trans-[Ru(NO)(NH3)4(py)](BF4)3.H2O encapsulated in PLGA microparticles for delivery of nitric oxide to B16-F10 cells: Cytotoxicity and phototoxicity

The NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (py=pyridine) was loaded into poly-lactic-co-glycolic acid (PLGA) microparticles using the double emulsification technique. Scanning electron microscopy (SEM) and dynamic light scattering revealed that the particles are spherical in shape, have a diameter of 1600nm, and have low tendency to aggregate. The entrapment efficiency was 25%. SEM analysis of the melanoma cell B16-F10 in the presence of the microparticles containing the complex trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (pyMP) showed that the microparticles were adhered to the cell surface after 2h of incubation. The complex with concentrations lower than 1x10(-4)M did not show toxicity in B16-F10 murine cells. The complex in solution is toxic at higher concentrations (>1x10(-3)M), with cell death attributed to NO release following the reduction of the complex. pyMP is not cytotoxic due to the lower bioavailability and availability of the entrapped complex to the medium and its reducing agents. However, pyMP is phototoxic upon light irradiation. The phototoxicity strongly suggests that cell death is due to NO release from trans-[Ru(NO)(NH(3))(4)(py)](3+). This work shows that pyMP can serve as a model for a drug delivery system carrying the NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O, which can release NO locally at the tumor cell by irradiation with light only.     

Molecular simulation of carbon nanotubes as sorptive materials: sorption effects towards retene,perylene and cholesterol to 100 degrees Celsius and above

New water purification technologies are being developed as the world’s water sources are increasingly being polluted and experience a dramatic consumption with the increasing world population. In this context, the emerging era of nanotechnology has introduced a series of innovations and materials with promising potential as sorptive materials for water decontamination. The application of nanomaterials for the purification of ground/surface water introduces nevertheless a series of important challenges, such as health and safety, cost, process-efficiency and chemical sorption properties. In this study, we consider the latter class and present a study of the sorptive properties of carbon nanotubes (CNTs) as potential water decontaminating materials. Molecular dynamics simulations are used and three molecular candidates of the water contaminants, cholesterol, perylene and retene were selected for interaction study with CNTs at different diameters. The results show that CNTs form densely packed clusters with retene, perylene and cholesterol, binding each strongly to their tubular surfaces, as well as in their hollow tubular spaces. Cholesterol and perylene bind more strongly than retene, accounting for the calculated binding energies in vacuo, however the planar geometries of polycyclics may in general favour binding to CNTs over semi-polar molecules and can require further studies. Our studies show furthermore that the CNTs retain the adsorbed molecules also at 100 degrees Celsius, and require therefore additional steps of separation for eventual recycling and reusing the nanomaterials for additional decontamination. This study is important in providing data for initiating studies and developments of water purification approaches based on using CNTs.     

Focusing of light by leaf epidermal cells

Leaf epidermal cells from a wide variety of plants focus light to surprisingly high levels. Using image analysis, the concentration and distribution of light was measured after it passed through epidermal cells within peels and epidermal cells attached to palisade cells in partially dissected leaves. In peels taken from Medicago sativa, Zea mays , and Impatiens sp., light was concentrated 15- to 20-fold by individual epidermal cells. When left attached to the mesophyll, which attenuated focusing by absorption and scattering, light was focused up to 5 times. The position of the focal spot beneath each epidermal cell was affected by the direction at which the light struck the cell. When the light was perpendicular to the leaf surface, individual focal spots fell beneath each epidermal cell. When the incident light was oblique, the focal spot shifted laterally and was positioned closer to the anticlinal cell wall. Focusing was observed when leaves were irradiated with collimated light but not with diffuse light. Focal lengths were relatively independent of wavelength within the visible region of the spectrum and there were only slight differences between focusing of blue vs red light. Epidermal lens properties can affect chlorophyll fluorescence and the photosynthetic performance of leaves. A survey of 47 species collected from a wide variety of habitats indicates that many plants have leaf epidermal cells with lens properties. The ability to measure epidermal focusing makes it possible to examine the adaptive and physiological significance of epidermal lens effects in plants.     

