Investigating the morphology and mechanical properties of blastomeres with atomic force microscopy

Atomic force microscopy (AFM) was used to directly investigate the morphology and mechanical properties of blastomeres during the embryo development. With AFM imaging, the surface topography of blastomeres from two‐cell, four‐cell, and eight‐cell stages was visualized, and the AFM images clearly revealed the blastomere's morphological changes during the different embryo developmental stages. The section measurements of the AFM topography images of the blastomeres showed that the axis of the embryos nearly kept constant during the two‐cell, four‐cell, and eight‐cell stages. With AFM indenting, the mechanical properties of living blastomeres from several embryos were measured quantitatively under physiological conditions. The results of mechanical properties measurements indicated that the Young's modulus of the two blastomeres from two‐cell embryo was different from each other, and the four blastomeres from the four‐cell embryo also had variable Young's modulus. Besides, the blastomeres from two‐cell embryos were significantly harder than blastomeres from four‐cell embryos. These results can improve our understanding of the embryo development from the view of cell mechanics. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a respirometric biochip for embryo assessment

A prototype respirometric biochip dedicated to monitoring oxygen consumption of preimplantation embryos has been developed. The biochip comprises a linear array of eight flow-through microchambers profiled on silicon substrate, and functions together with a phosphorescent oxygen sensitive probe and fluorescence plate reader detection. A high level of sensitivity to changes in dissolved oxygen was achieved through miniaturisation and optimization of biochip geometry, and incorporation of appropriate sealing and humidification systems. The biochips have allowed characterisation of oxygen consumption, by 2 cell and blastocyst stage preimplantation mouse embryos, through monitoring as few as ten preimplantation embryos over a one-hour time period. They provide a non-invasive, simple and convenient means for assessing preimplantation embryo metabolism.     

Three-dimensional imaging of sulfides in silicate rocks at submicron resolution with multiphoton microscopy

We report the first application of multiphoton microscopy (MPM) to generate three-dimensional (3D) images of natural minerals (micron-sized sulfides) in thick (～120 μm) rock sections. First, reflection mode (RM) using confocal laser scanning microscopy (CLSM), combined with differential interference contrast (DIC), was tested on polished sections. Second, two-photon fluorescence (TPF) and second harmonic signal (SHG) images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences.     

Imaging mitochondrial organization in living primate oocytes and embryos using multiphoton microscopy.

We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage-in the same embryo-thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.     

Resolving rotational motions of nano-objects in engineered environments and live cells with gold nanorods and differential interference contrast microscopy

Gold nanorods are excellent orientation probes due to their anisotropic optical properties. Their dynamic rotational motion in the 3D space can be disclosed with Nomarski-type differential interference contrast (DIC) microscopy. We demonstrate that by using the combination of gold nanorod probes and DIC microscopy, we are able to resolve rotational motions of nano-cargos transported by motor proteins at video rate not only on engineered surfaces but also on cytoskeleton tracks in live cells.     

Real-time dynamics of label-free single mast cell granules revealed by differential interference contrast microscopy

We demonstrate the capability of differential interference contrast (DIC) microscopy as a simple and useful tool for studying cellular events without fluorescence labeling. By coupling an advanced DIC microscope to a computer-controlled motorized vertical stage and a high-speed, high-resolution CCD camera, real-time three-dimensional monitoring is possible in a high-throughput manner. The performance among three modes of microscopy, bright-field, dark-field and DIC, in terms of horizontal resolving power and vertical sectioning was investigated. As a model, exocytosis of rat peritoneal mast cells was recorded on the subsecond time scale. Three-dimensional tracking of granules during degranulation was achieved and granule–granule fusion before plasma membrane fusion was recorded. Electronic Supplementary Material Supplementary material is available for this article at     

The effect of mixing methods on the dispersion of carbon nanotubes during the solvent-free processing of multiwalled carbon nanotube/epoxy composites

Several solvent-free processing methods to disperse multiwalled carbon nanotubes (MWCNTs) in bisphenol F-based epoxy resin were investigated, including the use of a microfluidizer (MF), planetary shear mixer (PSM), ultrasonication (US) and combinations. The processed mixture was cured with diethyl toluene diamine. Three complimentary techniques were used to characterize the dispersion of the MWCNTs in cured composite samples: optical microscopy, micro Raman spectroscopy, and scanning electron microscopy (SEM). For sample MF + PSM, optical micrographs and Raman images showed reduced agglomeration and a homogeneous distribution of MWCNTs in the epoxy matrix. SEM analysis of fractured specimen after tensile testing revealed breakage of nanotubes along the fracture surface of the composite. A comparison of the MWCNT dispersion in the epoxy samples processed using different methods showed that a combination of MF and PSM processing yields a more homogeneous sample than the PSM or US + PSM processed samples. Mechanical testing of the composites showed about 15% improvement in the tensile strength of samples processed by the MF + PSM method over other methods. Thermogravimetric analysis (TGA) results showed a small decrease in the onset degradation temperature for poorly dispersed samples produced by PSM compared with the well-mixed samples (MF + PSM). These results strongly suggest that the MF + PSM processing method yield better-dispersed and stronger MWCNT/epoxy composites. © 2012 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2013     

