一种昆虫过冷却点的简易测定装置

在前人的研究基础上改进了利用热敏电阻测定昆虫过冷却点和体液冰点的装置,经检验其准确度与常用的热电偶法基本相同,但其测定成功率较高,使用方便。本装置先录制热敏电阻值变化的全过程,便于随时调出并准确地读出过冷却点与体液冰点的热敏电阻值;开发了操作方便的昆虫夹,明显缩短了测定时间,提高了测定效率,可用于定期的、连续的昆虫过冷却点与体液冰点的批量测定。     

Controlling the freezing process: a robotic device for rapidly freezing biological tissues with millisecond time resolution

A robotic cryogenic device was developed which allows freezing of thick biological tissues with millisecond time resolution. The device consists of two horizontally oriented hammers (pre-cooled with liquid N(2)) driven by two linear servo-motors. The tissue sample is bathed in Ringers contained in a chamber which drops rapidly out of the way just as the hammers approach. A third linear motor is vertically oriented, and permits the rapidly dropping chamber to smoothly decelerate. All movements were performed by the three motors and four solenoids controlled by a PC. Mechanical adjustments, that change the size of the gap between the hammers at the end position, permit the final thickness of the frozen tissue to be varied. Here we show that the freezing time increased with the square of the final thickness of the frozen bundle. However, when bundles of different original thicknesses (up to at least 1mm) were compressed to the same final thickness (e.g., 0.2mm), they exhibited nearly equal freezing times. Hence, by being able to adjust the final thickness of the frozen bundles, the device not only speeds the rate of freezing, but standardizes the freezing time for different diameter samples. This permits the use of freezing for accurate determination of the kinetics of cellular processes in biological tissue.     

A simple technique for thermal stimulation of single semicircular canals.

Two simple thermodes for stimulating single semicircular canals have been designed in order to elicit eye nystagmus. The first, for warm stimulation, is based on the Joule effect: a silver needle is warmed up by means of a coiled nickrome wire. The temperature at the thermode tip is function of current intensity. The thermode for cold stimulation is a device in which a circulating freezing mixture cools a silver needle. The temperature at the thermode tip is proportional to the temperature of the freezing mixture.     

Effects of glycerol and freezing on the appearance and arrangement of microtubules in three different systems: A freeze-substitution study

Effects of glycerol and freezing on microtubules in three different systems viz. the ovaries of Notonecta glauca, the sperm tails of Notonecta glauca and the hyphae of the fungus Phanaerochaete velutina have been studied by means of freeze-substitution. The protofilamentous substructure of microtubules is visible in positive image whether or not the tissues are treated with glycerol prior to freezing, suggesting that it is the freezing which allows this by the weakening of hydrophobic bonding between protofilaments. Glycerol, used as a cryoprotectant, causes a re-arrangement of the microtubules which is proportional to the concentration of, and the duration of exposure to, glycerol. This rearrangement of microtubules is greater in systems where the microtubules are not inter-linked than those where microtubules have obvious intertubule linkages. Rates of cooling on freezing affected the degree of rearrangement and was less with propane than with Freon 22. Possible explanations for these effects have been discussed.     

Selective freezing of target biological tissues after injection of solutions with specific thermal properties

Cryosurgery is a minimally invasive surgical technique that employs the destructive effect of freezing to eradicate undesirable tissues. This paper proposes a flexible method to control the size and shape of the iceball by injecting solutions with specific thermal properties into the target tissues, to enhance freezing damage to the diseased tissues while preserving the normal tissues from injury. The cryosurgical procedure was performed using a minimally invasive cryoprobe cooled by liquid nitrogen (LN2) to obtain deep regional freezing. Several needle thermocouples were applied simultaneously to record the transient temperature to detect the freezing effect on the tissues. Simulation experiments on biological tissue (fresh pork) were performed in vitro and four different liquids were injected into the test materials; these were distilled water, an aqueous suspension of aluminum nanoparticles in water, ethanol, and a 10% solution of the cryoprotective agent dimethyl sulfoxide (Me2SO). The experimental results demonstrate that the localized injection of an appropriate solution could enhance the tumor-killing effect without altering the freezing conditions. The study also suggests the potential value of combining cryosurgery with other therapeutic methods, such as electrical, chemical, and thermal treatments, to develop new clinical modalities in the near future.     

