Cryomicroscopy of isolated plant protoplasts

It has been nearly 100 years since Müller-Thurgau (26) employed cryomicroscopy to identify the cooling rate dependency of intracellular ice formation. Since that time cryomicroscopy has advanced from the “ice age” when Molisch (23) packed his microscope in ice to the “space age” of today when computer hardware developed for space satellite imagery is used for cryomicroscopic image analysis. Although interest in cryomicroscopy has been sporadic in the intervening period, current interest is at a high level—largely as a result of the refinement in the cryomicroscope design by Diller and Cravalho (9). The increased sophistication in cryostage design and precision of temperature control allow for quantitative studies of cell behavior during a freeze-thaw cycle. Not only does quantitative video image analysis facilitate this task, but it provides for increased resolution of cellular and subcellular responses during the freeze-thaw cycle. Most importantly, cryomicroscopy presents a researcher with a panorama of cellular behavior within which existing facts can be placed in perspective and from which future experiments can be more accurately focused.     

Physiological thermoregulation in a crustacean? Heart rate hysteresis in the freshwater crayfish Cherax destructor

Differential heart rates during heating and cooling (heart rate hysteresis) are an important thermoregulatory mechanism in ectothermic reptiles. We speculate that heart rate hysteresis has evolved alongside vascularisation, and to determine whether this phenomenon occurs in a lineage with vascularised circulatory systems that is phylogenetically distant from reptiles, we measured the response of heart rate to convective heat transfer in the Australian freshwater crayfish, Cherax destructor. Heart rate during convective heating (from 20 to 30 degrees C) was significantly faster than during cooling for any given body temperature. Heart rate declined rapidly immediately following the removal of the heat source, despite only negligible losses in body temperature. This heart rate 'hysteresis' is similar to the pattern reported in many reptiles and, by varying peripheral blood flow, it is presumed to confer thermoregulatory benefits particularly given the thermal sensitivity of many physiological rate functions in crustaceans.     

Thermal conductance and its relation to thermal time constants

A large body of thermoregulation studies exists in which the proper relationship between the physiological variable, thermal conductance, and heating or cooling time is not recognized. This is owing, in part, to conflicting definitions of conductance which either fail to relate to mean heat flow through the body surface or which fail to separate body conductance from that of the environment. Here we analyse ectotherm heating and cooling experiments to relate properly, conductance to thermal time constants and to determine how time constants are scaled according to animal size. It appears that the scaling properties are intimately related to peripheral circulation.Since thermal time constants can be unambiguously defined only for objects of constant thermal conductance we have provided simple illustratons of some phenomena related to temperature-dependent conductors and used these to indicate some of the limits of applicability of the time constant concept and its connection to conductance.Conditions described here do not include large radiant heat exchanges, however, convective heat transport and evaporation are considered. The treatment of evaporation should be quite useful to others.     

Effects of cold and heat on behavior and cerebellar function in goldfish

Summary A similar sequence of behavioral effects was observed for either cooling or heating; most effects occurred on changing temperature of entire fish or of only the cerebellum. On moderate heating or cooling, fish are hyperexcitable, spontaneously hyperactive; on further heating or cooling swimming is uncoordinated; when the subcerebellar structures are heated or cooled, equilibrium is disturbed; on further heating or cooling coma and respiratory failure ensue. Critical temperatures are modifiable by acclimation. The behavioral effects of cerebellectomy are additive with temperature effects on motor centers.Electrical activity of Purkinje neurons changes in the same thermal ranges as behavior. Inhibition via cerebellar interneurons is most sensitive and can be modified by acclimation. Ongoing activity increases with warming up to a blocking temperature; interspike interval histograms show pattern changes during warming. Activation via mossy fibers-granule cells is more sensitive than that via climbing fibers, and antidromic impulses are most resistant.A neuronal model based on inhibitory actions of Purkinje neurons on motor centers and parallel feedback excitatory pathways can explain both behavioral and electrical observations.     

The seasonal pattern of thermal characteristics of four of the Bishoftu crater lakes, Ethiopia

Measurements of temperature/depth profiles in four Ethiopian crater lakes were made during 1964, 1965 and 1966 at fairly regular intervals. Several more intensive diurnal studies, with observations at 3-h intervals during periods of about 48 h, were also carried out. Results show a pattern of heating leading to thermal stratification from February to September and cooling from September to December with possible overturn at the end of that period. Thermal stability was never great and thermoclines were often poorly defined and multiple. Net daily changes in heat content during both heating and cooling can be a small fraction (c. 3%) of the diurnal heat flux and depend upon the relative magnitudes of solar radiation and evaporative heat loss. These are such that cooling may occur during periods of highest solar input and vice versa. However, meteorological conditions could easily reverse the cooling process before the December overturn so that complete mixing is by no means a predictably regular annual event.     

