a vitrification robot (37 °C and 100% relative humidity), these specific structural artefacts were nearly absent, illustrating the importance of controlled sample preparation. Moreover, data on glutaraldehyde‐fixed cells and obtained by using different vitrification methods suggested that chemical prefixation is not essential when vitrification is performed under controlled conditions. Conditioned vitrification therefore equals chemical fixation in preserving and imaging cellular fine structure. Unfixed, vitrified LSECs show fenestrae and fenestrae‐associated cytoskeleton rings, indicating that these structures are not artefacts resulting from chemical fixation.     

Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes

Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO₄ fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO₄ fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO₄ fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.     

An optical-axis freezing stage for laser-scanning microscopy of broad ice–water interfaces

This article presents a method to view a dynamic ice interface along the axis of ice growth using a laser‐scanning microscope. A deep liquid volume is chilled from below so that ice growth is directed upward toward the microscope objective. The interface is made visible by rejection of fluorescent dye from the solid phase into the liquid. Images of the interface morphology in water with solutes of interest to cryobiology illustrate the imaging capability. These images are processed to quantify the lamellar structure of the ice interface. The optical‐axis cryostage provides advantages over horizontal arrangements because (1) immersion objectives enhance, rather than disturb, the desired thermal gradient, and (2) features in the ice interface are not confined within a narrow capillary tube or microscope slide. This arrangement loses some of the thermal control found in planar freezing stages, and the dynamic, refractive interface presents challenges to confocal microscopy.     

Mathematical modelling and experimental investigation on sun and solar drying of white mulberry

The drying kinetics of white mulberry was investigated in a solar dryer with forced convection and under open sun with natural convection. The constant rate period is absent from the drying curve. The drying process took place in the falling rate period. The drying data were fitted to the different mathematical models. The performance of these models was investigated by comparing the determination of coefficient (R), reduced chi-square (χ ²) and root mean square error (RMSE) between the observed and predicted moisture ratios. Among these models, the drying model developed by Logarithmic model showed good agreement with the data obtained from the experiments in the solar dryer with forced convection drying mode. The Verma et al. model has shown a better fit to the experimental mulberries data for open sun drying with natural convection mode than the other models. The effective moisture diffusivity values were estimated from Fick’s diffusional model. These values were 3.56×10⁻⁹ m²/s for solar drying and 2.40×10⁻⁹ m²/s for open sun drying.