The prospect of utilizing stem cells for clinical applications has generated an enormous amount of enthusiasm in the stem cell research community and has led to a wealth of new data that offer the possibility for practical applications into clinical translation. Recent advances in stem cell imaging has contributed greatly to the ultimate goal to identify and culture specific cell types for regeneration of tissue that had been affected by disease such as Parkinson's, Alzheimer's, heart disease, muscle diseases, and others.

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.

At the beginning of the last century, reproductive biologists have discussed whether in mammalian species the fetal oocytes persist or are replaced by neo-oogenesis during adulthood. Currently the prevailing view is that neo-oogenesis is functional in lower vertebrates but not in mammalian species. However, contrary to the evolutionary rules, this suggests that females of lower vertebrates have a better opportunity to provide healthy offspring compared to mammals with oocytes subjected to environmental threats for up to several decades. During the last 15 years, a new effort has been made to determine whether the oocyte pool in adult mammals is renewed as well. Most recently, Ji Wu and colleagues reported a production of offspring from female germline stem cells derived from neonatal and adult mouse ovaries. This indicates that both neonatal and adult mouse ovaries carry stem cells capable of producing functional oocytes. However, it is unclear whether neo-oogenesis from ovarian somatic stem cells is physiologically involved in follicular renewal and why menopause occurs. Here we review observations that indicate an involvement of immunoregulation in physiological neo-oogenesis and follicular renewal from ovarian stem cells during the prime reproductive period and propose why menopause occurs in spite of persisting ovarian stem cells.

The role of centrosomes in stem cell division has recently been highlighted and further ascribes important functions to centrosomes in stem cell maintenance, cellular differentiation, and development. Advanced cell and molecular studies coupled with immunofluorescence, electron microscopy, and live cell imaging of specific centrosome proteins have contributed greatly to our knowledge of centrosome composition, structure, and dynamics and have uncovered new insights into mechanisms of centrosome functions in asymmetric cell division. The establishment of asymmetry and differential positioning of mother and daughter centrosomes during stem cell mitosis is important for allowing one cell to maintain stem cell characteristics while the sibling cell undergoes differentiation. Another key role for centrosomes has been revealed in primary cilia of embryonic stem cells that play significant roles in cellular signaling and are therefore critically important for stem cell decisions. Studies of signaling through primary cilia may contribute important information that may aid in the production of specific cells that are suitable for tissue repair and regeneration in the field of regenerative medicine.

Adult stem cells (SCs) are essential for tissue homeostasis and wound repair. They have the ability to both self-renew and differentiate into multiple cell types. They often reside in specialized microenvironments or niches that preserve their proliferative and tissue regenerative capacity. The murine hair follicle (HF) has a specialized and permanent compartment—the bulge, which safely lodges SCs and provides the necessary molecular cues to regulate their function. The HF undergoes cyclic periods of destruction, regeneration, and rest, making it an excellent system to study SC biology.

The process of neurogenesis and formation of neural stem cells is reported in human neurospheres (NS) and embryoid bodies (EB) derived from human embryonic stem cells, in vitro, and compared with neural tissue formed in human ectopic embryos in week 4 (stage 9), developed in vivo. This morphological study was done using digital imaging by light microscopy and routine transmission electron microscopy. Both NS and EB form neural rosettes from the surface epithelium much like the process of neural tube formation from ectoderm in the embryo. The rosette is the developmental signature of neuroprogenitors in cultures of differentiating embryonic stem cells and is a radial arrangement of columnar cells that express many of the proteins expressed in neuroepithelial cells in the neural tube. The NS produce all of the major classes of progeny of the neural tube, some of which have been documented here. Specific neural markers expressed in the NS and the clinical implications of this study in cell therapy are also discussed.