The Leeuwenhoek lecture 2006. Microscopy goes cold: frozen viruses reveal their structural secrets

The electron microscope provides a powerful tool for investigating the structure of biological complexes such as viruses. A modern instrument is fully capable of atomic resolution on suitable non-biological specimens, but biological materials are difficult to preserve, owing to their fragility, and to image, owing to their radiation, sensitivity. The act of imaging the specimen severely damages it. Originally, samples were prepared by staining with a heavy metal salt, which provides a stable specimen but limits the amount of details that can be retrieved. Now particulate specimens, such as viruses, are prepared by rapid freezing of unstained material and observed in a frozen state with low doses of electrons. The resulting images require extensive computer processing to extract fully detailed three-dimensional information about the specimen. The whole process is referred to as single-particle electron cryomicroscopy. Using this approach, the structure of the human hepatitis B virus core was solved at the level of the protein fold. By comparing maps of RNA- and DNA-containing cores, it was possible to propose a model for the maturation and control of the envelopment of the virus during assembly. These examples show that cryomicroscopy offers great potential for understanding the structure and function of complex biological assemblies.     

24-gauge ultrafine cryoprobe with diameter of 550 μm and its cooling performance

This paper describes the development of a novel cryoprobe with the same size as a 24-gauge injection needle and the evaluation of its cooling performance. This ultrafine cryoprobe was designed to reduce the invasiveness and extend application areas of cryosurgery. The ultrafine cryoprobe has a double-tube structure and consists of two stainless steel microtubes. The outer diameter of the cryoprobe is 550 μm, and the inner tube has a 70-μm inner diameter to depressurize the high-pressure refrigerant. By solving the bioheat transfer equation and considering freezing phenomena, the relationship between the size of the frozen region and the heat transfer coefficient of the refrigerant flow in an ultrafine cryoprobe was derived analytically. The results showed that the size of the frozen region is strongly affected by the heat transfer coefficient. A high heat transfer coefficient such as that of phase change heat transfer is required to generate a frozen region of sufficient size. In the experiment, trifluoromethane (HFC-23) was used as the refrigerant, and the cooling effects of the gas and liquid phase states at the inlet were evaluated. When the ultrafine cryoprobe was cooled using a liquid refrigerant, the surface temperature was approximately −50 °C, and the temperature distribution on the surface was uniform for a thermally insulated condition. However, for the case with vaporized refrigerant, the temperature distribution was not uniform. Therefore, it was concluded that the cooling mechanism using liquid refrigerant was suitable for ultrafine cryoprobes. Furthermore, to simulate cryosurgery, a cooling experiment using hydrogel was conducted. The results showed that the surface temperature of the ultrafine cryoprobe reached −35 °C and formed a frozen region with a radius of 4 mm in 4 min. These results indicate that the ultrafine cryoprobe can be applied in actual cryosurgeries for small affected areas.     

Convective water exchanges during differential cooling and heating: implications for dissolved constituent transport

Convective water exchange patterns, determined from water temperature variations, were examined in the Minky Creek embayment of Guntersville Reservoir, Alabama (USA), during the month of September, 1990. During periods of differential cooling, cooler water originating from shallow stations moved as an underflow current toward the center of the embayment, while warmer water moved as an overflow current toward the shore. During periods of differential heating, convective exchange was much more shallow, occurring in the upper 3 m of the water column. As warmer water from shallow regions moved out as an overrflow current, this water was replaced by a return flow of cooler water originating from the pelagic epilimnion. Wind speed appeared to influence convective exchange patterns during differential heating phases by affecting the depth of the surface mixed layer. The potential for convective exchanges during periods of differential cooling and heating occurred on 54% and 74% of the days, respectively, in September. This high potential for horizontal water exchanges in Guntersville Reservoir has strong implications for the lateral transport of dissolved constituents.     