Effects of footwear on three-dimensional tibiotalar and subtalar joint motion during running

Running is a popular form of recreation, but injuries are common and may be associated with abnormal joint motion. The objective of this study was to determine the effect of three footwear conditions – barefoot (BF), an ultraflexible training shoe (FREE), and a motion control shoe (MC) – on 3D foot and ankle motion. Dynamic, biplane radiographic images were acquired from 12 runners during overground running. 3D rotations of the tibiotalar and subtalar joints were quantified in terms of plantarflexion/dorsiflexion (PF/DF), inversion/eversion (IN/EV) and internal/external rotation (IR/ER). Across the early stance phase (defined as footstrike to heel-off), BF running demonstrated greater tibiotalar joint range of motion for PF/DF (28.2±8.3°) and IR/ER (7.0±1.4°) than the shod conditions (FREE: PF/DF=15.1±5.9°, IR/ER=4.8±2.1°; MC: PF/DF=15.0±6.2°, IR/ER=4.3±0.7°). Also at the tibiotalar joint, BF running resulted in a position significantly more plantarflexed (BF: 2.0±12.5°, FREE: 15.7±12.2°, MC: 16.5±9.3°) and internally rotated (BF: 12.9±4.5°, FREE: 10.7±4.3°, MC: 10.6±3.9°) at footstrike compared to both shod conditions. No differences were detected between the shod conditions at any point in the early stance phase at the tibiotalar joint. The MC condition demonstrated significant differences compared to FREE at several points throughout the early stance phase at the subtalar joint, with the greatest differences seen at 30% in PF/DF (MC −1.4±8.8°: FREE: −0.5±9.0°), IN/EV (MC −8.1±5.7°: FREE −6.3±5.5°) and IR/ER (MC −9.5±5.3°: FREE: −8.7±5.2°). These findings indicate that footwear has subtle effects on joint motion mainly between BF and shod conditions at the tibiotalar joint and between shod conditions at the subtalar joint.     

Cell analysis using a multiple internal reflection photonic lab-on-a-chip

Here we present a protocol for analyzing cell cultures using a photonic lab-on-a-chip (PhLoC). By using a broadband light source and a spectrometer, the spectrum of a given cell culture with an arbitrary population is acquired. The PhLoC can work in three different regimes: light scattering (using label-free cells), light scattering plus absorption (using stained cells) and, by subtraction of the two former regimes, absorption (without the scattering band). The acquisition time of the PhLoC is ～30 ms. Hence, it can be used for rapid cell counting, dead/live ratio estimation or multiparametric measurements through the use of different dyes. The PhLoC, including microlenses, micromirrors and microfluidics, is simply fabricated in a single-mask process (by soft lithographic methods) using low-cost materials. Because of its low cost it can easily be implemented for point-of-care applications. From raw substrates to final results, this protocol can be completed in 29 h.     

Simple method for quantification of fast plasma membrane movements.

We present a method for the quantification of the fast plasma membrane movements that are involved in ruffling, blebbing, fast shape change, and fast translocation. The method is based on the Kontron Vidas image analysis computer program. Video images from cells viewed through an inverted microscope were transmitted to the computer. The procedure was as follows: 4 consecutive video images were averaged (image 1); 28 s later a second set of 4 video images was averaged (image 2); image 2 was subtracted from image 1 and the grey level of each pixel of the resulting image was increased with 128 grey level units, resulting in the subtraction image, showing a uniform grey background speckled with brighter and darker spots corresponding to areas of movement. These spots were discriminated and turned into white objects against a black background. Interactive editing was used to delete artefacts that resulted from floating debris. The total area of the discriminated objects was measured, and the parameter motile area in micron2 per cell was calculated. We have applied our method to the study of motility induced in epithelial cell lines by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate and by epidermal growth factor.     