Tumors consist of a heterogeneous population of neoplastic cells infiltrated by an equally heterogeneous collection of nonneoplastic cells that comprise the tumor microenvironment. Tumor growth, invasion, and metastasis depend on multiple interactions between these cells. To assess their potential as therapeutic targets or vehicles for tumor specific delivery of therapeutic agents, we examined the contribution of bone marrow derived cells (BMDCs) to the intestinal tumor microenvironment. Hematopoietic stem cells expressing the enhanced green fluorescent protein (eGFP) were transplanted into lethally irradiated ApcMin/+ mice, and their engraftment was analyzed by confocal microscopy. The results showed abundant infiltration of eGFP cells into the small intestine, colon, and spleen compared to heart, muscle, liver, lung, and kidney. Within the intestine, there was a pronounced gradient of engraftment along the anterior to posterior axis, with enhanced infiltration into adenomas. Immunofluorescence analysis showed that osteopontin was expressed in tumor stromal cells but not in nontumor stromal populations, suggesting that gene expression in these cells is distinct. Tumor vasculature in ApcMin/+ mice was chaotic compared to normal intestinal regions. Our data suggest that BMDCs can be harnessed for tumor-targeted therapies to enhance antitumor efficacy.

Detection and tracking of stem cell state are difficult due to insufficient means for rapidly screening cell state in a noninvasive manner. This challenge is compounded when stem cells are cultured in aggregates or three-dimensional (3D) constructs because living cells in this form are difficult to analyze without disrupting cellular contacts. Multiphoton laser scanning microscopy is uniquely suited to analyze 3D structures due to the broad tunability of excitation sources, deep sectioning capacity, and minimal phototoxicity but is throughput limited. A novel multiphoton fluorescence excitation flow cytometry (MPFC) instrument could be used to accurately probe cells in the interior of multicell aggregates or tissue constructs in an enhanced-throughput manner and measure corresponding fluorescent properties. By exciting endogenous fluorophores as intrinsic biomarkers or exciting extrinsic reporter molecules, the properties of cells in aggregates can be understood while the viable cellular aggregates are maintained. Here we introduce a first generation MPFC system and show appropriate speed and accuracy of image capture and measured fluorescence intensity, including intrinsic fluorescence intensity. Thus, this novel instrument enables rapid characterization of stem cells and corresponding aggregates in a noninvasive manner and could dramatically transform how stem cells are studied in the laboratory and utilized in the clinic.

Little is known about how age influences the ways in which cardiac fibroblasts interact with the extracellular matrix. We investigated the deformation of collagen substrates by neonatal and adult rat cardiac fibroblasts in monolayer and three-dimensional (3D) cultures, and quantified the expression of three collagen receptors [discoidin domain receptor (DDR)1, DDR2, and β1 integrin] and the contractile protein alpha smooth muscle actin (α-SMA) in these cells. We report that adult fibroblasts contracted 3D collagen substrates significantly less than their neonate counterparts, whereas no differences were observed in monolayer cultures. Adult cells had lower expression of β1 integrin and α-SMA than neonate cultures, and we detected significant correlations between the expression of α-SMA and each of the collagen receptors in neonate cells but not in adult cells. Consistent with recent work demonstrating age-dependent interactions with myocytes, our results indicate that interactions between cardiac fibroblasts and the extracellular matrix change with age.

This work comprises the structural characterization of Eucalyptus and Pinus radiata pulp fibers and their corresponding fibrillated materials, based on quantitative electron microscopy techniques. Compared to hardwood fibers, the softwood fibers have a relatively open structure of the fiber wall outer layers. The fibrillation of the fibers was performed mechanically and chemi-mechanically. In the chemi-mechanical process, the pulp fibers were subjected to a TEMPO-mediated oxidation to facilitate the homogenization. Films were made of the fibrillated materials to evaluate some structural properties. The thicknesses and roughnesses of the films were evaluated with standardized methods and with scanning electron microscopy (SEM), in backscattered electron imaging mode. Field-emission SEM (FE-SEM) and transmission electron microscopy (TEM) were performed to quantify the nanofibril morphology. In this study, we give additional and significant evidences about the suitability of electron microscopy techniques for quantification of nanofibril structures. In addition, we conclude that standard methods are not suitable for estimating the thickness of films having relatively rough surfaces. The results revealed significant differences with respect to the morphology of the fibrillated material. The differences are due to the starting raw material and to the procedure applied for the fibrillation.

