A Computer-Controlled Spraying-Freezing Apparatus for Millisecond Time-Resolution Electron Cryomicroscopy

Apparatus is described for the kinetic investigation of biological reactions by electron cryomicroscopy with time resolution on the order of milliseconds. This involves layering a grid with one reactant and then spraying on a second reactant immediately before freezing. Two-stage mixing can be achieved by mixing two solutions, holding them in a delay line for a preset interval, and then spraying the aged solution onto a grid carrying a third reactant. The individual steps of these procedures are under software control and can be adjusted independently. Spray-freezing is widely applicable since solutions of small molecules, proteins, and protein assemblies can be delivered as aerosols. Thus the method can be used to study both the effects of small molecules on macromolecules and for monitoring protein–protein interactions. It may also be useful in other situations, for instance in light microscopy.     

An Inexpensive Microtome Attachment for Cutting 50-Micron Nonfrozen Sections for Electron Microscopic Histochemistry

The attachment is for an American Optical Co., Model 888 freezing microtome. It consists of right-angle bracket made from % inch metal rod, one end of which M clamped in the freezing stage holder; the other supports a tissue holder whose base lies at a right angle to the usual position. The fixed and blotted tissue specimen is enclosed (without infiltration) in parah, on a piece of filter paper which is attached to the base of the tissue holder, and sectioned across its long axis by 50 μ increments. After sectioning, the filter paper is removed from the base, the paraffin matrix opened, and the sections transferred to the appropriate processing fluid     

Freezing injury and uncoupling of phosphorylation from electron transport in chloroplasts

Freezing of chloroplast membranes uncouples photophosphorylation from electron transport and inactivates the light-dependent and thiol-requiring ATPase, conformational changes and the light-dependent proton uptake. All of these energy requiring activities can be protected against inactivation by addition of sucrose prior to freezing. The direct relation to photophosphorylation is demonstrated by the quantitatively similar response of photophosphorylation and the other activities to sucrose protection. Salts interfere with the protection afforded by sucrose.

In contrast to the light-dependent ATPase, the ATPase activities which are unmasked by digestion with trypsin show no significant response to freezing. Similarly, the chloroplast coupling factor, which is released from the membranes by ethylenediamine tetraacetic acid treatment, survives freezing. The membranes, which are depleted of the factor, are damaged by freezing.

The results suggest that uncoupling of phosphorylation from electron transport is caused by interference of freezing with a structure involved in the formation of a non-phosphorylated high energy state of chloroplasts. They are best explained on the basis of Mitchell's theory of phosphorylation. Since freezing alters the permeability properties of chloroplast membranes—frozen membrane vesicles no longer function as osmometers—it may be assumed that freezing uncouples phosphorylation from electron transport by preventing the formation of a pH gradient across the vesicle membranes owing to proton leakage through the membranes. From the results, the basic injury caused by freezing appears to consist in the alteration of permeability properties of biological membranes due to the dehydration which accompanies freezing.

     

Effects of freezing on biological membranes in vivo and in vitro

Membrane inactivation by freezing has been investigated using intact spinach leaves and isolated thylakoid membranes from chloroplasts of leaf cells as test material. During freezing in vitro in solutions containing neutral solute and a slight excess of inorganic salts such as NaCl, electron transport is stimulated while photophosphorylation is lost. Under more drastic freezing conditions damage increases, affecting dichlorophenolindophenol reduction, the rise in variable fluorescence, ferricyanide reduction and electron transport through Photosystem I, in that order. Semipolar compounds such as phenylalanine or phenylpyruvate exhibit a much higher membrane toxicity during freezing than inorganic salts. The profile of damage caused by this class of compounds is different from that caused by salts. Damage to membranes isolated rapidly from frost-killed leaves is similar to that produced by semipolar compounds during freezing in vitro. A few sites of damage could be identified, among them the site responsible for oxidation of water during photosynthesis. The results support the view that the sensitivity of their membranes limits the ability of cells to withstand freezing and suggest that freezing sensitivity is due to the accumulation in the cells of potentially membrane-toxic organic and norganic cell constituents.     

