A. G. Pogorelov

Evaluation of Collagen Gel Microstructure by Scanning Electron Microscopy

We performed qualitative comparison of freeze drying and chemical drying as methods of preparing 3D wet specimens for scanning electron microscopy. Human fibroblasts immobilized in collagen gel were used as a model system. Specimens fixed with glutaraldehyde were frozen in liquid nitrogen and freeze-dried at low temperature in high vacuum. In parallel experiments, glutaraldehyde-fixed samples were dehydrated in ascending ethanol solutions, absolute ethanol, and 100% hexamethyldisilazane and then dried at room temperature. Scanning electron microscopy microphotographs of collagen fibers and cells were characterized by high resolution and the absence of collapsed or deformed structures even at high magnification (×50,000) for both chemical drying and high-vacuum freeze drying. However, high-vacuum freeze drying is superior to chemical drying for the investigation of the internal space of 3D scaffolds, because sample fracture can be prepared directly in liquid nitrogen. These techniques are a part of the sample preparation process for scanning electron microscopy and can also be used for studies of cell adhesion, morphology, and arrangement in wet specimens (3D gels and flexible tissue engineering scaffolds).

Changes in intracellular potassium concentration in a one-cell mouse embryo after enucleation

Comparative analysis of potassium concentrations in the cytoplasm of intact and enucleated one-cell mouse embryos of showed that microsurgical manipulations during collection of pronuclei disordered potassium homeostasis in the embryonic cell.

Amino Acid Correction of Regulatory Volume Decrease Evoked by Hypotonic Stress in Mouse Oocytes In Vitro

Regulatory volume decrease in response to hypotonic stress is typical of the oocytes and early mouse embryos. Changes in the kinetics of osmotic reaction can be used as a marker of the modulating effect of the incubation medium on transmembrane transport in embryonic cells. Quantitative laser scanning microtomography (QLSM) was used to measure oocyte volume. In this paper, it is shown that addition of 5 μM glycine, taurine, or GABA, as well as ATP to Dulbecco’s medium abolished the regulatory volume decrease in mature mouse oocytes.

Scanning Electron Microscopy of Biofilms Adherent to the Inner Catheter Surface

The inner surface of the drainage catheter used in surgical interventions for biliary system pathologies was examined by scanning electron microscopy. Microfl ora in the catheter lumen refl ects etiological characteristics of the pathological process and helps to predict possible complications. The developed scanning electron microscopy imaging technique of visualization of the fi ne spatial structure of microbial biofi lm formed on the catheter surface allows describing the cell pool and structure of the biofi lm.

Scanning Electron Microscopy of Biosynthetic Wound Dressings Biocol

The surface of wound dressing Biocol was studied by scanning electron microscopy. This composite system consists of latex matrix with incorporated water-soluble polysaccharide. The peculiarities of the surface are important for manufacturing of the dressing and for modifi cation of its surface upon contact with fl uids, e.g. during de novo tissue reconstruction. The method for studying the fi ne structure of the polymeric fi lm surface was developed. The relief of the wound dressing changes during interaction with the fl uid and nanopores appear on the surface. Thus, scanning electron microscopy is an informative method for studying the surface of biosynthetic films.

Three dimensional reconstruction of two-cell mouse embryo by laser scanning microscopy

The direct method of volumetric parameters’ measurement of early mammal embryo was developed. The sample preparation was based on embryo cryofixation followed by laser scanning microscopy. Digital image processing and three dimensional reconstruction of embryos was performed with standard graphic software. The availability of a developed technique for the analysis of cell physiology at cultivation conditions was demonstrated on isolated two-cell mouse embryo.

Hypoxia during mammalian preimplantation development: Extreme circumstance vs. typical environment

The given paper summarizes the data on the early mammalian embryo development in culture media containing low oxygen concentration. Experimental results on in vitro modeling the hypoxia for preimplantation development are reviewed. Hypoxic conditions were shown to be available in the female reproductive tract of different mammalian species. The estimation of the embryo developing in vitro exhibits that lower oxygen level in culture media improves embryonic quality.

Laser-scanning microscopy as applied to mouse early embryos: Cytometry and analysis of cell morphology

This paper updates our knowledge on quantitative laser scanning microscopy and summarizes the capabilities of this method as applied to cytometry and analysis of cell structure of the mouse early embryo and the oocyte. This method requires a stack of optical sections obtained as Z-series with subsequent 3D reconstruction. This approach was used for visualization of the 3D cell model, measurement of the cell volume and surface area, as well as a study of the cell interior via optical sections. To maintain the dimensional characteristics of embryos or oocytes the method of sample preparation involved the following consequent steps: rapid cryofixation, low-temperature dehydration, infiltration by optically transparent mounting media, and laser-scanning microscopy. This strategy enables volume measurement, even in the case of a single cell within the multicellular system of the mouse early embryo.

Analysis of changes in cell volume of a mouse early embryo exposed to osmotic shock.

The impact of the osmotic component of the incubation medium for the volume of mouse early embryonic cell was studied by laser scanning microscopy. Common Dulbecco’s medium caused a prolonged hyperosmotic effect. Adaptive phase of regulatory compensation for the osmotic shock was observed under hypotonic conditions. From these data, water permeability of the blastomer membrane is evaluated as 0.4 μ/(min×atm).

Accelerated utilization of lactate under the effect of hypoxen after intensive exercise

Administration of substrates of energy metabolism in combination with hypoxen (sulfur-containing oligoquinone) promoted the increase in blood lactate concentration during maximum exercise, accelerated lactate utilization, and reduced the lactate/pyruvate ratio during recovery. The in vivo effects are in line with hypoxen capacity to accelerate in vitro oxidation of exogenous NADH in mitochondria by the non-rotenone-dependent pathway realized with participation of cytochrome C.