Karyotype analysis and classification of buckwheat chromosomes were performed without chemical banding or staining using atomic force microscopy (AFM). Fagopyrum esculentum (common buckwheat) and Fagopyrum tartaricum (Tartarian buckwheat) chromosomes were isolated from root tissues using an enzymatic maceration technique and spread over a glass substrate. Air-dried chromosomes had a surface with ridges, and the height of common and tartary buckwheat were approximately 350 and 150 nm. Volumes of metaphase sets of buckwheat chromosomes were calculated using three-dimensional AFM measurements. Chromosomes were morphologically characterized by the size, volume, arm lengths, and ratios. The calculated volumes of the F. esculentum and F. tartaricum chromosomes were in the ranges of 1.08–2.09 μm3 and 0.49–0.78 μm3, respectively. The parameters such as the relative arm length, centromere position, and the chromosome volumes measured using AFM provide accurate karyomorphological classification by avoiding the subjective inconsistencies in banding patterns of conventional methods. The karyotype evolutionary trend indicates that F. esculentum is an ancient species compared to F. tartaricum. This is the first report of a cytological karyotype of buckwheat using AFM.

We show in this article that it is possible to obtain elemental compositional maps and profiles with atomic-column resolution across an InxGa1−xAs multilayer structure from 5th-order aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images. The compositional profiles obtained from the analysis of HAADF-STEM images describe accurately the distribution of In in the studied multilayer in good agreement with Muraki's segregation model [Muraki, K., Fukatsu, S., Shiraki, Y. & Ito, R. (1992). Surface segregation of In atoms during molecular beam epitaxy and its influence on the energy levels in InGaAs/GaAs quantums wells. Appl Phys Lett61, 557–559].

To understand diffusion processes occurring inside Fe catalysts during multiwall carbon nanotube (MWCNT) growth, catalysts were studied using atomic-resolution scanning transmission electron microscopy combined with electron energy-loss spectroscopy. Nanotube walls emanate from structurally modified and chemically complex catalysts that consist of cementite and a 5 nm amorphous FeOx cap separated by a 2–3 nm thick carbon-rich region that also contains Fe and O (a-C:FexOy). Nonuniform distribution of carbon atoms throughout the catalyst base reveals that carbon molecules from the gas phase decompose near the catalyst multisection junction, where the MWCNT walls terminate. Formation of the a-C:FexOy region provides the essential carbon source for MWCNT growth. Two different carbon diffusion mechanisms are responsible for the growth of the inner and outer walls of each MWCNT.

We demonstrate that single-pass Kelvin force microscopy (KFM) and dC/dz measurements in different environments expand the compositional imaging with atomic force microscopy. The KFM and dC/dz studies were performed in the intermittent contact mode with force gradient detection of tip-sample electrostatic interactions. Both factors contribute to sensitive measurements of the surface potential and capacitance gradient with nanometer-scale spatial resolution as it was verified on a broad range of materials: metal alloys, polymers, organic layers, and liquid-like objects. For many samples the surface potential and dC/dz variations complement each other in identification of individual components of heterogeneous materials. In situ imaging in different humidity or vapors of various organic solvents further facilitate recognition of the constituents of multicomponent polymer samples due to selective swelling of components.

