Light Microscopy Approaches Research Articles

A Brief Overview to Ageing-Related Organ Damage: A Light and Electron Microscopic Approach to Several Systems

Ageing is thought to be a degenerative process caused by accumulated damage leading to cellular dysfunction, tissue, and organ failure, and eventually death. Although aging-related skin changes are considered the most important indicator of ageing, morphological and functional changes occur in all of the internal organs. Most of those changes are directly or indirectly associated with age-related decline of life quality. Histopathological features of the organs of old people are examined in postmortem tissues which are probably affected by diseases alongside ageing itself. Recent studies principally report the cellular changes obtained from rodents. The most commonly reported ageing-related ultrastructural changes are mitochondrial damage, lysosome and lipofuscin accumulation and dilatation or proliferation of endoplasmic reticulum. Here I summarize ageing-related changes in various organs such as skin, brain, heart, kidney, intestines etc. that have been revealed by light and electron microscopic examinations so far. Understanding ageing-related cell and tissue-based changes and related molecular mechanisms will contribute to the development of new strategies to prevent or eliminate age-related organ damage.

A Brief Overview to Ageing-Related Organ Damage: A Light and Electron Microscopic Approach to Several Systems

A Brief Overview to Ageing-Related Organ Damage: A Light and Electron Microscopic Approach to Several Systems

Assaying RIPK2 Activation by Complex Formation.

The receptor-interacting serine/threonine-protein kinase-2 (RIPK2, RIP2) is a key player in downstream signaling of nuclear oligomerization domain (NOD)-like receptor (NLR)-mediated innate immune response against bacterial infections. RIPK2 is recruited following activation of the pattern recognition receptors (PRRs) NOD1 and NOD2 by sensing bacterial peptidoglycans leading to activation of NF-κB and MAPK pathways and the production of pro-inflammatory cytokines. Upon NOD1/2 activation, RIPK2 forms complexes in the cytoplasm of human cells, also called RIPosomes. These can be induced by Shigella flexneri or by the inhibition of RIPK2 by small compounds, such as GSK583 and gefitinib.In this chapter, we describe fluorescent light microscopic and Western blot approaches to analyze the cytoplasmic aggregation of RIPK2 upon infection with the invasive, Gram-negative bacterial pathogen Shigella flexneri, or by the treatment with RIPK2 inhibitors. This method is based on HeLa cells stably expressing eGFP-tagged RIPK2 and describes a protocol to induce and visualize RIPosome formation. The described method is useful to study the deposition of RIPK2 in speck-like structures, also in living cells, using live cell imaging and can be adopted for the study of other inhibitory proteins or to further analyze the process of RIPosome structure assembly.

Analysis of the Localization of NLRs upon Shigella flexneri Infection Exemplified by NOD1.

NOD-like receptors (NLRs) are a family of pattern recognition receptors, able to respond to conserved microbial structures and endogenous danger signals. The NLR NOD1 responds to bacterial peptidoglycan, leading to recruitment of RIPK2, following activation of NFκB and MAPK pathways. In this chapter, we describe a fluorescent light microscopic approach to analyze the subcellular distribution of NOD1 upon infection with the invasive, Gram-negative bacterial pathogen Shigella flexneri. This method is based on exogenously expressed EGFP-tagged NOD1 and describes a protocol to obtain inducible cell lines with functional NOD1 signaling. The described protocol is useful to study NOD1 function, also in living cells, using live cell imaging and can be adopted for the study of other NLR proteins.

Placozoan fiber cells: mediators of innate immunity and participants in wound healing

Placozoa is a phylum of non-bilaterian marine animals. These small, flat organisms adhere to the substrate via their densely ciliated ventral epithelium, which mediates mucociliary locomotion and nutrient uptake. They have only six morphological cell types, including one, fiber cells, for which functional data is lacking. Fiber cells are non-epithelial cells with multiple processes. We used electron and light microscopic approaches to unravel the roles of fiber cells in Trichoplax adhaerens, a representative member of the phylum. Three-dimensional reconstructions of serial sections of Trichoplax showed that each fiber cell is in contact with several other cells. Examination of fiber cells in thin sections and observations of live dissociated fiber cells demonstrated that they phagocytose cell debris and bacteria. In situ hybridization confirmed that fiber cells express genes involved in phagocytic activity. Fiber cells also are involved in wound healing as evidenced from microsurgery experiments. Based on these observations we conclude that fiber cells are multi-purpose macrophage-like cells. Macrophage-like cells have been described in Porifera, Ctenophora, and Cnidaria and are widespread among Bilateria, but our study is the first to show that Placozoa possesses this cell type. The phylogenetic distribution of macrophage-like cells suggests that they appeared early in metazoan evolution.

