A ring-like nucleoid is not necessary for radioresistance in the <Emphasis Type="Italic">Deinococcaceae</Emphasis>

Background  

Transmission electron microscopy images of Deinococcus radiodurans R1 suggest that the nucleoid of this species exists as a "ring-like" body, and have led to speculation that this structure contributes to the radioresistance of the species. Since extreme radioresistance is characteristic of six other species of Deinococcus, we have attempted to correlate nucleoid morphology and radioresistance by determining whether the genomic DNA of each of these species exhibit similar structures.     

Biomedical potential of silver nanoparticles synthesized from calli cells of Citrullus colocynthis (L.) Schrad

Background

Transmission electron microscopy (TEM) remains an important technique to investigate the size, shape and surface characteristics of particles at the nanometer scale. Resulting micrographs are two dimensional projections of objects and their interpretation can be difficult. Recently, electron tomography (ET) is increasingly used to reveal the morphology of nanomaterials (NM) in 3D. In this study, we examined the feasibility to visualize and measure silica and gold NM in suspension using conventional bright field electron tomography.

Results

The general morphology of gold and silica NM was visualized in 3D by conventional TEM in bright field mode. In orthoslices of the examined NM the surface features of a NM could be seen and measured without interference of higher or lower lying structures inherent to conventional TEM. Segmentation by isosurface rendering allowed visualizing the 3D information of an electron tomographic reconstruction in greater detail than digital slicing. From the 3D reconstructions, the surface area and the volume of the examined NM could be estimated directly and the volume-specific surface area (VSSA) was calculated. The mean VSSA of all examined NM was significantly larger than the threshold of 60 m²/cm³. The high correlation between the measured values of area and volume gold nanoparticles with a known spherical morphology and the areas and volumes calculated from the equivalent circle diameter (ECD) of projected nanoparticles (NP) indicates that the values measured from electron tomographic reconstructions are valid for these gold particles.

Conclusion

The characterization and definition of the examined gold and silica NM can benefit from application of conventional bright field electron tomography: the NM can be visualized in 3D, while surface features and the VSSA can be measured.     

A nano-view of West Nile virus-induced cellular changes during infection

Background  

Microscopic imaging of viruses and their interactions with and effects on host cells are frequently held back by limitations of the microscope's resolution or the invasive nature of the sample preparation procedures. It is also difficult to have a technique that would allow simultaneous imaging of both surface and sub-surface on the same cell. This has hampered endeavours to elucidate virus-host interactions. Atomic Force Microscopy (AFM), which is commonly used in the physical sciences, is now becoming a good correlative form of microscopy used to complement existing optical, confocal and electron microscopy for biological applications     

The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells

Background  

Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied.     

<Emphasis Type="Italic">In vivo</Emphasis> molecular imaging of experimental joint inflammation by combined <Superscript>18</Superscript>F-FDG positron emission tomography and computed tomography

Introduction  

The purpose of this work was to establish and validate combined small animal positron emission tomography - computed tomography (PET/CT) as a new in vivo imaging method for visualisation and quantification of joint inflammation.     

Edge detection in microscopy images using curvelets

Background  

Despite significant progress in imaging technologies, the efficient detection of edges and elongated features in images of intracellular and multicellular structures acquired using light or electron microscopy is a challenging and time consuming task in many laboratories.     

Characterization of a Spontaneous Retinal Neovascular Mouse Model

Background

Vision loss due to vascular disease of the retina is a leading cause of blindness in the world. Retinal angiomatous proliferation (RAP) is a subgroup of neovascular age-related macular degeneration (AMD), whereby abnormal blood vessels develop in the retina leading to debilitating vision loss and eventual blindness. The novel mouse strain, neoretinal vascularization 2 (NRV2), shows spontaneous fundus changes associated with abnormal neovascularization. The purpose of this study is to characterize the induction of pathologic angiogenesis in this mouse model.