Visualization of the microtubules of glutaraldehyde-fixed cells by reflection-enhanced backscatter confocal microscopy.

Performing reflection-mode (backscatter-mode) confocal microscopy on cells growing on reflective substrates gives images that have improved contrast and are more easily interpreted than standard reflection-mode confocal micrographs (Keith et al., 1998). However, a number of factors degrade the quality of images taken with the highest-resolution microscope objectives in this technique. We here describe modifications to reflection-enhanced backscatter confocal microscopy that (partially) overcome these factors. With these modifications of the technique, it is possible to visualize structures the size-and refractility-of individual microtubules in intact cells. Additionally, we demonstrate that this technique, in common with fluorescence techniques such as standing wave widefield fluorescence microscopy and 4-Pi confocal microscopy, offers improved resolution in the Z-direction.     

Differential interference contrast microscopy for real-time dynamics and manipulation of single cells in microchannels

Nomarski differential interference contrast (DIC) microscopy was used for real-time dynamics of intact single cells in various microchannels for adaptation to microfluidic chip application. The cheek cell was chosen as a model, single cell and the dynamics was measured at the microchannels. The image resolution of single cell was shaper and more distinct in DIC than in conventional microscopy. The individual single living cells were also manipulated by both hydrodynamic and electrokinetic flow-driving forces at the microchannels. The DIC contrast was enhanced according to the order of round-, square-, and rectangle-type microchannels. The velocity of the single living cell was consistently increased with increasing electric field strength and pH. However, the velocity of cell was decreased with increasing run buffer concentration. The driving direction of the individual single cell was simply controlled by changing the polarity of the applied voltage and the electric field strength. The cells were consistently manipulated in the microchannel under the co-application of the low electric field of 2.44 V/cm, instead of the solo application of the hydrodynamic force.     

Endocytosis of a single mesoporous silica nanoparticle into a human lung cancer cell observed by differential interference contrast microscopy

The unique structural features of mesoporous silica nanoparticles (MSN) have made them very useful in biological applications, such as gene therapy and drug delivery. Flow cytometry, confocal microscopy, and electron microscopy have been used for observing the endocytosis of MSN. However, flow cytometry cannot directly observe the process of endocytosis. Confocal microscopy requires fluorescence labeling of the cells. Electron microscopy can only utilize fixed cells. In the present work, we demonstrate for the first time that differential interference contrast (DIC) microscopy can be used to observe the entire endocytosis process of MSN into living human lung cancer cells (A549) without fluorescence staining. There are three physical observables that characterize the locations of MSN and the stages of the endocytosis process: motion, shape, and vertical position. When it was outside the cell, the MSN underwent significant Brownian motion in the cell growth medium. When it was trapped on the cell membrane, the motion of the MSN was greatly limited. After the MSN had entered the cell, it resumed motion at a much slower speed because the cytoplasm is more viscous than the cell growth medium and the cellular cytoskeleton networks act as obstacles. Moreover, there were shape changes around the MSN due to the formation of a vesicle after the MSN had been trapped on the cell membrane and prior to entry into the cell. Finally, by coupling a motorized vertical stage to the DIC microscope, we recorded the location of the MSN in three dimensions. Such accurate 3D particle tracking ability in living cells is essential for studies of selectively targeted drug delivery based on endocytosis.     

Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells

We report on imaging living bacterial cells by using a correlated tapping-mode atomic force microscopy (AFM) and confocal fluorescence lifetime imaging microscopy (FLIM). For optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells, we explored different methods of bacterial sample preparation, such as spreading the cells on poly-L-lysine coated surfaces or agarose gel coated surfaces. We have found that the agarose gel containing 99% ammonium acetate buffer can provide sufficient local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and signal-to-noise ratio of the AFM images. Near-field AFM-tip-enhanced fluorescence lifetime imaging (AFM-FLIM) holds high promise on obtaining fluorescence images beyond optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging bacterial living cells, we demonstrated a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging of living bacterial cells to characterize cell polarity.     