Regional hyperthermia by magnetic induction in a beagle dog model: analysis of thermal dosimetry

We have investigated magnetic induction heating techniques for achieving normal tissue hyperthermia in a beagle dog model to clarify the physics and physiology of "regional heating," to develop an animal model of regional heating in humans, and to develop a method of rapid regional heating in dogs for a normal visceral tissue toxicity study. Heating was done with a concentric coil or a coaxial pair of coils applied to the abdominal region, and with or without surface cooling blankets in each case. Thermometers were placed at multiple visceral and subcutaneous sites including an intraarterial thermocouple at the aortic arch level. With either electrode arrangement and no surface cooling, whole-body hyperthermia ( WBH ) at 42 degrees C was produced within 30 to 55 min with 250 W applied power; the 42 degrees C state could be maintained with 40 to 60 W of power. Thermal gradients in these cases reflected nonuniform power deposition superimposed upon arterial temperature elevation. With surface cooling blankets added, systemic heating was significantly reduced, and temperature gradients again reflected the nonuniform power deposition. Regional heating in a dog produces WBH unless sufficient surface cooling is used to provide a heat dissipation rate balancing the heat absorption rate; this latter case best models the use of inductive techniques in humans. The coaxial pair of coils, without surface cooling, produced rapid WBH and the visceral temperature maximum and minimum were within Tesoph + 0.21 degrees C and Tesoph - 0.07 degrees C, respectively (95% confidence index; Tesoph = esophageal temperature). This is an appropriate technique for the proposed toxicity study.     

Simulation of gel phase formation and melting in lipid bilayers using a coarse grained model

The transformation between a gel and a fluid phase in dipalmitoyl-phosphatidylcholine (DPPC) bilayers has been simulated using a coarse grained (CG) model by cooling bilayer patches composed of up to 8000 lipids. The critical step in the transformation process is the nucleation of a gel cluster consisting of 20-80 lipids, spanning both monolayers. After the formation of the critical cluster, a fast growth regime is entered. Growth slows when multiple gel domains start interacting, forming a percolating network. Long-lived fluid domains remain trapped and can be metastable on a microsecond time scale. From the temperature dependence of the rate of cluster growth, the line tension of the fluid-gel interface was estimated to be 3+/-2 pN. The reverse process is observed when heating the gel phase. No evidence is found for a hexatic phase as an intermediate stage of melting. The hysteresis observed in the freezing and melting transformation is found to depend both on the system size and on the time scale of the simulation. Extrapolating to macroscopic length and time scales, the transition temperature for heating and cooling converges to 295+/-5 K, in semi-quantitative agreement with the experimental value for DPPC (315 K). The phase transformation is associated with a drop in lateral mobility of the lipids by two orders of magnitude, and an increase in the rotational correlation time of the same order of magnitude. The lipid headgroups, however, remain fluid. These observations are in agreement with experimental findings, and show that the nature of the ordered phase obtained with the CG model is indeed a gel rather than a crystalline phase. Simulations performed at different levels of hydration furthermore show that the gel phase is stabilized at low hydration. A simulation of a small DPPC vesicle reveals that curvature has the opposite effect.     

MHD Free Convective Boundary Layer Flow of a Nanofluid past a Flat Vertical Plate with Newtonian Heating Boundary Condition

Steady two dimensional MHD laminar free convective boundary layer flows of an electrically conducting Newtonian nanofluid over a solid stationary vertical plate in a quiescent fluid taking into account the Newtonian heating boundary condition is investigated numerically. A magnetic field can be used to control the motion of an electrically conducting fluid in micro/nano scale systems used for transportation of fluid. The transport equations along with the boundary conditions are first converted into dimensionless form and then using linear group of transformations, the similarity governing equations are developed. The transformed equations are solved numerically using the Runge-Kutta-Fehlberg fourth-fifth order method with shooting technique. The effects of different controlling parameters, namely, Lewis number, Prandtl number, buoyancy ratio, thermophoresis, Brownian motion, magnetic field and Newtonian heating on the flow and heat transfer are investigated. The numerical results for the dimensionless axial velocity, temperature and nanoparticle volume fraction as well as the reduced Nusselt and Sherwood number have been presented graphically and discussed. It is found that the rate of heat and mass transfer increase as Newtonian heating parameter increases. The dimensionless velocity and temperature distributions increase with the increase of Newtonian heating parameter. The results of the reduced heat transfer rate is compared for convective heating boundary condition and found an excellent agreement.     

