A cryo-SEM technique developed and applied to surfactant liposomes

1993 ◽  
Vol 51 ◽  
pp. 284-285
Author(s):  
Kiran Bhadriraju ◽  
Jayesh Bellare
Keyword(s):  
Sample Preparation ◽  
Liquid Nitrogen ◽  
Freeze Drying ◽  
Freezing Point ◽  
Freeze Fracture ◽  
Depth Of Field ◽  
Particle Sizes ◽  
Copper Sheet ◽  
Freeze Etching ◽  
Liquid Nitrogen Bath

Freeze-fracture replication TEM and Cryo-TEM are developed techniques for studying surfactant dispersions. Application of freeze-fracture cryo-SEM with direct imaging to such systems has the advantages of observing a greater range of particle sizes, large depth of field implying larger tilts together with rotation, and freeze-etching/freeze drying the sample while imaging it. A procedure for cryo-SEM of liquid colloids, which uses a simple sample preparation setup, and its results for liposomal dispersions, are described here.Samples are plunge-frozen by a freezing device (Fig.l) made from a standard desoldering tool (Fig.2) used as a plunging unit. Fracture plates (Fig.3) are made from 0.1 mm thin copper sheet made adhesive to the liquid by 400 mesh TEM grids that are bent over the two edges of the plates and stuck on the non-sample side with a rubber adhesive. The sample is sandwiched between a pair of fracture plates (Fig.3) and plunged into liquid Freon-22 kept at its freezing point (-160°C) in an electrically heated cup (Fig.l) cooled by a liquid liquid nitrogen bath.

scholarly journals Freeze-fracturing in normal vacuum reveals ringlike yeast plasmalemma structures

1978 ◽  
Vol 79 (1) ◽  
pp. 276-280 ◽  
Author(s):  
UB Sleytr ◽  
P Messner
Keyword(s):  
Liquid Nitrogen ◽  
Freeze Fracture ◽  
Ultrahigh Vacuum ◽  
Drastic Reduction ◽  
Preparation Conditions ◽  
Vacuum Technology ◽  
Significant Difference ◽  
Specimen Area ◽  
Freeze Etching ◽  
Regular Arrays

The fine structure of the regular arrays of subunits seen on both plasmalemma fracture faces in resting and starved Saccharomyces cerevisiae (baker's yeast) has been compared using different freeze-fracture replication methods. Freeze-cleaving was carried out at 173 degrees, 133 degrees, and 108 degrees K under a vacuum of 2 X 10(-7) torr (2.6 X 10(- 7)mbar) or under liquid nitrogen at atmosphereic pressure. Independent of the preparation conditions (fracturing temperature, and whether cleaved under vacuum or liquid nitrogen), resting and starved yeast show a significant difference in the morphology of the subunits forming the regular arrays. The regularly arranged particles of the P face of the plasmalemma of starved yeast have a clear craterlike structure which has previously been reported to be demonstrated only by freeze-etching at very low temperatures in ultrahigh vacuum. A complementary structure is seen on the plasmalemma E face. Prolonged exposures of fracture faces under the protection of liquid nitrogen-cooled shrouds have shown that, because of the consequent drastic reduction of condensable gases in the specimen area, no detectable condensation contamination of exposed fracture faces occurs within 15 min at a specimen temperature of 108 degrees K. This shows that a complicated ultrahigh vacuum technology is not required for high resolution freeze- etching.

scholarly journals Porous PLGA Microspheres Effectively Loaded with BSA Protein by Electrospraying Combined with Phase Separation in Liquid Nitrogen

2010 ◽  
Vol 6 ◽  
pp. 1-18 ◽  
Author(s):  
G. Liu ◽  
X. Miao ◽  
W. Fan ◽  
Ross Crawford ◽  
Yin Xiao
Keyword(s):  
Porous Structure ◽  
Liquid Nitrogen ◽  
Encapsulation Efficiency ◽  
Freeze Drying ◽  
Double Emulsion ◽  
Polymer Microspheres ◽  
Particle Sizes ◽  
Plga Microspheres ◽  
Loading Efficiency ◽  
Highly Porous