Super-resolution microscopy: a closer look at synaptic dysfunction in Alzheimer disease.

The synapse has emerged as a critical neuronal structure in the degenerative process of Alzheimer disease (AD), in which the pathogenic signals of two key players - amyloid-β (Aβ) and tau - converge, thereby causing synaptic dysfunction and cognitive deficits. The synapse presents a dynamic, confined microenvironment in which to explore how key molecules travel, localize, interact and assume different levels of organizational complexity, thereby affecting neuronal function. However, owing to their small size and the diffraction-limited resolution of conventional light microscopic approaches, investigating synaptic structure and dynamics has been challenging. Super-resolution microscopy (SRM) techniques have overcome the resolution barrier and are revolutionizing our quantitative understanding of biological systems in unprecedented spatio-temporal detail. Here we review critical new insights provided by SRM into the molecular architecture and dynamic organization of the synapse and, in particular, the interactions between Aβ and tau in this compartment. We further highlight how SRM can transform our understanding of the molecular pathological mechanisms that underlie AD. The application of SRM for understanding the roles of synapses in AD pathology will provide a stepping stone towards a broader understanding of dysfunction in other subcellular compartments and at cellular and circuit levels in this disease.

Application of multimicroscopic techniques (LM and SEM) in comparative palynological study of Asteroideae members, inhabited in Pakistan.

In the present study light and scanning electron microscopic approaches were used to study the palynological variations among 30 Asteroideae members. Variety of qualitative and quantitative palynological characters like pollen shape and type, pollen sculpturing, pollen size, P/E ratio, exine and intine thickness, interspecific difference, colpi size, pore size, spines length, spine width, and pollen fertility were observed. Results reported remarkable variations among pollen characters of studied taxa. The shape of pollen grains in polar and equatorial views varied from specie to specie such as spheroidal, prolate, oblate, subspheroidal, oblate spheroidal, prolate spheroidal, suboblate, and sub prolate. Bidens pilosa L. showed maximum pollen size in both polar and equatorial views, whereas minimum pollen size in polar view was found in Tetraneuris scaposa (DC.) Greene. (i.e., 30.5 μm) and in equatorial view was found in Cosmos sulphureus Cav. (25.5 μm).The highest P/E ratio (1.39 μm) was measured in Glebionis coronaria L. The values of exine and intine thickness also varied. Tricolporate, tetracolporate, trizonocolporate, pentoporate, and polypentoporate pollen types were examined. Spinateand echinate pollen sculpturingobserved under SEM. Variations in spine length and width also recorded. The maximum pollen fertility (98%) was measured in Chrysanthemum morifolium Ramat. and the lowest (56%) was recorded in Thymophylla tenuiloba (DC.) Small. It was concluded that the variations in qualitative and quantitative characters were seemed to be valuable for the taxonomic investigations of Asteroideae taxa.

Scanning electron microscopic screening; Can it be a taxonomic tool for identification of traditional therapeutic plants.