Effects of freezing and thawing on mammalian oocyte

In an attempt to study the deleterious effects which occur during the freezing and thawing of mammalian oocytes, we developed a cryomicroscope controlled by digital programmable equipment. The program permits any cooling rate between 0.1 and 60 degrees C/min with a precision of 0.6 degrees C. Using a precooled stage, it is possible to obtain rapid cooling (100 degrees C/min). The maximum thawing rate is about 60 degrees C/min. A copper-- constantan microthermocouple allows precise measurement of the specimen temperature. All information (specimen, temperature of the specimen, date, hour, and minutes) is recorded at the same time on photographic film by a camera fitted with a " Recordata Back" and a motor drive which allows three frames per second. Our preliminary results show that: (1) rapid cooling yields a supercooling with simultaneous extra- and intracellular crystallization; (2) slow cooling with seeding at -8 degrees C gives an extracellular crystallization which is achieved by -9 degrees C, followed by an extracellular recrystallization occurring at almost -8 degrees C which alters the morphology of the oocyte and the zona pellucida, without any visible intracellular crystallization; (3) during continued slow cooling the oocytes dehydrate without any intracellular freezing; and (4) during rewarming a partial rehydration of the cell occurs with a swelling of the oocytes to their original volumes after the thawing has been achieved.     

Studies on the three-dimensional temperature transients in the canine prostate during transurethral microwave thermal therapy

Thermal therapy of benign prostatic hyperplasia requires accurate prediction of the temperature distribution induced by the heating within the prostatic tissue. In this study, the Pennes bioheat transfer equation was used to model the transient heat transfer inside the canine prostate during transurethral microwave thermal therapy. Incorporating the specific absorption rate of microwave energy in tissue, a closed-form analytical solution was obtained. Good agreement was found between the theoretical predictions and in-vivo experimental results. Effects of blood perfusion and the cooling at the urethral wall on the temperature rise were investigated within the prostate during heating. The peak intraprostatic temperatures attained by application of 5, 10, or 15 W microwave power were predicted to be 38 degrees C, 41 degrees C, and 44 degrees C. Results from this study will help optimize the thermal dose that can be applied to target tissue during the therapy.     

Redistribution of blood within the body is important for thermoregulation in an ectothermic vertebrate (<Emphasis Type="Italic">Crocodylus porosus</Emphasis>)

Changes in blood flow are a principal mechanism of thermoregulation in vertebrates. Changes in heart rate will alter blood flow, although multiple demands for limited cardiac output may compromise effective thermoregulation. We tested the hypothesis that regional differences in blood flow during heating and cooling can occur independently from changes in heart rate. We measured heart rate and blood pressure concurrently with blood flow in the crocodile, Crocodylus porosus. We measured changes in blood flow by laser Doppler flowmetry, and by injecting coloured microspheres. All measurements were made under different heat loads, with and without blocking cholinergic and β-adrenergic receptors (autonomic blockade). Heart rates were significantly faster during heating than cooling in the control animals, but not when autonomic receptors were blocked. There were no significant differences in blood flow distribution between the control and autonomic blockade treatments. In both treatments, blood flow was directed to the dorsal skin and muscle and away from the tail and duodenum during heating. When the heat source was switched off, there was a redistribution of blood from the dorsal surface to the duodenum. Blood flow to the leg skin and muscle, and to the liver did not change significantly with thermal state. Blood pressure was significantly higher during the autonomic blockade than during the control. Thermal time constants of heating and cooling were unaffected by the blockade of autonomic receptors. We concluded that animals partially compensated for a lack of differential heart rates during heating and cooling by redistributing blood within the body, and by increasing blood pressure to increase flow. Hence, measures of heart rate alone are insufficient to assess physiological thermoregulation in reptiles.     

Effects of cooling rate on thermal shock hemolysis

The increase in thermal shock hemolysis in hypertonic sodium chloride with increasing cooling rate was confirmed. Thermal shock damage was also induced by hypertonic solutions of sucrose but it decreased with increasing cooling rate. The effect of cooling rate on thermal shock hemolysis appears to be due to the time that the cells are in the hypertonic solutions. The extent of the stress of the temperature reduction was independent of the cooling rate. In hypertonic sodium chloride susceptibility to thermal shock damage increased with increasing time of exposure at +25 °C (0–5 min) before decreasing with time (5–50 min). In contrast, with hypertonic sucrose, thermal shock damage increased gradually with time of exposure. The protective effects of sucrose on thermal shock hemolysis at a given osmolality can be explained by the different solution properties (e.g., ionic strength) of hypertonic sodium chloride and sucrose. These results suggest that the role of thermal shock damage during slow freezing should be reexamined.     