Developmental phases of individual mouse preimplantation embryos characterized by lipid signatures using desorption electrospray ionization mass spectrometry

Knowledge of the lipids present in individual preimplantation embryos is of interest in fundamental studies of embryology, in attempts to understand cellular pluripotency and in optimization of in vitro culture conditions necessary for the application and development of biotechnologies such as in vitro fertilization and transgenesis. In this work, the profiles of fatty acids and phospholipids (PL) in individual mouse preimplantation embryos and oocytes were acquired using an analytical strategy based on desorption electrospray ionization mass spectrometry (DESI-MS). The methodology avoids sample preparation and provides information on the lipids present in these microscopic structures. Differences in the lipid profiles observed for unfertilized oocytes, two- and four-cell embryos, and blastocysts were characterized. For a representative set of embryos (N?=?114) using multivariate analysis (specifically principal component analysis) unfertilized oocytes showed a narrower range of PL species than did blastocysts. Two- and four-cell embryos showed a wide range of PLs compared with unfertilized oocytes and high abundances of fatty acids, indicating pronounced synthetic activity. The data suggest that the lipid changes observed in mouse preimplantation development reflect acquisition of a degree of cellular membrane functional and structural specialization by the blastocyst stage. It is also noteworthy that embryos cultured in vitro from the two-cell through the blastocyst stage have a more homogeneous lipid profile as compared with their in vivo-derived counterparts, which is ascribed to the restricted diversity of nutrients present in synthetic culture media. The DESI-MS data are interpreted from lipid biochemistry and previous reports on gene expression of diverse lipids known to be vital to early embryonic development.     

Roles of gangliosides in mouse embryogenesis and embryonic stem cell differentiation

Gangliosides have been suggested to play important roles in various functions such as adhesion, cell differentiation, growth control, and signaling. Mouse follicular development, ovulation, and luteinization during the estrous cycle are regulated by several hormones and cell-cell interactions. In addition, spermatogenesis in seminiferous tubules of adult testes is also regulated by several hormones, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and cell-cell interactions. The regulation of these processes by hormones and cell-cell interactions provides evidence for the importance of surface membrane components, including gangliosides. During preimplantation embryo development, a mammalian embryo undergoes a series of cleavage divisions whereby a zygote is converted into a blastocyst that is sufficiently competent to be implanted in the ma ternal uterus and continue its development. Mouse embryonic stem (mES) cells are pluripotent cells derived from mouse embryo, specifically, from the inner cell mass of blastocysts. Differentiated neuronal cells are derived from mES cells through the formation of embryonic bodies (EBs). EBs recapitulate many aspects of lineage-specific differentiation and temporal and spatial gene expression patterns during early embryogenesis. Previous studies on ganglioside expression during mouse embryonic development (including during in vitro fertilization, ovulation, spermatogenesis, and embryogenesis) reported that gangliosides were expressed in both undifferentiated and differentiated (or differentiating) mES cells. In this review, we summarize some of the advances in our understanding of the functional roles of gangliosides during the stages of mouse embryonic development, including ovulation, spermatogenesis, and embryogenesis, focusing on undifferentiated and differentiated mES cells (neuronal cells).     

Supravital fluorometric apoptosis detection in a single mouse embryo using lab-on-a-chip

Detection of apoptosis is one of the main criteria of preimplantation embryo growth potential assessment. Recent developments in lab-on-a-chip techniques has led to apoptosis detection and monitoring on a single cell or embryo level. However, single embryo apoptosis detection without a change in embryo developmental competence and post-examination "recovery" still remains a challenge. In this paper we present a lab-on-a-chip, co-working with miniaturized optical instrumentation, which allows supravital examination of single embryos for the presence of apoptotic blastomers with full after lab-on-a-chip study "recovery" and maintenance of their further developmental capacity.     

Atomic force microscopy and magnetic force microscopy study of model colloids

Atomic force microscopy (AFM) is used to study the size, shape, and polydispersity of a variety of magnetic and nonmagnetic model colloids, previously imaged by transmission electron microscopy (TEM) only. Both height and phase images are analyzed and special attention is given to 3D morphology and softness of particles, as well as structures and presence of secondary components in the colloid, difficult to investigate with TEM. Several methods of tip characterization followed by deconvolution were applied in order to improve the accuracy of lateral diameter determination. In the case of magnetite particles dispersed in conventional ferrofluids, we explore both experimentally and theoretically the possibility of using magnetic force microscopy (MFM). We propose and discuss several models which allow to estimate the magnetic moment of a single domain superparamagnetic sphere using MFM, which cannot be done with other techniques; alternatively the tip magnetization can be determined.     