The limiting factor in the utilization of herbal medicine is the risk of replacement or substitution of their plant resources. Therefore, the authentication of medicinal plants before its use in herbal medicines is essentially required. Hence, the prime objective of this study was to provide some reliable morpho-anatomical and morpho-palynological tools for the identification of nine traditional therapeutic plants of district Lahore. For this study, scanning electron microscopic and light microscopic approaches has been employed. Results reported great variations in qualitative and quantitative morpho-anatomical features of the epidermal cell, stomatal complexes, trichomes, silica bodies and oil droplets. For example, Aegle marmelos L. can be distinguished from Cestrum diurnum L. based on epidermal cell shapes, as irregular cells in former and lobed cells shape in later one. Similarly unicellular, bicellular, and multicellular trichomes could be an identification basis for Cynodon dactylon L., Lantana camara L., and Pongamia pinnata L., respectively. In combination with these morpho-anatomical characters, SEM-based morpho-palynological characterization also reported diagnostic value. Both the Aegle marmelos and Lantana camara possessed similar cell shape (irregular) but can be delimited based on their pollen morphology as spheroidal pollen was reported in Aegle marmelos whereas prolate in Lantana camara. Moreover, quantitative characters also aid well for species delimitation, such as prolate pollen found in both Cestrum diurnum and Terminalia chebula Retz. but both of these varied in their P/E ratio, that is, 0.97 and 0.88, respectively. The leaf epidermal characters in correlation with morph-palynological traits showed potential for taxonomic resolution at the species level. RESEARCH HIGHLIGHTS: This study provides valuable morpho-anatomical and morpho-palynological tools for the nine traditional therapeutic plants of the district, Lahore, Pakistan. Light and scanning electron microscopic studies provided useful systematic data for the identification of studied plants. This systematic data include moprho-anatomical and morpho-palynological keys for the studied plants. This data can be used for future identification purposes.

An accelerated procedure for approaching and imaging of optically branded region of interest in tissue.

Many areas of biology have benefited from advances in light microscopy (LM). However, one limitation of the LM approach is that numerous critically important aspects of subcellular machineries are well beyond the resolution of conventional LM. For studying these, electron microscopy (EM) remains the technique of choice to visualize and identify macromolecules at the ultrastructural level. The most powerful approach is combining both techniques, LM and EM (i.e., to apply correlative light/electron microscopy, CLEM) to image exactly the same region of interest. This combination allows, for example, to immuno-localize proteins by LM and then to visualize the ultrastructural context of the same region of the sample. However, the identification and correlation of the regions of interest (ROIs) at the levels of LM and EM remains a major challenge, mostly due to the difficulties with correlation along the Z-axis for both modalities. In this chapter, we address this difficulty and describe an approach for performing CLEM in tissue samples using marks from near-infrared branding as indicators of a ROI, and then using serial block face-scanning electron microscopy (SBF-SEM) to identify and approach this ROI. Once a ROI has been approached, serial sections are collected on grids for high-resolution imaging by transmission EM, and subsequent correlation with LM images showing labeled proteins.

Exogenous Ochronosis as an Elastotic Disease: A Light-Microscopic Approach.

Exogenous ochronosis (EO) is a deposition disease associated with application of hydroquinone-containing preparations. Characteristic ochronotic bodies (OBs) arise from endogenous connective tissues, most often reported as collagen. We highlight a significant role for elastic fibers as a precursor tissue. To evaluate elastic tissue pathology in EO, specifically as it relates a precursor role in ochronotic body formation. In this retrospective observational study, a literature review using PubMed/MEDLINE database was conducted to ascertain the most commonly ascribed precursor connective tissue. Eleven histopathologic cases of EO were identified. Patient demographics and clinical characteristics were recorded. Slides were reviewed for the presence and grade of solar elastosis (SE), the relationship of OBs to elastotic material, the presence of elastotic fibers transitioning to OBs, and positivity of bodies with Verhoeff-van Gieson elastic tissue stain. Elastic fibers are uncommonly reported as the major precursor tissue of OBs. SE was uniformly present in our cases, and the majority demonstrated heavy/high-grade elastosis. Elastotic fibers transitioning to OBs were observed in all cases, and the bodies demonstrated Verhoeff-van Gieson positivity. Small sample size. Ochronotic body formation is associated with SE, and bodies appear to arise from damaged elastic fibers.

Pollen micromorphological analysis of tribe Acacieae (Mimosaceae) with LM and SEM techniques.

