Contrast Microscopy Images Research Articles

A New 3D Simulation Method for the Construction of Optical Phase Contrast Images of Gold Nanoparticle Clusters in Biological Cells

A new 3D simulation method based on the finite‐difference time domain (FDTD) approach in combination with Fourier optic techniques is applied to the modeling of optical phase contrast microscope (OPCM) imaging of gold nanoparticles (NPs) in singe biological cells. We consider a realistic size 3D cell model at optical immersion conditions, that is, when the refractive index values of the cytoplasm and of the extracellular medium are equal. For the first time, an FDTD‐based OPCM model is applied to visualize the presence of a cluster of gold NPs in the cytoplasm at both resonant and nonresonant conditions. The results demonstrate the capability to model OPCM image enhancement by optically controlling the resonant properties of the NPs. Our research study extends the applicability of the FDTD modeling approach into a new biomedical optics research area.

Measurement of flow speed in the channels of novel threadlike structures on the surfaces of mammalian organs

There have been several reports on novel threadlike structures (NTSs) on the surfaces of the internal organs of rats and rabbits since their first observation by Bonghan Kim in 1963. To confirm this novel circulatory function, it is necessary to observe the flow of liquid through the NTS as well as the structurally corroborating channels in the NTS. In this article, we report on the measurement of the flow speed of Alcian blue solution in the NTSs on the organ surfaces of rabbits, and we present electron microscopic images depicting the cribrous cross-section with channels. The speed was measured as 0.3 +/- 0.1 mm/s, and the flow distance was up to 12 cm. The flow was unidirectional, and the phase contrast microscopic images showed that the NTSs were strongly stained with Alcian blue. The ultrastructure of the NTSs revealed by cryo-scanning electron microscopy and high-voltage electron microscopy showed that (1) there were cell-like bodies and globular clumps of matter inside the sinus of the channel with thin strands of segregated zones which is a microscopic evidence of the liquid flow, (2) the sinuses have wall structures surrounded with extracellular matrices of collagenous fibers, and (3) there exists a cribriform structure of sinuses. To understand the mechanism for the circulation, a quantitative analysis of the flow speed has been undertaken applying a simplified windkessel model. In this analysis, it was shown that the liquid flow through the NTSs could be due to peristaltic motion of the NTS itself.

A system for measuring cell division patterns of early Caenorhabditis elegans embryos by using image processing and object tracking

AbstractCell division patterns provide crucial information for understanding the mechanisms of development of multicellular organisms. These patterns are measured manually by long‐term microscopic observation or by studying large numbers of images taken by using time‐lapse microscope systems. Because manual measurement limits the objectivity and productivity of measurement, the comprehensive cell division pattern analysis of gene knockdown embryos has been difficult. Here, we present a system that automatically measures the cell division pattern of Caenorhabditis elegans embryos from the 1‐ to 24‐cell stages with high levels of objectivity and productivity. The system automatically detects the nuclei in a set of four‐dimensional differential interference contrast microscope images of a C. elegans embryo by using image processing and measures the cell division pattern by tracking the detected nuclei over time. This system enables a comprehensive cell division pattern analysis of gene knockdown C. elegans embryos. © 2007 Wiley Periodicals, Inc. Syst Comp Jpn, 38(11): 12–24, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/scj.20783

Electrospray in the dripping mode for cell microencapsulation

Entrapment of living cells in microbeads is to protect the encapsulated cells from the host's immune system, which can be used as drug delivery vehicles, immunotherapies and engineered tissues. The main objective of the present study was to investigate the droplet formation and to better develop mono-dispersed microencapsulation of living cells with controllable size. The uniformity of microencapsulation size was realized by performing electrospray in the dripping mode and also stabilized by an additional ring electrode. Reduction of droplet diameter and increase in the dripping frequency were observed with increasing applied voltage to the nozzle using a conventional electrospray setup. The vibration of the needle was found to reduce when high voltage was applied to the nozzle. With increasing voltage applied to the ring electrode, the dripping frequency was found to decrease with the formation of slightly larger sizes of droplets. Hep G2 cell line was taken as the model cell line for encapsulation in calcium alginate microbeads. Relatively uniform microbeads could be achieved when operating under low flow rates with high voltages applied to the nozzle by using a conventional electrospray setup. In contrast, uniform microbeads can not be obtained using a similar setup under high flow rates unless the ring electrode is applied with voltage to stabilize the electrospray in the dripping mode. In this modified electrospray, microbeads with narrow size distribution and slightly larger size can be obtained even for cases under high flow rates. Phase contrast microscope images showed that the diameter of microbeads from around 200 μm to 2 mm could be finely tuned by adjusting various operating parameters.