Imaging and differentiation of mouse embryo tissues by ToF-SIMS

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) equipped with a gold ion gun was used to image mouse embryo sections and differentiate tissue types (brain, spinal cord, skull, rib, heart and liver). Embryos were paraffin-embedded and then deparaffinized. The robustness and repeatability of the method was determined by analyzing ten tissue slices from three different embryos over a period of several weeks. Using principal component analysis (PCA) to reduce the spectral data generated by ToF-SIMS, histopathologically identified tissue types of the mouse embryos can be differentiated based on the characteristic differences in their mass spectra. These results demonstrate the ability of ToF-SIMS to determine subtle chemical differences even in fixed histological specimens.     

A method for visualization of biomolecules labeled by a single quantum dot in living cells by a combination of total internal reflection fluorescence microscopy and intracellular fluorescence microscopy

We developed a simple fluorescence microscopy for acquisition of high-resolution images of single quantum dots (QDs) labeled to biomolecules on apical plasma membrane, in cell interior and on basal plasma membrane of living cells. The method was a combination of total internal reflection fluorescence microscopy (TIRFM) at apical cell surface and intracellular microscopy coupled with focusing objective. Insulin conjugated to single QD (insulin-QD) was chosen as the model system. In order to bind insulin-QDs to insulin receptors on the plasma membrane through the interaction between insulin and its receptor, as well as internalize them, the cells attached on a coverslip were incubated with biotinylated insulin and QD-streptavidin conjugate at 37 °C. Next, fluorescent molecules in the cells were photobleached by illuminating the cells using a 100-W mercury lamp with the wavelengths from 460 to 490 nm. Then, the incident angle of a laser beam was adjusted to produce total internal reflection at the apical surface of a single cell. In this case, the insulin-QDs in the whole cell were excited, and the fluorescent molecules outside the cell were not illuminated. Finally, the images of single insulin-QDs on the apical plasma membrane, in the cell interior and on the basal plasma membrane of the cell were taken by focusing the objective to different positions, respectively. The resolution and contrast of the fluorescent spots in the images were much higher than those obtained by using epi-fluorescence microscopy and comparable to those obtained by using the conventional TIRFM. The method improved the image acquisition speed for the images on the apical and basal plasma membrane using the conventional TIRFM, and could acquire the high-resolution images in the cell interior quickly.     

Atomic force microscopy investigation of phage infection of bacteria

Atomic force microscopy (AFM) was used to study the process of infection of bacterial cells by bacteriophages, for which purpose experimental protocols were elaborated. Three types of bacteriophages were characterized with AFM and transmission electron microscopy (TEM). Bacteriophage interaction with cells was studied for three bacterial hosts: Gram-negative Escherichia coli 057 and Salmonella enteritidis 89 and Gram-positive Bacillus thuringiensis 393. Depending on the phase of lytic cycle, different cell surface changes are observed in AFM images of infected cells in comparison with intact cells: from phage adsorption on the cells and flagella to complete lysis of the cells, accompanied by the release of a large number of newly formed phages. Control experiments (cells without phages and cells with nonspecific phages) did not reveal any surface changes. Penetration of phages inside obligate aerobe Bacillus thuringiensis was shown to be oxygen-dependent and required aeration in laboratory conditions. Our results show great potential of using AFM for numerous fundamental and applied tasks connected with pathogen-host interaction.     

A Set of Homo‐Oligomeric Standards Allows Accurate Protein Counting

Techniques based on fluorescence microscopy are increasingly used to count proteins in cells, but few stoichiometrically well‐defined standards are available to test their accuracy. A selection of bacterial homo‐oligomers were developed that contain 10–24 subunits and fully assemble when expressed in mammalian cells, and they can be used to easily validate/calibrate molecular counting methods. The utility of these standards was demonstrated by showing that nuclear pores contain 32 copies of the Nup107 complex.     

Intracellular concentration map of magnesium in whole cells by combined use of X-ray fluorescence microscopy and atomic force microscopy

We report a novel experimental approach to derive quantitative concentration map of light elements in whole cells by combining two complementary nano-probe methods: X-ray fluorescence microscopy (XRFM) and atomic force microscopy (AFM). The concentration is derived by normalizing point-by-point the elemental (here Mg) spatial distribution obtained by XRFM, by the thickness measured using AFM. The considerable difference between the elemental distribution and the concentration maps indicates that this procedure is essential to obtain reliable information on the role and function of elements in whole cells.