The morphology of pollen grains of the 06 species of tribe Acacieae (Mimosaceae) belonging to one genus were investigated using scanning electron microscopic (SEM) and light microscopic (LM) approach. Lactic acid acetolysis method was used for (LM) while non-acetolysis for (SEM). The species were examined for the both qualitative and quantitative palynological features. Qualitatively the pollen was studied for the shape, exine sculpturing, pollen shape, and pollen type. Oblate spheroidal shape of pollen was observed in Accacia modesta. Subprolate shape in Accaca nilotica. In Accacia farnesiana, prolate pollen was examined while prolate spheroidal in Acacia tortilis and spheroidal pollen grains were studied in Acacia catechu, respectively. Pollen exine sculpturing of the studied tribe possesses novel variation. Foveolate, reticulate, foveolate-psilate, faint reticulate, fossulate, perforate, and scrabate exine sculpturing were noticed. Colpi were absent in all species. Excessive variation was observed in both qualitative and quantitative character of pollen. SPSS software was use for the quantitative measurement of the pollen grains. The current study will be helpful for the identification of the problematic species and phylogenetic studies of family Mimosaceae.

Micro-computed tomographic analysis of the radial geometry of intrarenal artery-vein pairs in rats and rabbits: Comparison with light microscopy.

We assessed the utility of synchrotron-radiation micro-computed tomography (micro-CT) for quantification of the radial geometry of the renal cortical vasculature. The kidneys of nine rats and six rabbits were perfusion fixed and the renal circulation filled with Microfil. In order to assess shrinkage of Microfil, rat kidneys were imaged at the Australian Synchrotron immediately upon tissue preparation and then post fixed in paraformaldehyde and reimaged 24hours later. The Microfil shrank only 2-5% over the 24hour period. All subsequent micro-CT imaging was completed within 24hours of sample preparation. After micro-CT imaging, the kidneys were processed for histological analysis. In both rat and rabbit kidneys, vascular structures identified in histological sections could be identified in two-dimensional (2D) micro-CT images from the original kidney. Vascular morphology was similar in the two sets of images. Radial geometry quantified by manual analysis of 2D images from micro-CT was consistent with corresponding data generated by light microscopy. However, due to limited spatial resolution when imaging a whole organ using contrast-enhanced micro-CT, only arteries ≥100 and ≥60μm in diameter, for the rat and rabbit respectively, could be assessed. We conclude that it is feasible and valid to use micro-CT to quantify vascular geometry of the renal cortical circulation in both the rat and rabbit. However, a combination of light microscopic and micro-CT approaches are required to evaluate the spatial relationships between intrarenal arteries and veins over an extensive range of vessel size.

Ultrastructural Insights into the Biological Significance of Persisting DNA Damage Foci after Low Doses of Ionizing Radiation

Intensity-modulated radiotherapy (IMRT) enables the delivery of high doses to target volume while sparing surrounding nontargeted tissues. IMRT treatment, however, substantially increases the normal tissue volume receiving low-dose irradiation, but the biologic consequences are unclear. Using mouse strains that varied in genetic DNA repair capacity, we investigated the DNA damage response of cortical neurons during daily low-dose irradiation (0.1 Gy). Using light and electron microscopic approaches, we enumerated and characterized DNA damage foci as marker for double-strand breaks (DSBs). During repeated low-dose irradiation, cortical neurons in brain tissues of all mouse strains had a significant increase of persisting foci with cumulative doses, with the most pronounced accumulation of large-sized foci in repair-deficient mice. Electron microscopic analysis revealed that persisting foci in repair-proficient neurons reflect chromatin alterations in heterochromatin, but not persistently unrepaired DSBs. Repair-deficient SCID neurons, by contrast, showed high numbers of unrepaired DSBs in eu- and heterochromatin, emphasizing the fundamental role of DNA-PKcs in DSB rejoining, independent of chromatin status. In repair-deficient ATM-/- neurons, large persisting damage foci reflect multiple unrepaired DSBs concentrated at the boundary of heterochromatin due to disturbed KAP1 phosphorylation. Repeated low-dose irradiation leads to the accumulation of persisting DNA damage foci in cortical neurons and thus may adversely affect brain tissue and increase the risk of carcinogenesis. Multiple unrepaired DSBs account for large-sized foci in repair-deficient neurons, thus quantifying foci alone may underestimate extent and complexity of persistent DNA damage. Clin Cancer Res; 22(21); 5300-11. ©2016 AACR.