Carbon nanotubes in cancer diagnosis and therapy

During the past years, great progress has been made in the field of nanomaterials given their great potential in biomedical applications. Carbon nanotubes (CNTs), due to their unique physicochemical properties, have become a popular tool in cancer diagnosis and therapy. They are considered one of the most promising nanomaterials with the capability of both detecting the cancerous cells and delivering drugs or small therapeutic molecules to these cells. Over the last several years, CNTs have been explored in almost every single cancer treatment modality, including drug delivery, lymphatic targeted chemotherapy, thermal therapy, photodynamic therapy, and gene therapy. In this review, we will show how they have been introduced into the diagnosis and treatment of cancer. Novel SWNT-based tumor-targeted drug delivery systems (DDS) will be highlighted. Furthermore, the in vitro and in vivo toxicity of CNTs reported in recent years will be summarized.     

Imaging the living plant cell: From probes to quantification

At the center of cell biology is our ability to image the cell and its various components, either in isolation or within an organism. Given its importance, biological imaging has emerged as a field of its own, which is inherently highly interdisciplinary. Indeed, biologists rely on physicists and engineers to build new microscopes and imaging techniques, chemists to develop better imaging probes, and mathematicians and computer scientists for image analysis and quantification. Live imaging collectively involves all the techniques aimed at imaging live samples. It is a rapidly evolving field, with countless new techniques, probes, and dyes being continuously developed. Some of these new methods or reagents are readily amenable to image plant samples, while others are not and require specific modifications for the plant field. Here, we review some recent advances in live imaging of plant cells. In particular, we discuss the solutions that plant biologists use to live image membrane-bound organelles, cytoskeleton components, hormones, and the mechanical properties of cells or tissues. We not only consider the imaging techniques per se, but also how the construction of new fluorescent probes and analysis pipelines are driving the field of plant cell biology.

Specific examples are used to illustrate some of the challenges of live cell imaging, from designing genetically encoded probes to choosing a pipeline for image analysis and quantification.     

A patch-based method for repetitive and transient event detection in fluorescence imaging

Automatic detection and characterization of molecular behavior in large data sets obtained by fast imaging in advanced light microscopy become key issues to decipher the dynamic architectures and their coordination in the living cell. Automatic quantification of the number of sudden and transient events observed in fluorescence microscopy is discussed in this paper. We propose a calibrated method based on the comparison of image patches expected to distinguish sudden appearing/vanishing fluorescent spots from other motion behaviors such as lateral movements. We analyze the performances of two statistical control procedures and compare the proposed approach to a frame difference approach using the same controls on a benchmark of synthetic image sequences. We have then selected a molecular model related to membrane trafficking and considered real image sequences obtained in cells stably expressing an endocytic-recycling trans-membrane protein, the Langerin-YFP, for validation. With this model, we targeted the efficient detection of fast and transient local fluorescence concentration arising in image sequences from a data base provided by two different microscopy modalities, wide field (WF) video microscopy using maximum intensity projection along the axial direction and total internal reflection fluorescence microscopy. Finally, the proposed detection method is briefly used to statistically explore the effect of several perturbations on the rate of transient events detected on the pilot biological model.     

Image analysis for understanding embryo development: a bridge from microscopy to biological insights

The digital reconstruction of the embryogenesis of model organisms from 3D+time data is revolutionizing practices in quantitative and integrative Developmental Biology. A manual and fully supervised image analysis of the massive complex data acquired with new microscopy technologies is no longer an option and automated image processing methods are required to fully exploit the potential of imaging data for biological insights. Current developments and challenges in biological image processing include algorithms for microscopy multiview fusion, cell nucleus tracking for quasi-perfect lineage reconstruction, segmentation, and validation methodologies for cell membrane shape identification, single cell gene expression quantification from in situ hybridization data, and multidimensional image registration algorithms for the construction of prototypic models. These tools will be essential to ultimately produce the multilevel in toto reconstruction that combines the cell lineage tree, cells, and tissues structural information and quantitative gene expression data in its spatio-temporal context throughout development.     