Origin of contrast in STM images of oxygen on Pd(111) and its dependence on tip structure and tunneling parameters

The dependence of the contrast and symmetry of scanning tunneling microscope images of $\mathrm{O}∕\mathrm{Pd}(111)\ensuremath{-}2\ifmmode\times\else\texttimes\fi{}2$ on the structure of the tunneling tip and on tunneling parameters is explained using first-principles density functional theory. Experimentally, the contrast changes in different ways when a metal-terminated tip over hcp and top sites changes its bias and tip-sample distance. These changes are also reflected in the symmetry of the image. A detailed analysis of the tunneling contributions indicates that for the metallic tips, the $\mathrm{Pd}\phantom{\rule{0.2em}{0ex}}d$ orbitals are determining the image symmetry at close range and low bias, while at larger separations and high bias the $\mathrm{Pd}\phantom{\rule{0.2em}{0ex}}{p}_{z}$ orbitals are the ones that control the image contrast. For oxygen-terminated tips, we predict a positive image contrast, associated with the tip oxygen bonds, as opposed to the negative contrast images obtained with metallic tips.

Detection of nuclei in 4D Nomarski DIC microscope images of early Caenorhabditis elegans embryos using local image entropy and object tracking

BackgroundThe ability to detect nuclei in embryos is essential for studying the development of multicellular organisms. A system of automated nuclear detection has already been tested on a set of four-dimensional (4D) Nomarski differential interference contrast (DIC) microscope images of Caenorhabditis elegans embryos. However, the system needed laborious hand-tuning of its parameters every time a new image set was used. It could not detect nuclei in the process of cell division, and could detect nuclei only from the two- to eight-cell stages.ResultsWe developed a system that automates the detection of nuclei in a set of 4D DIC microscope images of C. elegans embryos. Local image entropy is used to produce regions of the images that have the image texture of the nucleus. From these regions, those that actually detect nuclei are manually selected at the first and last time points of the image set, and an object-tracking algorithm then selects regions that detect nuclei in between the first and last time points. The use of local image entropy makes the system applicable to multiple image sets without the need to change its parameter values. The use of an object-tracking algorithm enables the system to detect nuclei in the process of cell division. The system detected nuclei with high sensitivity and specificity from the one- to 24-cell stages.ConclusionA combination of local image entropy and an object-tracking algorithm enabled highly objective and productive detection of nuclei in a set of 4D DIC microscope images of C. elegans embryos. The system will facilitate genomic and computational analyses of C. elegans embryos.

P2Y Receptor-Mediated Ca2+ Signaling Increases Human Vascular Endothelial Cell Permeability

We investigated the effects of P2-receptor agonists on cell size, intracellular calcium levels ([Ca2+]i), and permeation of FITC-labeled dextran (FD-4) as well as the relationship between these effects in human umbilical vein endothelial cells (HUVEC). FD-4 concentration, cell size, and [Ca2+]i were analyzed by HPLC with fluorescence, phase contrast microscopic imaging, and fluorescent confocal microscopic imaging, respectively. The P2Y1-receptor agonists 2-methylthio ATP (2meS-ATP) and ADP decreased cell size and increased [Ca2+]i in HUVEC. The P2Y2-receptor agonist UTP increased [Ca2+]i, but did not influence cell size. The P2X-receptor agonist α,β-methylene ATP did not induce either response. The decrease in size and increase in [Ca2+]i by 2meS-ATP were blocked by pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, P2Y1-antagonist), thapsigargin (Ca2+-pump inhibitor), and U73122 (phospholipase C inhibitor). Furthermore, 2meS-ATP (P2Y1-receptor agonist) enhanced permeation of FD-4 through the endothelial cell monolayer. The 2meS-ATP-induced enhancement of the permeation was also prevented by PPADS, thapsigargin, and U73122. These results indicate that activation of P2Y receptors induces a decrease in cell size, an increase in [Ca2+]i, and may participate in facilitating macromolecular permeability in HUVEC.

Scanning tunneling microscopy image contrast variation on a copper (II) complex adlayer with tip–sample separation

Contrast variation of scanning tunneling microscopy (STM) image has been investigated as a function of tip–sample separation on a bis[1,3-di( p- n-tetradecyloxyphenyl)propane-1,3-dionato]copper(II) (abbreviated as C 14O–Cu) adlayer on highly oriented pyrolytic graphite with STM. Submolecular features of the molecular core and interdigitated alkyl chain are clearly revealed. A preliminary theoretical calculation is carried out to simulate the image variation. The simulated results are in agreement with STM observation.