Polymer microspheres loaded with bioactive particles, biomolecules, proteins, and/or growth factors play important roles in tissue engineering, drug delivery, and cell therapy. The conventional double emulsion method and a new method of electrospraying into liquid nitrogen were used to prepare bovine serum albumin (BAS)-loaded poly(lactic-co-glycolic acid) (PLGA) porous microspheres. The particle size, the surface morphology and the internal porous structure of the microspheres were observed using scanning electron microscopy (SEM). The loading efficiency, the encapsulation efficiency, and the release profile of the BSA-loaded PLGA microspheres were measured and studied. It was shown that the microspheres from double emulsion had smaller particle sizes (3-50 m), a less porous structure, a poor loading efficiency (5.2 %), and a poor encapsulation efficiency (43.5%). However, the microspheres from the electrospraying into liquid nitrogen had larger particle sizes (400-600 m), a highly porous structure, a high loading efficiency (12.2%), and a high encapsulation efficiency (93.8%). Thus the combination of electrospraying with freezing in liquid nitrogen and subsequent freeze drying represented a suitable way to produce polymer microspheres for effective loading and sustained release of proteins.

THE EFFECT OF METHOD OF SAMPLE PREPARATION UPON THE DETERMINATION OF SOLUBILIZED INTRAMUSCULAR COLLAGEN FROM BOVINE MUSCLE BY QUANTITATION OF ITS HYDROXYPROLINE CONTENT

1980 ◽  
Vol 60 (3) ◽  
pp. 627-634 ◽  
Author(s):  
L. E. JEREMIAH ◽  
A. C. MURRAY ◽  
A. H. MARTIN
Keyword(s):  
Sample Preparation ◽  
Liquid Nitrogen ◽  
Muscle Tissue ◽  
Recovery Rate ◽  
Freeze Drying ◽  
Soluble Fraction ◽  
Collagen Content ◽  
Hydroxyproline Content ◽  
Bovine Muscle

A total of 91 muscle samples were utilized in a series of three separate but related experiments to evaluate possible effects of method of sample preparation upon the yields of intramuscular hydroxyproline from the heat-soluble, insoluble and combined fractions of bovine muscle tissue. The combined results of these experiments indicate that yields of intramuscular hydroxyproline, and thereby yields of intramuscular collagen, obtained in different studies can not be compared unless the same muscle is evaluated and the same method of sample preparation is employed in both and/or all studies in which yields are to be compared. Results from these experiments may also suggest that sample preparation, in which samples are frozen in liquid nitrogen, results in underestimation of intramuscular hydroxyproline content, and thereby intramuscular collagen content, from the heat-soluble fraction as evidenced by: (1) the substantially lower yields of intramuscular hydroxyproline from the heat-soluble fraction of samples frozen in liquid nitrogen; (2) the fact that added hydroxyproline was completely recovered in the heat-soluble fraction of muscle during preliminary experiments; and (3) the fact that method of sample preparation (freeze drying vs. direct homogenization) did not influence the recovery rate or result in overestimation of added hydroxyproline.

Ca-Histochemistry in slime mold plasmodia

1978 ◽  
Vol 36 (2) ◽  
pp. 130-131
Author(s):  
Ulrich Dierkes
Keyword(s):  
Physarum Polycephalum ◽  
Carbon Coating ◽  
Freeze Drying ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Slime Mold ◽  
Staining Procedure ◽  
Protoplasmic Streaming ◽  
Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

Freeze-Fracture Replication of Frozen Outer Segments of Rat Retinal Rods

1969 ◽  
Vol 27 ◽  
pp. 392-393
Author(s):  
Thomas S. Leeson ◽  
C. Roland Leeson
Keyword(s):  
Electron Microscopy ◽  
Cross Section ◽  
Outer Segment ◽  
Freeze Fracture ◽  
X Ray Diffraction ◽  
X Ray ◽  
Outer Segments ◽  
Retinal Rods ◽  
Temperature Electron ◽  
Freeze Etching

Numerous previous studies of outer segments of retinal receptors have demonstrated a complex internal structure of a series of transversely orientated membranous lamellae, discs, or saccules. In cones, these lamellae probably are invaginations of the covering plasma membrane. In rods, however, they appear to be isolated and separate discs although some authors report interconnections and some continuities with the surface near the base of the outer segment, i.e. toward the inner segment. In some species, variations have been reported, such as longitudinally orientated lamellae and lamellar whorls. In cross section, the discs or saccules show one or more incisures. The saccules probably contain photolabile pigment, with resulting potentials after dipole formation during bleaching of pigment. Continuity between the lamina of rod saccules and extracellular space may be necessary for the detection of dipoles, although such continuity usually is not found by electron microscopy. Particles on the membranes have been found by low angle X-ray diffraction, by low temperature electron microscopy and by freeze-etching techniques.