利用飞秒激光研究微液滴的冻结相变特性

对微液滴冻结行为的认识在低温生物学、分析化学等方面具有重要意义.引入飞秒激光实验手段研究液滴及微量生物材料(蛋白)的冻结相变特性.实验考察了样品在多次冻结过程中荧光光谱的变化规律,结果表明:生物材料与非生物材料在冻结及复温过程中的荧光光谱变化趋势存在差异,非生物试剂在冻结过程中光谱下降,经历复温后,其光谱可回复到初始状态;而蛋白在冻结过程中光谱上升,经历复温后,由于降温/升温过程对其造成的不可逆损伤,光谱无法回复到初始状态.基于此提出了用以评估生物样品活性的非接触式飞秒激光测量方法.     

Green fluorescent protein: A novel viability assay for cryobiological applications

Assessment of tissue viability following the application of a freezing protocol is challenging due to the paucity of viability assays that can be used dynamically, in situ. Cells transfected with a green fluorescent protein (GFP) vector actively produce GFP, which is retained intracellularly. Because of its constitutive and heritable expression, GFP fluorescence of transfected cells may have significant utility as a viability assay for cells within tissues. As a first step toward application to tissues, this work seeks to establish the validity of this GFP-based assay in cell suspensions by comparing the results to other accepted measures of viability. To the authors' knowledge, this is the first use of GFP in cryobiology applications. Intracellular GFP fluorescence was evaluated following slow freezing. Nontransfected and GFP-transfected rat 3230 adenocarcinoma (R3230AC) cells were frozen at 1 degrees C/min to minimum temperatures between -5 and -30 degrees C and then immediately thawed in a 37 degrees C water bath. Samples were assayed using the common viability indicators trypan blue and ethidium bromide (EtBr). A regression analysis of recovery measured with the GFP assay as a function of recovery measured with a trypan blue assay gave a correlation coefficient of 0.97. A similar correlation coefficient, 0.95, was determined for recovery assessed by the GFP assay as a function of recovery measured by an EtBr assay. Nontransfected and GFP-transfected cells responded similarly to slow freezing, indicating that GFP transfection did not significantly alter the response of cells to typical freezing conditions. The excellent correlation of GFP assay results with those of two common viability assays suggests that the GFP-based assay is valid for cells and that further development of a tissue viability assay based on transfection is appropriate.     

Abscission: quantitative measurement with a recording abscissor

The construction, operation, and effectiveness of an abscission measuring instrument called an abscissor is described. The device measured the force required for a spring-opposed plunger to shear abscission zone explants and was capable of automatically recording break strength data. Examples of data obtained with the abscissor are presented to demonstrate its capability of rapidly measuring significant changes in explant break strength.     

A randomised controlled trial evaluating the effect of an individual auditory cueing device on freezing and gait speed in people with Parkinson's disease

Background  

Parkinson's disease is a progressive neurological disorder resulting from a degeneration of dopamine producing cells in the substantia nigra. Clinical symptoms typically affect gait pattern and motor performance. Evidence suggests that the use of individual auditory cueing devices may be used effectively for the management of gait and freezing in people with Parkinson's disease. The primary aim of the randomised controlled trial is to evaluate the effect of an individual auditory cueing device on freezing and gait speed in people with Parkinson's disease.     

The thermodynamic principles of isochoric cryopreservation

The goal of this study is to introduce the fundamental thermodynamic principles of isochoric (constant volume) cryopreservation for low temperature preservation of biological materials. Traditionally, cryopreservation is performed in an isobaric process (constant pressure) at 1 atm, because this is our natural environment and it is most convenient experimentally. More than half a century of studies on cryopreservation shows that the major mechanism of damage during isobaric cryopreservation is the increase in intracellular ionic concentration during freezing, which presumably causes chemical damage to the components of cells. Cryoprotectants as well as hyperbaric pressures have been developed as methods to reduce the extent of chemical damage during freezing. The theoretical studies in this paper show that in isochoric cryopreservation, the increase in solution concentration during freezing is lower at each temperature by almost an order of magnitude from that in isobaric cryopreservation. This suggests that isochoric cryopreservation could be a preferential alternative to isobaric cryopreservation. The technology for isochoric cryopreservation is very simple; freezing in a constant volume chamber. Using a simple isochoric cryopreservation device, we confirm the theoretical thermodynamic predictions.     