Effect of tip morphology on AFM images

The effect of tip morphology on the atomic force microscope (AFM) image contrast for a GaAs(110) surface is investigated theoretically by considering three different tip apexes of a Si tip: (1) Si apex with a half-filled dangling bond; (2) Ga apex with an empty dangling bond; and (3) As apex with a fully filled dangling bond. It is shown that the dangling-bond state of the tip apex has significant effects on the image contrast: the Ga apex will image the As sublattice, and the As apex will image the Ga sublattice, and in the case of the Si apex, it is possible to image only the As sublattice or both the As and Ga sublattices, depending on the tip–sample separation.

Scanning tunneling microscopy image contrast of monolayer platinum on graphite

Platinum particles supported on graphite have been investigated by scanning tunneling microscopy (STM). For one monolayer thick Pt particles the individual Pt atoms form a characteristic intensity pattern due to a mismatch between the Pt and graphite lattice. Based on density functional theory calculations and model structures of Pt on graphite it is argued that the observed STM imaging contrast has its origin in the tip induced elastic deformation of the graphite underneath the Pt particle. The Pt–graphite potential is much stiffer than the graphite–graphite potential. The calculations furthermore indicate that Pt adsorption is favored over top rather than hole sites and that the barrier for diffusion is very low.

Interface-Induced Coarsening Process in Polymer Blends

The coarsening process of the droplets in a two-phase polymer blend (PP/EVAc) was studied under two-dimensional and three-dimensional conditions using a phase contrast microscope and computer image analyzer. The results showed that under three-dimensional conditions the growth of the droplet's radius with time follows r3∼t1.01, corresponding to the evaporation–condensation theory of Lifshitz–Slyozov, r3∼t, while under two-dimensional conditions the growth law is r3∼t1.31. The growth rate of the droplets under two-dimensional conditions is faster than that under three-dimensional conditions. This difference is caused by an interfacial interaction (wetting effects) between the substrates and polymer blend. The existence of the interface promoted the coarsening process of the polymer blend under two-dimensional conditions.

Interpretation of GaAs(110) scanning tunneling microscopy image contrast by the symmetry of the surface Bloch wave functions

A simple qualitative correlation between the corrugation anisotropy observed in scanning tunneling microscope (STM) images of GaAs(110) surfaces and the symmetry properties of the surface states is presented. We show that as a function of bias, tunneling from different electronic states near high-symmetry points of the surface Brillouin zone gives rise to a distinct corrugation along [11̄0] and [001] in STM images. Existing models of the surface band structure are used to identify these states. We show that at small bias, due to band bending effects, the same surface state near the conduction-band edge determines the image corrugation in both filled and empty states images of n-type GaAs.

Atomic Force Microscope Image Contrast Mechanisms on Supported Lipid Bilayers

This work presents a methodology to measure and quantitatively interpret force curves on supported lipid bilayers in water. We then use this method to correlate topographic imaging contrast in atomic force microscopy (AFM) images of phase-separated Langmuir-Blodgett bilayers with imaging load. Force curves collected on pure monolayers of both distearoylphosphatidylethanolamine (DSPE) and monogalactosylethanolamine (MGDG) and dioleoylethanolamine (DOPE) deposited at similar surface pressures onto a monolayer of DSPE show an abrupt breakthrough event at a repeatable, material-dependent force. The breakthrough force for DSPE and MGDG is sizable, whereas the breakthrough force for DOPE is too small to measure accurately. Contact-mode AFM images on 1:1 mixed monolayers of DSPE/DOPE and MGDG/DOPE have a high topographic contrast at loads between the breakthrough force of each phase, and a low topographic contrast at loads above the breakthrough force of both phases. Frictional contrast is inverted and magnified at loads above the breakthrough force of both phases. These results emphasize the important role that surface forces and mechanics can play in imaging multicomponent biomembranes with AFM.

Using the Hilbert transform for 3D visualization of differential interference contrast microscope images.

Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.

High resolution transmission electron microscopy to study very thin crystalline layers buried at an amorphous–crystalline interface

Structure characterisation of interfaces is a field of widespread application of high resolution transmission electron microscopy for its very high spatial resolution. Specimen thickness and electron optical condition have a deep influence on the high resolution electron transmission microscopy image contrast. Hence, in many cases, the real structure of the sample can be understood from experimental images only by comparison with the relevant simulation. Moreover, the understanding of the contrast variation of a few Å at an interface is a task in which even the use of simulation could not produce an unequivocal solution of the experimental result. In this paper high resolution transmission electron microscopy image simulations show that two monolayers of crystalline material buried at an amorphous–crystalline interface can be successfully revealed and interpreted. The simulated images reproduce the experimental results as obtained from the Al/Si–As/n-GaAs (001) heterostructure.