Effects of single and multi walled carbon nanotubes on macrophages: cyto and genotoxicity and electron microscopy

Production of nanotechnology-based materials is increasing worldwide: it is essential to evaluate their potential toxicity. Among these nanomaterials, carbon nanotubes (CNTs) have tremendous potential in many areas of research and applications. We have investigated the cyto- and genotoxic effects of single and multi-walled CNTs (SWCNTs, MWCNTs) and carbon black (CB) on the mouse macrophage cell line RAW 264.7. Specifically we have investigated inflammatory response, release of tumor necrosis factor-α (TNF-α), intracellular reactive oxygen species (ROS) production, cell death (both necrosis and apoptosis), chromosomal aberrations and cellular ultrastructural alteration caused by CB, MWCNTs and SWCNTs. Our data confirm that both CNTs and CB are cyto and geno-toxic to RAW 264.7 mouse macrophages. CNTs exposure induced ROS release, necrosis and chromosomal aberrations but did not cause an inflammatory response. In addition CNTs induce ultrastructural damage and apoptosis. CNTs penetrate the cell membrane and individual MWCNTs are seen associated with the nuclear envelope.     

Coherent anti-stokes Raman scattering microscopy: a biological review.

Microscopic imaging of cells and tissues are generated by the interaction of light with either the sample itself or contrast agents that label the sample. Most contrast agents, however, alter the cell in order to introduce molecular labels, complicating live cell imaging. The interaction of light from multiple laser sources has given rise to microscopy, based on Raman scattering or vibrational resonance, which demonstrates selectivity to specific chemical bonds while imaging unmodified live cells. Here, we discuss the nonlinear optical technique of coherent anti-Stokes Raman scattering (CARS) microscopy, its instrumentation, and its status in live cell imaging.     

Adaptive Geometric Tessellation for 3D Reconstruction of Anisotropically Developing Cells in Multilayer Tissues from Sparse Volumetric Microscopy Images

The need for quantification of cell growth patterns in a multilayer, multi-cellular tissue necessitates the development of a 3D reconstruction technique that can estimate 3D shapes and sizes of individual cells from Confocal Microscopy (CLSM) image slices. However, the current methods of 3D reconstruction using CLSM imaging require large number of image slices per cell. But, in case of Live Cell Imaging of an actively developing tissue, large depth resolution is not feasible in order to avoid damage to cells from prolonged exposure to laser radiation. In the present work, we have proposed an anisotropic Voronoi tessellation based 3D reconstruction framework for a tightly packed multilayer tissue with extreme z-sparsity (2–4 slices/cell) and wide range of cell shapes and sizes. The proposed method, named as the ‘Adaptive Quadratic Voronoi Tessellation’ (AQVT), is capable of handling both the sparsity problem and the non-uniformity in cell shapes by estimating the tessellation parameters for each cell from the sparse data-points on its boundaries. We have tested the proposed 3D reconstruction method on time-lapse CLSM image stacks of the Arabidopsis Shoot Apical Meristem (SAM) and have shown that the AQVT based reconstruction method can correctly estimate the 3D shapes of a large number of SAM cells.     

In vivo19F MRI for Cell Tracking

In vivo¹⁹F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.     

DNA‐Functionalized Plasmonic Nanomaterials for Optical Biosensing

Plasmonic nanomaterials, especially Au and Ag nanomaterials, have shown attractive physicochemical properties, such as easy functionalization and tunable optical bands. The development of this active subfield paves the way to the fascinating biosensing platforms. In recent years, plasmonic nanomaterials–based sensors have been extensively investigated because they are useful for genetic diseases, biological processes, devices, and cell imaging. In this account, a brief introduction of the development of optical biosensors based on DNA‐functionalized plasmonic nanomaterials is presented. Then the common strategies for the application of the optical sensors are summarized, including colorimetry, fluorescence, localized surface plasmon resonance, and surface‐enhanced resonance scattering detection. The focus is on the fundamental aspect of detection methods, and then a few examples of each method are highlighted. Finally, the opportunities and challenges for the plasmonic nanomaterials–based biosensing are discussed with the development of modern technologies.     

Saturated two‐photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near‐infrared spectrum can improve the present situation. Here, the capability of saturated two‐photon saturated excitation (TP‐SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering‐enlarged point spread function (PSF), we find that TP‐SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single‐photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering‐enlarged PSF, TP‐SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.      