A simple procedure to obtain one-micrometer sections of routinely embedded paraffin material

By freezing blocks of paraffin-embedded tissues to a convenient temperature it is possible to obtain routinely 1 micron sections that can be further processed as normal thicker sections. Normal and disposable steel knives can be used and the staining time should be increased in most procedures. Gradual freezing of blocks to the temperature of dry ice is the simplest and safest way to obtain an adequate temperature. The best results were obtained using as fixative 4% paraformaldehyde in phosphate buffered saline solution.     

A Simple Procedure to Obtain One-Micrometer Sections of Routinely Embedded Paraffin Material

By freezing blocks of paraffin-embedded tissues to a convenient temperature it is possible to obtain routinely 1 µm sections that can be further processed as normal thicker sections. Normal and disposable steel knives can be used and the staining time should be increased in most procedures. Gradual freezing of blocks to the temperature of dry ice is the simplest and safest way to obtain an adequate temperature. The best results were obtained using as fixative 4% paraformaldehyde in phosphate buffered saline solution.     

Numerical simulation of selective freezing of target biological tissues following injection of solutions with specific thermal properties

Recently, we proposed a method for controlling the extent of freezing during cryosurgery by percutaneously injecting some solutions with particular thermal properties into the target tissues. In order to better understand the mechanism of the enhancement of freezing by these injections, a new numerical algorithm was developed to simulate the corresponding heat transfer process that is involved. The three-dimensional phase change processes in biological tissues subjected to cryoprobe freezing, with or without injection, were compared numerically. Two specific cases were investigated to illustrate the selective freezing method: the injection of solutions with high thermal conductivity; the injection of solutions with low latent heat. It was found that the localized injection of such solutions could significantly enhance the freezing effect and decrease the lowest temperature in the target tissues. The result also suggests that the injection of these solutions may be a feasible and flexible way to control the size of the ice ball and its direction of growth during cryosurgery, which will help to optimize the treatment process.     

石楠和蜡梅茎结冰动力学过程的差热扫描分析

在（0.66 ±0.2）℃/min（0℃~-20℃）的降温速度下,采用高分辨率差热分析法分别对石楠（Photinia serrulata Lindl.）和蜡梅（Chimonanthus praecox（L.）Link）活体幼茎和经过10 min高温煮沸的幼茎在结冰过程中的热力学行为进行分析,并根据茎的形态解剖结构对他们的结冰特征进行研究。结果显示：石楠和蜡梅的活体幼茎在结冰过程中的差热扫描曲线均出现3个放热峰;而经过高温杀死后的茎仅出现1个单放热峰。分析结果表明,2种植物活体幼茎的3个放热峰可能与其木质部、质外体、韧皮部、形成层的结冰、脱水以及髓组织的结冰、脱水过程有关。进一步采用生理盐水浸湿的滤纸进行模拟实验,结果发现差热扫描曲线出现与高温杀死的茎类似的放热峰。实验结果表明,采用高分辨率差热分析法可以探测植物组织结冰过程中的放热强度、结冰温度及其与结冰动力学过程相关的大量细节,适用于植物的结冰动力学分析。     

Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)

The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 °C/day) of dormant buds to −50 °C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with LN and then rewarming to −20 °C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to −30 °C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to −50 °C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds.     

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.     

Freezing behaviours in wintering Cornus florida flower bud tissues revisited using MRI

How plant tissues control their water behaviours (phase and movement) under subfreezing temperatures through adaptative strategies (freezing behaviours) is important for their survival. However, the fine details of freezing behaviours in complex organs and their regulation mechanisms are poorly understood, and non‐invasive visualization/analysis is required. The localization/density of unfrozen water in wintering Cornus florida flower buds at subfreezing temperatures was visualized with high‐resolution magnetic resonance imaging (MRI). This allowed tissue‐specific freezing behaviours to be determined. MRI images revealed that individual anthers and ovules remained stably supercooled to ?14 to ?21 °C or lower. The signal from other floral tissues decreased during cooling to ?7 °C, which likely indicates their extracellular freezing. Microscopic observation and differential thermal analyses revealed that the abrupt breakdown of supercooled individual ovules and anthers resulted in their all‐or‐nothing type of injuries. The distribution of ice nucleation activity in flower buds determined using a test tube‐based assay corroborated which tissues primarily froze. MRI is a powerful tool for non‐invasively visualizing unfrozen tissues. Freezing events and/or dehydration events can be located by digital comparison of MRI images acquired at different temperatures. Only anthers and ovules preferentially remaining unfrozen are a novel freezing behaviour in flower buds. Physicochemical and biological mechanisms/implications are discussed.     