Sequential sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase chromatography of unfolded proteins

Sequential sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography on a fluorocarbon packing (poly F column, DuPont) provide fully denatured but highly purified protein, which is free of low-molecular-weight substances and directly amenable to structural analysis. Conditions for the preparative elution of four test proteins (bovine serum albumin, carbonic anhydrase, myoglobin, cytochrome c) blotted to Immobilon membranes have been optimized. Phenol saturated with 50 mM Tris-HCl (pH 8.25) and containing 2% SDS and 1% 2-mercaptoethanol is able to elute proteins that have been blotted to Immobilon membranes, stained with Coomassie R-250, and stored as dry sheets, largely irrespective of their molecular mass. Polypeptides that are not degraded by exposure to low pH can also be efficiently extracted directly from stained gels with 70% formic acid. If further separation of polypeptides is not needed, a simple run of a protein sample dissolved in 1-2% SDS on the poly F column will efficiently remove low-molecular-weight substances, including SDS.     

A New Methylene Blue Technic for Permanent Preparations

1. Tissues stained intra vitam with methylene blue are fixed in a 10% ammonium molybdate solution in physiological saline (or sea water if the tissue is from a marine animal). Fixation time is kept to a minimum. Washing also is reduced to a minimum.

2. Excess fluids are removed from tissues by blotting with a paper or cloth towel before they are put into the succeeding solution. Tissues are taken from the wash water, blotted and placed in a mixture of equal parts of absolute ethyl alcohol and n-butyl alcohol for 30 minutes. They are then blotted and transferred to n-butyl alcohol for 30 minutes. After blotting they are placed in a mixture of one part methyl salicylate and four parts xylene until cleared. Tissues may be mounted whole or prepared for sectioning by embedding in paraffin in the usual way.

3. Tissues fixed, washed, dehydrated and cleared as described retain nearly all of the stain; the time required is greatly reduced; there is no need to chill the dehydrating solutions; cell distortion is much reduced.     

Rapid Removal of Tissue Culture Monolayers by Collodion for Subsequent Staining

Removing cultures from roller tubes before staining eliminates the destaining which often occurs when the cells are first stained and then removed by embedding in collodion. The cells are fixed in situ, dehydrated, and covered with collodion (Merk's flexible) for 10 min. The collodion is poured off, the fluid residue lining the tube allowed to dry for 10 min, and the tube is filled with tap water. The collodion cast containing the cells is loosened and removed, cut into strips, placed on slides and blotted into firm contact. The collodion is then dehydrated and dissolved with absolute alcohol followed by a 1:1 mixture of alcohol and ether. The slides can then be rehydrated and stained by conventional methods.     

Scanning and Transmission Electron Micrographs of the Same Frozen Fractured Surface

The freeze-etch technique was applied to scanning and transmission electron-microscopy by freezing samples of tissue from 3-wk-old laboratory white mice in Freon 12 cooled with liquid nitrogen. The samples were fractured and placed into a rotary stage cold-block device and rotary shadowed with platinum and then carbon at a 45° angle. They were dried overnight in a vacuum at approximately 5 × 10^-6 torr and then exposed to OsO₄ vapors for 48 hr. After being viewed in a scanning electron microscope, the carbon-platinum replica was removed from the sample by dissolving away the tissue with Clorox. The replica was mounted on a grid and viewed in a transmission electron microscope.     

The pleated sheet: an unusual negative-staining method for transmission electron microscopy of biological macromolecules