Calcium and the regulation of mammalian ciliary beating

This report summarizes our recent work on the role of intracellular Ca2+ ([Ca2+]i) in regulating mammalian ciliary beat frequency (CBF). CBF from a single ovine cilium and [Ca2+]i from the same cell were measured by digital video phase contrast microscopy and fura-2 ratiometric imaging video microscopy, respectively. Cells were stimulated with two exposures to 10 μM acetylcholine (ACh). CBF was recorded during the first and [Ca2+]i during the second stimulation. ACh increased [Ca2+]i and CBF transiently with indistinguishable kinetics and, early in culture, even induced [Ca2+]i oscillations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 μM thapsigargin, an inhibitor of the endoplasmic-reticulum Ca2+-ATPase, showed transient [Ca2+]i and CBF increases, again with similar kinetics, which often remained at an elevated plateau. Application of ACh to cells pretreated with thapsigargin produced decreases in both [Ca2+]i and CBF. Finally, changing extracellular Ca2+-concentrations induced corresponding changes in [Ca2+]i that were associated with kinetically similar CBF changes. These data strongly suggested that [Ca2+]i is a critical signal to regulate CBF in mammalian tracheal epithelial cells. In an initial effort to provide constraints on the number and type of reactions that link changes in [Ca2+]i to changes in CBF, simultaneous recordings of both signals from a single cell were analyzed. Such recordings provided higher resolution of the kinetic responses of CBF and [Ca2+]i to ACh as well as they allowed direct assessment of the coupling between [Ca2+]i and CBF. Simultaneous measurements revealed that [Ca2+]i and CBF were perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial ACh exposure. There, changes in CBF lagged the changes in [Ca2+]i by 1–3 ciliary beat cycles (ca. 150–450 ms).

Transformation of molecular oxygen on a platinum surface: A theoretical calculation of STM images

Using the recently modified electron scattering quantum chemical approach, we have performed simulations of the scanning tunneling microscope (STM) image contrast associated with elementary steps of ${\mathrm{O}}_{2}$ dissociation on platinum(111). Two metastable molecular precursors, binding to different adsorption sites, and the stable atomic species have been studied. The two molecular chemisorbed structures have been identified very recently with a low-temperature STM. From density-functional-theory calculations, which allow a full geometry optimization of the adsorbate and the surface, two molecular precursors adsorbed at the fcc hollow and bridge sites have very similar chemisorption energy. In the calculated STM image, with a tip ending with a Pt atom, the ${\mathrm{O}}_{2}$ molecule and the O atom appear as a depression, which only agrees with the experiment in the case of the O atom. However, the image is found to be strongly dependent on the value of the gap resistance and on the termination of the tip. A tip ending with a CO molecule, which models a tip contaminated with an adsorbate, gives images in good agreement with the experimental results.

Contrast of scanning ion microscope images compared with scanning electron microscope images for metals?

Y. Sakai, T. Y. T. S. T. I., T. Y. T. S. T. I., T. Suzuki and T. Ichinokawa, J. Anal. At. Spectrom., 1999, 14, 419 DOI: 10.1039/A806833J

Scanning Tunneling Microscopy Image Contrast as a Function of Scan Angle in Hydrogen-Bonded Self-Assembled Monolayers

Scanning tunneling microscopy (STM) has been performed on monolayers of the nucleic acid base, adenine, adsorbed to molybdenum disulfide (MoS2) surfaces. Analysis of the real space images of the adsorbates, together with molecular mechanics simulations suggest that the molecules form monolayers stablized by cyclic hydrogen bonds. We present data showing the effect on image contrast of varying scan angles of the STM tip. These indicate an anisotropic response of components of the monolayer to the scan direction and support a structural model implicating cyclic hydrogen bonds in both the stability of the monolayer and in the STM image contrast mechanism.

Interpretation of High Resolution Transmission Electron Microscope Images of Short Range Ordered Ni<SUB>4</SUB>Mo

High resolution transmission electron microscope (HRTEM) image contrast of short-range order (SRO) in Ni 4 Mo alloys has been investigated by means of multi-slice simulations. The HRTEM image contrast of SRO in Ni 4 Mo coincides with a projection contrast of an SRO structure which involves D1 a , D0 22 and Pt 2 Mo-type subunit cells in a disordered matrix. Multi-slice simulations revealed that the SRO structure exhibit a projection contrast in the HRTEM image when the fundamental fcc lattice reflections contribute sufficiently to HRTEM imaging. The results indicate that the image of SRO in Ni 4 Mo can be interpreted in terms of projected potential of atomic columns under an appropriate imaging condition.