Freezing Characteristics of Rigid Plant Tissues (Development of Cell Tension during Extracellular Freezing)

The freezing characteristics and development of cell tension during extracellular freezing were examined in supercooling stem tissues of riverbank grapes (Vitis riparia) and cold-hardened leaves of live oak (Quercus virginiana) and mountain cranberry (Vaccinium vitis-idaea). Dormant stem xylem and pith tissues of river-bank grapes were resistant to freeze-induced dehydration above the homogeneous nucleation temperature, and they developed cell tension reaching a maximum of 27 MPa. Similarly, extracellular freezing induced cell tension in the leaves of live oak and mountain cranberry. Maximum cell tension in the leaves of live oak was 16.8 MPa and 8.3 MPa in the leaves of mountain cranberry. Following peak tensions in the leaves, a decline in the pressure was observed with progressive freezing. The results suggest that resistance to cell deformation during extracellular freezing due to cell-wall rigidity can lead to reduced cell dehydration and increased cell tension. A relationship to predict freezing behavior in plant tissues based on cell rigidity is presented. Based on cell-water relations and ice nucleation rates, cell-wall rigidity has been shown to effect the freezing characteristics of plant tissues, including freeze-induced dehydration, supercooling, and homogeneous nucleation temperatures.     

Avoidance of intracellular freezing by the freezing-tolerant New Zealand Alpine weta Hemideina maori (Orthoptera: Stenopelmatidae)

Calorimetric analysis indicates that 82% of the body water of Hemideina maori is converted into ice at 10 degrees C. This is a high proportion and led us to investigate whether intracellular freezing occurs in H. maori tissue. Malpighian tubules and fat bodies were frozen in haemolymph on a microscope cold stage. No fat body cells, and 2% of Malpighian tubule cells froze during cooling to -8 degrees C. Unfrozen cells appeared shrunken after ice formed in the extracellular medium. There was no difference between the survival of control tissues and those frozen to -8 degrees C. At temperatures below -15 degrees C (lethal temperatures for weta), there was a decline in survival, which was strongly correlated with temperature, but no change in the appearance of tissue. It is concluded that intracellular freezing is avoided by Hemideina maori through osmotic dehydration and freeze concentration effects, but the reasons for low temperature mortality remain unclear. The freezing process in H. maori appears to rely on extracellular ice nucleation, possibly with the aid of an ice nucleating protein, to osmotically dehydrate the cells and avoid intracellular freezing. The lower lethal temperature of H. maori (-10 degrees C) is high compared to organisms that survive intracellular freezing. This suggests that the category of 'freezing tolerance' is an oversimplification, and that it may encompass at least two strategies: intracellular freezing tolerance and avoidance.     

The ability of the Antarctic nematode Panagrolaimus davidi to survive intracellular freezing is dependent upon nutritional status

The Antarctic nematode Panagrolaimus davidi is the best documented example of an animal surviving intracellular freezing and the only animal so far shown to survive such freezing throughout its tissues. However, a recent study found that after exposure to a freezing stress that produced intracellular freezing in a proportion of nematodes, the resulting survival levels could be explained if those nematodes that froze intracellularly had died. We have thus re-examined the survival of intracellular freezing in this nematode. The ability to survive a freezing exposure that is likely to produce intracellular freezing (freezing at ?10 °C) declines with culture age. In cultures that are fed regularly, the ability to survive freezing at ?10 °C increases, but in starved cultures freezing survival declines. Survival of intracellular freezing in fed cultures was confirmed using cryomicroscopy, staining of cells with vital dyes and by freeze substitution and transmission electron microscopy. We have thus confirmed that P. davidi can survive intracellular freezing and shown that this ability is dependent upon them being well fed. The effect of culture conditions on the nutrient status of the nematodes should thus be an important factor in the design of experiments.