A novel method for preparing negatively stained specimens is described which appears to improve the routine resolution of biological structure in direct images obtained by transmission electron microscopy. In the new method, which we term the pleated sheet technique, macromolecules are adsorbed to a carbon film by the Valentine procedure (R. Valentine, B. Shapiro, and E. Stadtman (1968) Biochemistry, 7, 2143-2152), and the film then carefully pleated while in contact with a 1% uranyl formate solution to trap stain within the folds of pleats. A grid is placed on the compressed film, and film plus grid retrieved with a Saran Wrap drum. Subsequent dehydration produces a filmed grid containing negatively stained macromolecules within the folds of pleated regions and positively stained macromolecules in single sheet regions. The effect of sandwiching sample and stain between carbon layers is to produce exceedingly uniform negative staining so that stain contours more accurately and more reproducibly reflect true molecular contours. Electron micrographs of IgG and IgA molecules prepared by these methods are exhibited that permit unambiguous comparison of structure imaged in the electron microscope against known structures solved by single-crystal X-ray diffraction. Correlation is excellent; the smallest resolvable element in micrographs is an immunoglobulin domain, whose molecular weight is 12 000.     

A Monoclonal Antibody to Rabbit Brain GABA Transaminase

A monoclonal antibody of class IgG (subclass IgG1) has been prepared to rabbit brain GABA transaminase (GABA-T). This antibody reveals a single band of molecular weight 52,000 on a nitrocellulose filter blotted with purified GABA-T. On a filter blotted with unfractionated rabbit brain supernatant a major band of molecular weight 58,000 is revealed. An immunoaffinity column was prepared by coupling proteins from ascites fluid containing anti-rabbit GABA-T antibody to Bio-Rad Affi-Gel 15. This column bound purified GABA-T and extracted from unfractionated rabbit brain supernatant a protein of molecular weight 58,000, which was almost homogeneous and which had GABA-T enzyme activity. Using immunoaffinity chromatography, therefore, a high degree of purification of GABA-T may be achieved in a single step. Further, this technique may preserve an authentic form of the enzyme that is lost during the conventional purification procedure. The antibody inhibits GABA-T enzyme activity, up to a maximum of 35%.     

Ultrastructural and thermocouple evaluation of rapid freezing techniques

The quality of freeze-fixation for electron microscopy is dependent upon the size of intracellular ice crystals. In the absence of cryoprotectants, ice crystal growth is thought to be related to the speed with which the specimen is cooled. The purpose of this study was to investigate the relationship between the cooling rate and ultrastructural preservation in commonly used freezing techniques. The techniques studied included immersion in stirred and unstirred forms of five quenching fluids: liquid nitrogen, isopentane, Freon 12, Freon 22, and propane. Also studied were freezing in a flowing stream of coolant using liquid nitrogen and liquid helium and freezing on a metal surface using cooper and mercury chilled to liquid nitrogen temperature. For each technique a cooling curve was obtained with a 0.360-mm thermocouple which was dropped into the quenching fluids or brought into contact with the metal surfaces. From oscilloscope tracings, the cooling rates were determined in degrees centigrade per second to −100 °C. To evaluate ultrastructural preservation 0.5-mm-thick slices of rat kidney were frozen by each of the techniques and dried in an all glass freeze-drier. The final evaluation was made from electron micrographs of the best morphological preservation yielded by each technique. The results indicate that the copper and mercury surfaces and propane gave the highest cooling rates and the best morphological preservation. The other techniques cooled at decreasing rates and correspondingly showed decreasing abilities to preserve ultrastructure. This work demonstrates that the preservation of cellular ultrastructure by freezing is dependent upon the cooling rate and that as the cooling rate is increased, ultrastructural preservation is enhanced.     

Agar-Screen Specimen Carrier for Bulk Processing of Biopsy Materials for Electron Microscopy

A specimen carrier for processing large numbers of biopsy materials for epoxy embedding and electron microscopy is described. Commercially available 18-mesh stainless steel or 16-mesh aluminum wire screening is used. The screening is cut into 1 × 3-inch strips. One corner is snipped off for orientation purposes. Four drops of warm 4% agar is placed on a prewarmed standard microscopic glass slide. A thin agar support film is formed on the bottom side of the horizontally held wire screen by lightly running it against the agar. Tissue blocks trimmed to 1 mm³ are blotted on filter paper and placed in a prearranged order on the top surface of the support film. A thin top coating of agar is applied on the specimen by touching it with the tip of a pasteur pipette containing warm 4% agar. The agar-screen unit with the mounted specimens is stabilized in 4% buffered formalin and rinsed with Sorenson's phosphate buffer, pH 7.4, with 6.8% sucrose. It is then processed as a unit through routine osmium tetroxide postfixation, alcohol dehydration, and Epon 812 infiltration. The tissue blocks are plucked off the agar support film with fine-tipped tweezers and embedded in individual capsules. No difficulty in thin sectioning was encountered and examination of the sections under the electron microscope showed good infiltration by the epoxy resin.     