Freeze-fracture cytochemistry: A goal set by Russell Steere in 1957

1993 ◽  
Vol 51 ◽  
pp. 60-61
Author(s):  
Nicholas J Severs
Keyword(s):  
Chemical Nature ◽  
Biological Systems ◽  
Freeze Fracture ◽  
Structural Components ◽  
Major Goal ◽  
Membrane Biology ◽  
Routine Application ◽  
The Future ◽  
Freeze Etching

In his pioneering demonstration of the potential of freeze-etching in biological systems, Russell Steere assessed the future promise and limitations of the technique with remarkable foresight. Item 2 in his list of inherent difficulties as they then stood stated “The chemical nature of the objects seen in the replica cannot be determined”. This defined a major goal for practitioners of freeze-fracture which, for more than a decade, seemed unattainable. It was not until the introduction of the label-fracture-etch technique in the early 1970s that the mould was broken, and not until the following decade that the full scope of modern freeze-fracture cytochemistry took shape. The culmination of these developments in the 1990s now equips the researcher with a set of effective techniques for routine application in cell and membrane biology.Freeze-fracture cytochemical techniques are all designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied.

HVEM Cryomicroscopy of Frozen Hydrated PTK1 Cells

1990 ◽  
Vol 48 (1) ◽  
pp. 500-501
Author(s):  
E.T. O’Toole ◽  
G.P. Wray ◽  
J.R. Kremer ◽  
J.R. Mcintosh
Keyword(s):  
Liquid Nitrogen ◽  
Phase Contrast ◽  
Culture Medium ◽  
Low Dose ◽  
Ammonium Acetate ◽  
Freezing Point ◽  
Voltage Electron ◽  
Cold Stage ◽  
Humidity Chamber ◽  
Coated Carbon

Ultrarapid freezing and cryomicroscopy of frozen hydrated material makes it possible to visualize samples that have never been exposed to chemical fixatives, dehydration, or stains. In principle, freezing and cryoimaging methods avoid artifacts associated with chemical fixation and processing and allow one to visualize the specimen in a condition that is close to its native state. Here we describe a way to use a high voltage electron microscope (HVEM) for the cryoimaging of frozen hydrated PTK1 cells.PTK1 cells were cultured on formvar-coated, carbon stabilized gold grids. After three days in culture, the grids were removed from the culture medium and blotted in a humidity chamber at 35° C. In some instances, the grids were rinsed briefly in 0.16 M ammonium acetate buffer (pH 7.2) prior to blotting. After blotting, the grids were transferred to a plunging apparatus and plunged into liquid ethane held directly above its freezing point. The plunging apparatus consists of a vertical slide rail that guides the fall of a mounted pair of forceps that clamp the specimen. The forceps are surrounded by a plexiglass humidity chamber mounted over a dewar of liquid nitrogen containing an ethane chamber. After freezing, the samples were transferred to liquid nitrogen and viewed in a JEOL JEM 1000 equipped with a top entry cold stage designed and built by Mr. George Wray (Univ. Colorado). The samples were routinely exposed to electron doses of 1 e/Å2/sec, and viewed at a temperature of −150° C. A GATAN video system was used to enhance contrast and to estimate the correct amount of underfocus needed to obtain phase contrast at various magnifications. Low dose micrographs were taken using two second exposures of Kodak 4463 film. The state of the solid water in the specimen was determined by diffraction using a 30/μm field limiting aperture and a camera length of 1 meter.

Rate of Sublimation of Ice by Radiative Heating in Freeze-Etching

1980 ◽  
Vol 38 ◽  
pp. 618-619
Author(s):  
Yeshayahu Talmon
Keyword(s):  
Heat Flux ◽  
Freeze Fracture ◽  
Constant Heat Flux ◽  
Radiative Heating ◽  
Constant Heat ◽  
Entire Sample ◽  
Simplified Method ◽  
Freeze Etching ◽  
Sublimation Rates ◽  
Fractured Surface

To bring out details in the fractured surface of a frozen sample in the freeze fracture/freeze-etch technique,the sample or part of it is warmed to enhance water sublimation.One way to do this is to raise the temperature of the entire sample to about -100°C to -90°C. In this case sublimation rates can be calculated by using plots such as Fig.1 (Talmon and Thomas),or by simplified formulae such as that given by Menold and Liittge. To achieve higher rates of sublimation without heating the entire sample a radiative heater can be used (Echlin et al.). In the present paper a simplified method for the calculation of the rates of sublimation under a constant heat flux F [W/m2] at the surface of the sample from a heater placed directly above the sample is described.

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