The days of being able to ascertain instrument performance by simply peering through the eye pieces at a specimen are gone. However, users and granting agencies need to be confident that data collected on these instruments is uniform and quantifiable both over time and between instruments. Ideally, a LASER should not fluctuate, illumination should be completely uniform, and colors should be perfectly aligned. To check the current performance of imaging equipment, we conducted a worldwide research study utilizing three image-based tests: long-/short-term illumination stability, co-registration of signals across various wavelengths, and field illumination uniformity. To differentiate between “acceptable” and “unacceptable” performance, the deviation in illumination power could not exceed 10% (long term) or 3% (short term), the difference in the center-of-mass of imaged multicolored beads could not exceed >1 pixel between different wavelengths, and field illumination values could not exceed 10% (horizontal) or 20% (diagonal) deviation. This study established the current state of microscope performance through simple, efficient, and robust tests, while defining relative standards to assist cores in maintaining their instruments in optimal operating conditions. We developed cross-platform performance standards that will improve the validity of quantitative measurements made using various light microscopes.

Light microscopy images suffer from poor contrast due to light absorption and scattering by the media. The resulting decay in contrast varies exponentially across the image along the incident light path. Classical space invariant deconvolution approaches, while very effective in deblurring, are not designed for the restoration of uneven illumination in microscopy images. In this article, we present a modified radiative transfer theory approach to solve the contrast degradation problem of light sheet microscopy (LSM) images. We confirmed the effectiveness of our approach through simulation as well as real LSM images.

In scattering specimens, multiphoton excitation and nondescanned detection improve imaging depth by a factor of 2 or more over confocal microscopy; however, imaging depth is still limited by scattering. We applied the concept of clearing to deep tissue imaging of highly scattering specimens. Clearing is a remarkably effective approach to improving image quality at depth using either confocal or multiphoton microscopy. Tissue clearing appears to eliminate the need for multiphoton excitation for deep tissue imaging.

A confocal scanning laser holography (CSLH) microscope that uniquely combines the concepts of confocal microscopy with holography has been validated for making nonintrusive, full three-dimensional (3D) intensity and phase measurements of objects from a single viewpoint of observation without loss of object information. The phase measurements have been used to determine the 3D refractive indices of a point source heated silicone oil. The refractive indices are converted to 3D temperature measurements, which are useful for heat transfer studies. An important feature of CSLH is its nonintrusive 3D scanning method, which enables its application to the study of Marangoni convection in microgravity with minimal operational vibrations affecting the motion of fluid in the specimen.

In recent years, novel ion sources have been designed and developed that have enabled focused ion beam machines to go beyond their use as nano-fabrication tools. Secondary electrons are usually taken to form images, for their yield is high and strongly dependent on the surface characteristics, in terms of chemical composition and topography. In particular, the secondary electron yield varies characteristically with the angle formed by the beam and the direction normal to the sample surface in the point of impact. Knowledge of this dependence, for different ion/atom pairs, is thus the first step toward a complete understanding of the contrast mechanism in scanning ion microscopy. In this article, experimentally obtained ion-induced secondary electron yields as a function of the incidence angle of the beam on flat surfaces of Al and Cr are reported, for usual conditions in Ga+ and He+ microscopes. The curves have been compared with models and simulations, showing a good agreement for most of the angle range; deviations from the expected behavior are addressed and explanations are suggested. It appears that the maximum value of the ion-induced secondary electron yield is very similar in all the studied cases; the yield range, however, is consistently larger for helium than for gallium, which partially explains the enhanced topographical contrast of helium microscopes over the gallium focused ion beams.

As the miniaturization of semiconductor devices continues, characterization of dopant distribution within the structures becomes increasingly challenging. One potential solution is the use of the secondary electron signal produced in scanning electron (SEMs) or helium ion microscopes (HeIMs) to image the changes in electrical potential caused by the dopant atoms. In this article, the contrast mechanisms and resolution limits of secondary electron dopant contrast are explored. It is shown that the resolution of the technique is dependent on the extent of electrical potential present at a junction and that the resolution of dopant contrast can be improved in the HeIM after an in-situ plasma cleaning routine, which causes an oxide to form on the surface altering the contrast mechanism from electrical potential to material contrast.