Effect of N-acetyl-D-glucosamine, and glycerol concentration and equilibration time on acrosome morphology and motility of frozen-thawed boar sperm.

A series of experiments were conducted to determine the effect of N-acetyl-D-glucosamine, glycerol concentration and equilibration time for the freezing of boar spermatozoa in 5 ml maxi-straws. The optimum final glycerol concentration in the diluent with 0.05% N-acetyl-D-glucosamine in the first diluent was 2-3% and the optimum glycerol equilibration time was 2-3h. In conclusion, we recommend the first diluent containing 11% lactose hydrate, 20% egg yolk and 0.05% N-acetyl-D-glucosamine in 100ml distilled water, and the second diluent containing 11% lactose hydrate, 20% egg yolk, 4% glycerol and 1% orvus es paste for the diluents of boar sperm freezing. Also, we found out that 0.05% soluble N-acetyl-D-glucosamine was the optimum concentration in the first diluent and a concentration of 0.05% soluble N-acetyl-D-glucosamine significantly enhanced the cryopreservation of boar spermatozoa.     

Thin-layer chromatography blotting for the fluorescence detection of phospholipid hydroperoxides and cholesteryl ester hydroperoxides

A blotting technique was developed to specifically detect lipid hydroperoxides in thin-layer chromatography. Phosphatidylcholine hydroperoxides and cholesteryl linoleate hydroperoxides ranging from 0.1 to 0.5 nmol, which were prepared by reaction with soybean lipoxygenase, were visualized as fluorescent spots on the blotted membrane by immersing the plate into a blotting solvent containing 0.01% (w/v) diphenyl-1-pyrenylphosphine. This technique was applied successfully to monitor lipid peroxidation in human low-density lipoprotein in vitro.     

Properties of the primary electron acceptor complex of photosystem I in the blue green alga Chlorogloea fritschii.

Photooxidation of P700 at low temperatures in membrane fractions from the blue-green alga $\text{Chlorogloea fritschii}$ may be coupled irreversibly to the reduction of a bound ferredoxin. If this ferredoxin is reduced before freezing, P700 photooxidation at low temperatures becomes reversible. This reversible photooxidation is coupled to the reduction of a component with an EPR signal at g = 2.08, 1.88 and 1.78. A complete spectrum of this component has been obtained for the first time. We propose that as in higher plants this component is the primary electron acceptor of Photosystem I, the bound ferredoxin is a secondary electron acceptor. Using ⁵⁷Fe enriched preparations we have shown that the ERP signals attributed to the bound ferredoxin are due to iron containing centres. This experiment did not show the presence of iron in the primary electron acceptor.     

Structural damages in adsorbed vaccines affected by freezing

This study was planned to evaluate structural damages in adsorbed vaccines affected by freezing using scanning electron microscopy and X-ray analysis of the elements. Randomly selected 42 vials of eight different types of WHO pre-qualified adsorbed freeze-sensitive vaccines from 10 manufacturers were included in the study. Vaccines were kept at 5 °C. Selected numbers of vials from each type were then exposed to ?25 °C for 24 h periods. All samples were evaluated for their structure using scanning electron microscopy, X-ray analysis of the elements and precipitation time. Scanning electron microscopy of vaccines affected by freezing showed either smooth or rough surfaced conglomerates associated with phosphate content of the precipitate. These vaccines precipitated 2–15 times faster compared to non-frozen samples. Non-frozen samples showed uniform flocculent structure either dense or dispersed. X-ray analysis of precipitates in frozen samples confirmed that the precipitate is mainly aluminium clutters. Scanning electron microscopy confirmed that the lattice structure of bonds between adsorbent and the antigen is broken and aluminium forms conglomerates that grow in size and weight. The precipitation time of vaccines affected by freezing is 4.5 times faster on average compared to non-frozen samples. These facts form the basis of the "shake test".     