Purification and Localization of Intraflagellar Transport Particles and Polypeptides.

The growth and maintenance of almost all cilia and flagella are dependent on the proper functioning of the process of intraflagellar transport (IFT). This includes the primary cilia of most human cells that are in the Go phase of the cell cycle. The model system for the study of IFT is the flagella of the biflagellate green alga Chlamydomonas. It is in this organism that IFT was first discovered, and genetic data from a Chlamydomonas mutant first linked the process of IFT to polycystic kidney disease in humans. The information provided in this chapter addresses procedures to purify IFT particles from flagella and localize these particles, and their associated motor proteins, in flagella using light and electron microscopic approaches.

Dissecting tripartite synapses with STED microscopy.

The concept of the tripartite synapse reflects the important role that astrocytic processes are thought to play in the function and regulation of neuronal synapses in the mammalian nervous system. However, many basic aspects regarding the dynamic interplay between pre- and postsynaptic neuronal structures and their astrocytic partners remain to be explored. A major experimental hurdle has been the small physical size of the relevant glial and synaptic structures, leaving them largely out of reach for conventional light microscopic approaches such as confocal and two-photon microscopy. Hence, most of what we know about the organization of the tripartite synapse is based on electron microscopy, which does not lend itself to investigating dynamic events and which cannot be carried out in parallel with functional assays. The development and application of superresolution microscopy for neuron-glia research is opening up exciting experimental opportunities in this regard. In this paper, we provide a basic explanation of the theory and operation of stimulated emission depletion (STED) microscopy, outlining the potential of this recent superresolution imaging modality for advancing our understanding of the morpho-functional interactions between astrocytes and neurons that regulate synaptic physiology.

Altered parvalbumin basket cell inputs in the dorsolateral prefrontal cortex of schizophrenia subjects

Cortical circuitry dysfunction in schizophrenia has been studied at many different levels of resolution, but not at the most basic unit of network organization--synaptic inputs. Multi-label electron or confocal light microscopy is required to examine specific types of synaptic inputs, and application of these methods to quantitatively study disease-related changes in human postmortem tissue has not been feasible for technical reasons. We recently developed a multi-label confocal light microscopic approach that makes possible the systematic identification and quantification of synaptic inputs, and of the relative levels of proteins localized to these inputs, in human postmortem tissue. We applied this approach to quantify parvalbumin basket cell (PVBC) inputs in area 9 of the dorsolateral prefrontal cortex from schizophrenia and matched comparison subjects. Tissue sections were triple-labeled for the 65 kD isoform of glutamic acid decarboxylase (GAD65), PV and the GABA(A) receptor α1 subunit. PVBC axonal boutons were defined as PV/GAD65 dual-labeled puncta, and PVBC inputs were defined as a PVBC bouton that overlapped a GABA(A) receptor α1 subunit punctum. The density of PVBC inputs was unchanged in subjects with schizophrenia, but levels of PV protein were lower in PVBC boutons. In concert with prior reports, these findings indicate that PVBC dysfunction in schizophrenia reflects molecular and not structural alterations in these cells and their axon terminals.

Efficient and accurate analysis of mitochondrial morphology in a whole cell with a high-voltage electron microscopy

Mitochondria in all eukaryotes are essential organelles responsible for adenosine triphosphate synthesis, calcium homeostasis and steroidogenesis. Because the structure and distribution of mitochondria are highly diverse depending on their function and cellular conditions, it is important to develop a rapid and accurate method to assess their morphology. In this study, we visualize whole mitochondria in cultured cells using high-voltage electron microscopy (HVEM). Compared with conventional transmission electron microscopic approaches, the present method does not require thin sectioning and thus requires less time for image acquisition and processing. Furthermore, compared with fluorescence-based light microscopic approaches, our method provides more accurate size information. Thus, we propose that HVEM is a useful tool for rapid and accurate analysis of mitochondrial morphology and distribution in a cell.