Methods

The NRV2 mice were examined from postnatal day 12 (p12) to 3 months. The phenotypic changes within the retina were evaluated by fundus photography, fluorescein angiography, optical coherence tomography, and immunohistochemical and electron microscopic analysis. The pathological neovascularization was imaged by confocal microscopy and reconstructed using three-dimensional image analysis software.

Results

We found that NRV2 mice develop multifocal retinal depigmentation in the posterior fundus. Depigmented lesions developed vascular leakage observed by fluorescein angiography. The spontaneous angiogenesis arose from the retinal vascular plexus at postnatal day (p)15 and extended toward retinal pigment epithelium (RPE). By three months of age, histological analysis revealed encapsulation of the neovascular lesion by the RPE in the photoreceptor cell layer and subretinal space.

Conclusions

The NRV2 mouse strain develops early neovascular lesions within the retina, which grow downward towards the RPE beginning at p15. This retinal neovascularization model mimics early stages of human retinal angiomatous proliferation (RAP) and will likely be a useful in elucidating targeted therapeutics for patients with ocular neovascular disease.     

From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

Background

In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM).

Methodology

To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression.

Conclusion

Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.     

Reprint of “Stereology meets electron tomography: towards quantitative 3D electron microscopy” [J. Struct. Biol. 159 (2007) 443–450]

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists.This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2–5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method.Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.     

ScanImage: Flexible software for operating laser scanning microscopes

Background  

Laser scanning microscopy is a powerful tool for analyzing the structure and function of biological specimens. Although numerous commercial laser scanning microscopes exist, some of the more interesting and challenging applications demand custom design. A major impediment to custom design is the difficulty of building custom data acquisition hardware and writing the complex software required to run the laser scanning microscope.     

PTools: an opensource molecular docking library

Background  

Macromolecular docking is a challenging field of bioinformatics. Developing new algorithms is a slow process generally involving routine tasks that should be found in a robust library and not programmed from scratch for every new software application.     

Detection, mapping, and quantification of single walled carbon nanotubes in histological specimens with photoacoustic microscopy

Aims

In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds).

Materials and Methods

Optical-resolution (OR) and acoustic-resolution (AR) - Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy).

Results

Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections.

Conclusions

The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs.     

Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration

Purpose

To investigate the safety and efficacy of subretinal injection of human Wharton’s Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats.

Methods

RCS rats were divided into 2 groups: hWJ-MSCs treated group (n = 8) and placebo control group (n = 8). In the treatment group, hWJ-MSCs from healthy donors were injected into the subretinal space in one eye of each rat at day 21. Control group received saline injection of the same volume. Additional 3 animals were injected with nanogold-labelled stem cells for in vivo tracking of cells localisation using a micro-computed tomography (microCT). Retinal function was assessed by electroretinography (ERG) 3 days before the injection and repeated at days 15, 30 and 70 after the injection. Eyes were collected at day 70 for histology, cellular and molecular studies.

Results

No retinal tumor formation was detected by histology during the study period. MicroCT scans showed that hWJ-MSCs stayed localised in the eye with no systemic migration. Transmission electron microscopy showed that nanogold-labelled cells were located within the subretinal space. Histology showed preservation of the outer nuclear layer (ONL) in the treated group but not in the control group. However, there were no significant differences in the ERG responses between the groups. Confocal microscopy showed evidence of hWJ-MSCs expressing markers for photoreceptor, Müller cells and bipolar cells.

Conclusions

Subretinal injection of hWJ-MSCs delay the loss of the ONL in RCS rats. hWJ-MSCs appears to be safe and has potential to differentiate into retinal-like cells. The potential of this cell-based therapy for the treatment of retinal dystrophies warrants further studies.     