Stoichiometry of the oligomycin-sensitivity-conferring protein (OSCP) in the mitochondrial F0F1-ATPase determined by an immunoelectrotransfer blot technique

The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the and β subunits of F₁-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with ¹²⁵I-labelled purified monoclonal antibodies specific to various peptides. The ¹²⁵I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H₂O₂ and -naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F₁.     

A two-step dilution tris-egg yolk extender containing Equex STM significantly improves sperm cryopreservation in the African wild dog (Lycaon pictus)

Conservation management of endangered African wild dogs (AWD; Lycaon pictus) can benefit greatly from development of sperm freezing and artificial insemination. Previous freezing attempts yielded nearly 0% motile sperm within 2 h of thawing. In this study, two canine freezing protocols were tested: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution. Semen was collected by electroejaculation from n = 24 AWDs, of which eight ejaculates of sufficient quality (four good quality with initial sperm motility of 75.0 ± 4.4% and four poor quality; showing rapid decrease in sperm motility to 3.3 ± 3.3% prior to freezing) were frozen. For good quality samples, motility and sperm motility index persisted for up to 8 h for Protocol 2, and was higher between 2 and 6 h after thawing with a decrease from 4 h of incubation. Motility dropped to nearly 0% after 2 h incubation for Protocol 1. Viability was higher for Protocol 2 throughout the 8 h of incubation, with a decrease after 6 h, compared to 4 h for Protocol 1. Acrosome integrity was higher for Protocol 2 throughout post-thaw incubation, with a decrease after 2 h for both protocols. Protocols did not differ in normal sperm morphology or DNA integrity. Poor quality samples yielded similar results, except for acrosome integrity, which declined for Protocol 2. In conclusion, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it suitable for sperm banking and artificial insemination initiatives.     

Commercial freezing of bovine embryos in straws

Bovine embryos were frozen commercially in clear double length $\text{1}\text{2}$ cc French straws with the wick and powder plug in the center of the straw. One-half of the double length straw serves as a handle and contains a color coded $\text{1}\text{4}$ cc straw around which an adhesive backed label has been applied. After plunging into liquid nitrogen, straws are transferred into goblets on canes while under liquid nitrogen. The straws are stored in the liquid phase of a nitrogen tank and canes containing straws are not transferred from one container to another unless the goblet containing the straws is full of liquid nitrogen.Embryos held for longer than 4 hours after collection prior to freezing showed a steady decline in pregnancy rate related to the length of time held prior to freezing. The percentage of embryos thawed and then evaluated as being transferrable was related to the quality of the embryos prior to freeze (Grade 1–93.6%, Grade 2–87.0%, Grade 3–63.8%). There was no statistical difference in pregnancy rates obtained from prefreeze Grade 1 embryos when comparing advanced blastocysts (45.2%), blastocysts (38.7%), early blastoclyst (43.1%) and advanced morula (41.6%).     

Multiple freezing points as a test for viability of plant stems in the determination of frost hardiness

A technique is presented for a simple, rapid, and reliable means of determining the viability of plant tissue subjected to freezing temperatures. Freezing curves of excised stems of Cornus stolonifera Michx., and several other genera were studied. Tissue temperature was recorded during freezing of plant stem sections. The heat of crystallization deflected the resultant freezing curves at points where tissue froze. Living stem sections of all genera studied revealed 2 freezing points, while dead tissue exhibited only 1. The influence of variables such as moisture content, sample size, thermocouple placement, and cooling rate on freezing curves was analyzed. Stem samples wrapped in moisture-proof film with a thermocouple inserted into the pith were frozen to a predetermined test temperature, thawed, and subjected to a second freezing cycle. The presence or absence of 2 freezing points in the second freezing cycle was used as a criterion for establishing viability. The results were immediately available and identical to results from regrowth tests which took about 20 days.