A novel approach for 3D reconstruction of mice full-grown oocytes by time-of-flight secondary ion mass spectrometry.

Currently two techniques exist for 3D reconstruction of biological samples by time-of-flight secondary ion mass spectrometry (ToF-SIMS). The first, based on microtomy and combining of successive section images, is successfully applied for tissues, while the second, based on sputter depth profiling, is widely used for cells. In the present work, we report the first successful adaptation of sectioning technique for ToF-SIMS 3D imaging of a single cell-fully grown mouse germinal vesicle (GV) oocyte. In addition, microtomy was combined with sputter depth profiling of individual flat sections for three-dimensional reconstruction of intracellular organelles. GV oocyte sectioning allowed us to obtain molecule-specific 3D maps free from artifacts associated with surface topography and uneven etching depth. Sputter depth profiling of individual flat slices revealed fine structure of specific organelles inside the oocyte. Different oocyte organelles (cytoplasm, germinal vesicle, membranes, cumulus cells) were presented on the ion images. Atypical nucleoli referred to as "nucleolus-like body" (NLB) was detected inside the germinal vesicle in PO3- and CN- ions generated by nucleic acids and proteins respectively. Significant difference in PO3- intensity in the NLB central area and NLB border was found. This difference appears as a bright halo around the center area. The NLB size calculated for PO3- and CN- ion images is 12.9 ± 0.2 µm and 11.9 ± 0.2 µm respectively, which suggests that bright halo of PO3- ions is a chromatin compaction on the NLB surface. Areas of approximately 1.0-2.5 µm size inside nucleoplasm with increased PO3- and CN- signal were registered in germinal vesicle. Observed compartments have different sizes and shapes, and they are likely attributed to chromocenters or chromosomes.

Correlating microscopy techniques and ToF-SIMS analysis of fully grown mammalian oocytes.

The 2D-molecular thin film analysis protocol for fully grown mice oocytes is described using an innovative approach. Time-of-flight secondary ion mass spectrometry (ToF-SIMS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and optical microscopy imaging were applied to the same mice oocyte section on the same sample holder. A freeze-dried mice oocyte was infiltrated into embedding media, e.g. Epon, and then was cut with a microtome and 2 µm thick sections were transferred onto an ITO coated conductive glass. Mammalian oocytes can contain "nucleolus-like body" (NLB) units and ToF-SIMS analysis was used to investigate the NLB composition. The ion-spatial distribution in the cell components was identified and compared with the images acquired by SEM, AFM and optical microscopy. This study presents a significant advancement in cell embryology, cell physiology and cancer-cell biochemistry.

High resolution 3D microscopy study of cardiomyocytes on polymer scaffold nanofibers reveals formation of unusual sheathed structure.

Building functional and robust scaffolds for engineered biological tissue requires a nanoscale mechanistic understanding of how cells use the scaffold for their growth and development. A vast majority of the scaffolds used for cardiac tissue engineering are based on polymer materials, the matrices of nanofibers. Attempts to load the polymer fibers of the scaffold with additional sophisticated features, such as electrical conductivity and controlled release of the growth factors or other biologically active molecules, as well as trying to match the mechanical features of the scaffold to those of the extracellular matrix, cannot be efficient without a detailed knowledge of how the cells are attached and strategically positioned with respect to the scaffold nanofibers at micro and nanolevel. Studying single cell - single fiber interactions with the aid of confocal laser scanning microscopy (CLSM), scanning probe nanotomography (SPNT), and transmission electron microscopy (TEM), we found that cardiac cells actively interact with substrate nanofibers, but in different ways. While cardiomyocytes often create a remarkable "sheath" structure, enveloping fiber and, thus, substantially increasing contact zone, fibroblasts interact with nanofibers in the locations of focal adhesion clusters mainly without wrapping the fiber. STATEMENTS OF SIGNIFICANCE: We found that cardiomyocytes grown on electrospun polymer nanofibers often create a striking "sheath" structure, enveloping fiber with the formation of a very narrow (∼22 nm) membrane gap leading from the fiber to the extracellular space. This wrapping makes the entire fiber surface available for cell attachment. This finding gives a new prospective view on how scaffold nanofibers may interact with growing cells. It may play a significant role in effective design of novel nanofiber scaffolds for tissue engineering concerning mechanical and electrical properties of scaffolds as well as controlled drug release from "smart" biomaterials.