Thermoregulatory responses to thermal stimulation of the preoptic anterior hypothalamus in the red fox (Vulpes vulpes)

The preoptic anterior hypothalamus (POAH) thermoregulatory controller can be characterized by two types of control, an adjustable setpoint temperature and changing POAH thermal sensitivity. Setpoint temperatures for shivering (T_shiver) and panting (T_pant) both increased with decreasing ambient temperature (T_a), and decreased with increasing T_a. The POAH controller is twice as sensitive to heating as to cooling. Metabolic rate (MR) increased during both heating and cooling of the POAH. Resting temperature of the POAH was lower than internal body temperature (T_b) at all temperatures. This indicates the presence of some form of brain cooling mechanism. Decreased T_b during POAH heating was a result of increased heat dissipation, while higher T_b during POAH cooling was a result of increased heat production and reduced heat dissipation. The surface temperature responses indicated that foxes can actively control heat flow from body surface. Such control can be achieved by increased peripheral blood flow and vasodilation during POAH heating, and reduced peripheral blood flow and vasoconstriction during POAH cooling. The observed surface temperature changes indicated that the thermoregulatory vasomotor responses can occur within l min following POAH heating or cooling. Such a degree of regulation can be achieved only by central neural control. Only surface regions covered with relatively short fur are used for heat dissipation. These thermoregulatory effective surface areas account for approximately 33% of the total body surface area, and include the area of the face, dorsal head, nose, pinna, lower legs, and paws.     

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.     

How to build an inexpensive cyclotherm instrument for automated polymerase chain reaction

The Cyclotherm instrument is a functionally fully equivalent but inexpensive alternative to commercial instruments for automated polymerase chain reaction (PCR). It can be rebuilt under conditions of a biochemical laboratory for less than +1000. A Peltier element is used for heating and cooling of the reaction vials and the temperature and timing of the PCR cycles are controlled by a BASIC program in a SHARP PC 1600 low cost computer.     

Hyperquenching and cold equilibration strategies for the study of liquid--liquid and protein folding transitions

In this paper we consider the extension of the recent quantitative studies of hyperquenched glassformers to include (1). systems that exhibit first order liquid-liquid phase transitions, and (2). systems that contain molecules, which, during normal cooling, undergo internal structural changes above the glass temperature. The general aim of these studies is to trap-in a high enthalpy, high entropy, state of the system and then observe it evolving in time at low temperatures during a controlled annealing procedure. In this manner events that normally occur during change of temperature may be observed occurring during passage of time, at much lower temperatures. At such low temperatures the smearing effects of vibrations are greatly reduced. While the case of most interest in the second class is the refolding of thermally denatured protein molecules, any reconstructive molecular or chemical exchange process is a potential subject for investigation. Processes that occur in stages can be studied in greater detail, and any stage of interest can be frozen when desired, by drop of temperature, for more detailed spectroscopic examination. We review an electrospray method for hyperquenching liquids at approximately 10(5) K/s, and discuss some results of such experiments in order to illustrate a calorimetric approach to exploiting the hyperquenching-and- cold-equilibration strategy. To apply the idea to the study of proteins, the following protein solvent requirements must be met: (1). the solvents must not crystallize ice on cooling or heating, yet must not denature the proteins; (2). the solvents must support thermally denatured molecules without permitting aggregation. We describe two solvent systems, the first of which meets the first requirement, but the second only partially. The second solvent system apparently meets both. Preliminary results, only at the proof of concept stage, are reported for cold refolding of lysozyme, which, it seems, can be trapped in our solvent in the unfolded but refoldable state, with only moderate (approx. 120 K/s) quenching rates.     

The use of cryomicroscopy in guppy sperm freezing

The present study employed cryomicroscopy to derive an optimal sperm freezing protocol for guppy (Poecilia reticulata) sperm. Evaluation criteria during the freezing-thawing process were assessed for nucleation temperature (Tn), temperature when more than 50% of sperm display bending mid-piece (Tb), temperature when more than 80% of sperm stop moving (Tm), thawing temperature (Tt), and post-thaw motility. We compared four different cryoprotectants: 5% N-dimethyl formamide (DMF), 6% methanol (MEOH), 10% dimethyl sulfoxide (DMSO), and 14% glycerol, as well as glycerol at different concentrations of 7-50%; cooling and rewarming rates ranged from 5 to 100 °C/min. The protocol that yielded the highest post-thaw motility was samples suspended in 14% glycerol, cooled at 25 °C/min, and thawed at 100 °C/min, which was in complete agreement with our previous findings derived from a controlled-rate freezer. In addition, Tb and Tm were found to be negatively correlated with post-thaw motility, suggesting their possible role in predicting freezing success. The present study for the first time demonstrated the usefulness of cryomicroscopy in deriving an optimal sperm freezing protocol for aquatic species.