Self assembly of amphiphilic C60 fullerene derivatives into nanoscale supramolecular structures

Background  

The amphiphilic fullerene monomer (AF-1) consists of a "buckyball" cage to which a Newkome-like dendrimer unit and five lipophilic C₁₂ chains positioned octahedrally to the dendrimer unit are attached. In this study, we report a novel fullerene-based liposome termed 'buckysome' that is water soluble and forms stable spherical nanometer sized vesicles. Cryogenic electron microscopy (Cryo-EM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) studies were used to characterize the different supra-molecular structures readily formed from the fullerene monomers under varying pH, aqueous solvents, and preparative conditions.     

Marker-free image registration of electron tomography tilt-series

Background  

Tilt series are commonly used in electron tomography as a means of collecting three-dimensional information from two-dimensional projections. A common problem encountered is the projection alignment prior to 3D reconstruction. Current alignment techniques usually employ gold particles or image derived markers to correctly align the images. When these markers are not present, correlation between adjacent views is used to align them. However, sequential pairwise correlation is prone to bias and the resulting alignment is not always optimal.     

Safety assessment of near infrared light emitting diodes for diffuse optical measurements

Background  

Near infrared (NIR) light has been used widely to monitor important hemodynamic parameters in tissue non-invasively. Pulse oximetry, near infrared spectroscopy, and diffuse optical tomography are examples of such NIR light-based applications. These and other similar applications employ either lasers or light emitting diodes (LED) as the source of the NIR light. Although the hazards of laser sources have been addressed in regulations, the risk of LED sources in such applications is still unknown.     

Cationic hydrous thorium dioxide colloids - a useful tool for staining negatively charged surface matrices of bacteria for use in energy-filtered transmission electron microscopy

Background  

Synthesis of cationic hydrous thorium dioxide colloids (ca. 1.0 to 1.7 nm) has been originally described by Müller [22] and Groot [11] and these have been used by Groot to stain acidic glucosaminoglycans for ultrastructure research of different tissues by conventional transmission electron microscopy.     

Thiolactomycin analogues as potential anti-Toxoplasma gondii agents

The discovery of new compounds active against Toxoplasma gondii is extremely important due to the severe disease caused by this pathogen in immunocompromised hosts and to congenital infection. Type II fatty acid biosynthesis has shown to be a promising target for drug intervention in toxoplasmosis. Here we describe the inhibitory effect of 8 thiolactomycin (TLM) analogues against tachyzoite-infected LLC-MK₂ cells. The TLM analogues demonstrated anti-T. gondii activity, arresting tachyzoite proliferation with IC₅₀ values in the micromolar level after 24 h and 48 h of treatment. Metabolic labelling of extracellular parasites treated with TLM analogues using [³H]acetate demonstrated that these drugs affected acylglycerol synthesis. The rapid reduction of parasite load suggests that these compounds have selective cytotoxic effects against T. gondii. Transmission electron microscopy demonstrated that TLM analogues interfered with membrane-bounded organelles and parasite division and this in turn affected parasite development and survival.     

VisualRepbase: an interface for the study of occurrences of transposable element families

Background  

Repbase is a reference database of eukaryotic repetitive DNA, which includes prototypic sequences of repeats and basic information described in annotations. Repbase already has software for entering new sequence families and for comparing the user's sequence with the database of consensus sequences.     

Ultrastructural changes during early infection of Vigna unguiculata and Phaseolus vulgaris leaves by Xanthomonas axonopodis pv. phaseoli and an unexpected association between chloroplast and mitochondrion

Common bacterial blight, caused by Xanthomonas axonopodis pv. phaseoli, is an important disease affecting beans causing considerable yield losses in many producing areas. Our aim was to investigate Xap–beans interactions in the first 48 h after infection analyzing structurally relevant changes by electron microscopy. Scanning electron microscopy analysis demonstrated morphological changes on leaf surfaces of both species. Transmission electron microscopy showed different types of chloroplast structural disorganization in both parenchyma and bundle sheath cells of Vigna unguiculata which were only observed in parenchyma cells of Phaseolus vulgaris. Additionally, we report an unexpected physical association between the chloroplasts and mitochondria observed only to P. vulgaris-infected plants.