Influence of exposure to vitrification solutions on 2-cell mouse embryos: II. Osmotic effects or chemical toxicity?

In the companion paper (CryoLetters, 2007, 28:403-408), we reported effects of exposure of two-cell mouse embryos to vitrification solutions containing different vitrificants (EG, PG and DMSO) on the intracellular potassium and sodium content. We also compared exposure of 30% (v:v) ethylene glycol for 1.5 min to the similar experiments with 3-min exposure reported previously (CryoLetters, 2006, 27:87-98). In all experiments, four step protocols (2 steps of addition and 2 steps of washing) were used. Here we present mathematical modeling of the cell osmotic response using the relativistic permeability (RP) approach, which allows calculation of the osmotic curves without using simulation software but by direct calculations of the cell volume, intracellular concentration, and amount of the permeable vitrificants (Cryobiology, 2006, 53:402-3). Magnitude of the maximum cell volume excursion and other important osmotic characteristics were calculated for each step of the protocol, and the results of the mathematical modeling were superimposed onto the experimental data reported and discussed in the companion paper. The osmotic damage vs. specific chemical toxicity of the vitrificants as the major cause of the elemental disturbance of intracellular potassium and sodium content are discussed.

Influence of exposure to vitrification solutions on 2-cell mouse embryos: I. Intracellular potassium and sodium content.

Intracellular concentrations of potassium and sodium in two-cell mouse embryos in G1/S phase after exposure to vitrification solutions containing vitrificant agents (VFAs) ethane-1,2-diol (ethylene glycol, EG), propane-1,2-diol (propylene glycol, PG), or dimethyl sulfoxide (DMSO), and sucrose (S) after incubation in Dulbecco solution were measured by electron probe microanalysis (EPMA) as described earlier (CryoLetters, 2006, 27: 87-98). The 4-step protocol was as followed: 10% VFA for 10 min => 30% VFA + 0.7 M S for 1.5 min ==> 0.5 M S for 10 min ==> 10 min pure Dulbecco's. The cytoplasmic concentration of potassium and sodium in immediately flashed out from the oviduct embryos was in range of 120 +/- 2 mM, with good concordance with the previous data (CryoLetters, 2006, 27:87-98). Exposure in Dulbecco's for 30 min did not alter elemental composition, neither did exposure in PG or DMSO for 1.5 min. In contrast, exposure for 1.5 min in EG dropped the level of potassium to 96 +/- 2 mM while elevating level of cytoplasmic sodium to 136 +/- 3 mM. Further exposure to 30%-EG for 3 min led to a two-fold decrease of both elements (60 +/- 3 mM and 66 +/- 2 mM for K and Na, respectively).

Changes in intracellular potassium and sodium content of 2-cell mouse embryos induced by exposition to vitrification concentrations of ethylene glycol.

Intracellular concentration of potassium and sodium in two-cell mouse embryos in G1/S phase after exposition to vitrification solutions containing ethylene glycol (EG) and sucrose or after incubation in Dulbecco solution were measured by electron probe microanalysis (EPMA). The embryos at room temperature were treated in 10 percent EG for 10 min, transferred into mixture of EG and 1.0 M sucrose in ratio of 3:7 (v/v) for 3 min, then to 0.5 M sucrose for 10 min followed by washing the cells with Dulbecco;s solution for 10 min prior to analysis. The cytoplasmic concentration of potassium and sodium in controlled untreated with EG embryos were in a range of 116-130 mM of potassium and 120 mM of sodium, with good concordance in two identical experiments. After exposition that mimicked vitrification protocols, the intracellular potassium dropped almost two-three-fold (47 + 3 mM in one experiment and to 70 mM in the second experiment. The intracellular sodium concentration also decreased two-fold in range 60-70 mM after treatment with EG. Possible mechanisms of changes in the intracellular elemental concentrations including the high intracellular sodium observed in intact embryos are discussed.