Development and evaluation of realistic optical cell models for rapid and label‐free cell assay by diffraction imaging

Methods for rapid and label‐free cell assay are highly desired in life science. Single‐shot diffraction imaging presents strong potentials to achieve this goal as evidenced by past experimental results using methods such as polarization diffraction imaging flow cytometry. We present here a platform of methods toward solving these problems and results of optical cell model (OCM) evaluations by calculations and analysis of cross‐polarized diffraction image (p‐DI) pairs. Four types of realistic OCMs have been developed with two prostate cell structures and adjustable refractive index (RI) parameters to investigate the effects of cell morphology and index distribution on calculated p‐DI pairs. Image patterns have been characterized by a gray‐level co‐occurrence matrix (GLCM) algorithm and four GLCM parameters and linear depolarization ratio δ_L have been selected to compare calculated against measured data of prostate cells. Our results show that the irregular shapes of and heterogeneity in RI distributions for organelles play significant roles in the spatial distribution of scattered light by cells in comparison to the average RI values and their differences among the organelles. Discrepancies in GLCM and δ_L parameters between calculated and measured p‐DI data provide useful insight for understanding light scattering by single cells and improving OCM.      

Nanoscale imaging and quantification of local proteolytic activity

Proteolytic cleavage of extracellular matrix (ECM) is a critical feature of tumor cell invasion, and affects cancer cell growth, differentiation, apoptosis, and migration. Malignant cells secrete most proteases as inactive proenzymes that undergo proteolytic cleavage for activation, and proteolytic activity is elevated in close proximity to these cells. Therefore, local activity rather than protease concentration determines ECM proteolysis. Precise quantification of local proteolytic activity, functional investigation, and high resolution imaging of morphological ECM alterations have proven difficult. In this study, we present a novel approach for measuring proteolytic activity in the microenvironment of cells by using atomic force microscopy (AFM). Amelanotic melanoma cells (A7-clone) were seeded on fluorescent gelatin or collagen-IV coatings. Proteolysis reduced fluorescence of these coatings. Fluorescence microscopy (FM) in combination with AFM was used to maneuver the AFM-tip to tumor cell induced proteolytic spots. AFM enabled nanoscale volume measurement, three-dimensional reconstruction of single proteins and demonstrated that ECM cleavage is restricted to the proteolytic microenvironment of cancer cells. This method detected significant decreases in molecular weight of protein clusters (-76.6%), matrix volume (-46.6%), and height (-38.1%) between intact and proteolyzed gelatin. Similar parameter changes were demonstrated without FM, by AFM-scanning gelatin in close proximity to invasive cells. Furthermore, AFM depicted significantly stronger local degradation of gelatin than collagen-IV by A7-cells. Taken together, AFM allows specific quantification and imaging of local proteolytic processes at a nanometer level, thus providing a unique method for the functional evaluation of invasiveness and metastatic potential of tumor cells in small scale samples.     

Quantitative Analysis of Centromeric FISH Spots during the Cell Cycle by Image Cytometry

Two-color fluorescence in situ hybridization (FISH) with chromosome enumeration DNA probes specific to chromosomes 7, 11, 17, and 18 was applied to CAL-51 breast cancer cells to examine whether the fluorescence intensity of FISH spots was associated with cell cycle progression. The fluorescence intensity of each FISH spot was quantitatively analyzed based on the cell cycle stage determined by image cytometry at the single-cell level. The spot intensity of cells in the G₂ phase was larger than that in the G_0/1 phase. This increased intensity was not seen during the early and mid S phases, whereas the cells in the late S phase showed significant increases in spot intensity, reaching the same level as that observed in the G₂ phase, indicating that alpha satellite DNA in the centromeric region was replicated in the late S phase. Thus, image cytometry can successfully detect small differences in the fluorescence intensities of centromeric spots of homologous chromosomes. This combinational image analysis of FISH spots and the cell cycle with cell image cytometry provides insights into new aspects of the cell cycle. This is the first report demonstrating that image cytometry can be used to analyze the fluorescence intensity of FISH signals during the cell cycle.