Allelopathy in Compositae plants. A review

Allelopathy plays an major role in agricultural management such as weed control, crop protection, and crop re-establishment. Compositae plants have potent allelopathic activity, and the activity is confirmed through (a) bioassays with aqueous or various solvent extracts and residues, (b) fractionation, identification, and quantification of causative allelochemicals, and (c) mechanism studies on the allelochemicals. Most assessments of allelopathy involve bioassays of plant or soil extracts, leachates, fractions, and residues based on seed germination and seedling growth in laboratory and greenhouse experiments. Plant growth may be stimulated below the allelopathic threshold, but severe growth reductions may be observed above the threshold concentration depending upon the sensitivity of the receiving species. Generally germination is less sensitive than is seedling growth, especially root growth. Some approaches showed that field soil collected under donor plants significantly reduced or somewhat promoted growth of the test plants. Petri-dish bioassays with methanol extracts or fractions and causative phenolic allelochemicals showed significant phytotoxic activities in concentration-dependent manner. Delayed seed germination and slow root growth due to the extracts could be confounded with osmotic effects on rate of imbibition, delayed initiation of germination, and especially cell elongation; the main factor that affects root growth before and after the tip penetrates the seed coat. Light and electron microscopic approaches extract evaluation at the ultrastructural level have been precisely investigated. Many Compositae plants have allelopathic potentials, and the activities and types and amount of causative compounds differ depending on the plant species. The incorporation of allelopathic substances into agricultural management may reduce the use of pesticides and lessen environmental deterioration.

Distribution of hypothalamic, hippocampal and other limbic peptidergic neuronal cell bodies giving rise to retinopetal fibers: Anterograde and retrograde tracing and neuropeptide immunohistochemical studies

In our present work utilizing the retrograde or anterograde transport of tracers (biotinylated dextran amine and Fluorogold, respectively) we have provided direct evidence for the cells of origin of the limboretinal pathway in rats and their termination in the retina using light microscopic approach. Administration of biotinylated dextran amine into the vitreous body resulted in nerve cell body labeling in several structures: the supraoptic and paraventricular nuclei, the hippocampus (CA1, CA3), the dentate gyrus, the indusium griseum, the olfactory tubercle, and the medial habenula, all of them belong to the limbic system. We estimated that the total number of retrogradely labeled cells is 1495±516. We have seen fiber labeling in the retinorecipient suprachiasmatic nucleus and in the primary visual center, the lateral geniculate body, but labeled nerve cell bodies in these structures were never seen. Iontophoretic application of Fluorogold into the hippocampal formation, where the major part of the biotinylated dextran amine-labeled cell bodies was observed, resulted in labeled fibers in the optic nerve and in the retina indicating that the retrogradely labeled cells in the hippocampus and the dentate gyrus among others are the cells of origin of the centrifugal visual fibers. Sections showing biotinylated dextran amine labeling were stained for vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity using immunohistochemistry. Some biotinylated dextran amine-labeled cells also showed vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity. We conclude that the limboretinal pathway exists and that the cells of origin are partially vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactive.

Nano-Sizing of Specific Gene Domains in Intact Human Cell Nuclei by Spatially Modulated Illumination Light Microscopy

Although light microscopy and three-dimensional image analysis have made considerable progress during the last decade, it is still challenging to analyze the genome nano-architecture of specific gene domains in three-dimensional cell nuclei by fluorescence microscopy. Here, we present for the first time chromatin compaction measurements in human lymphocyte cell nuclei for three different, specific gene domains using a novel light microscopic approach called Spatially Modulated Illumination microscopy. Gene domains for p53, p58, and c-myc were labeled by fluorescence in situ hybridization and the sizes of the fluorescence in situ hybridization "spots" were measured. The mean diameters of the gene domains were determined to 103 nm (c-myc), 119 nm (p53), and 123 nm (p58) and did not correlate to the genomic, labeled sequence length. Assuming a spherical domain shape, these values would correspond to volumes of 5.7 x 10(-4) microm(3) (c-myc), 8.9 x 10(-4) microm(3) (p53), and 9.7 x 10(-4) microm(3) (p58). These volumes are approximately 2 orders of magnitude smaller than the diffraction limited illumination or observation volume, respectively, in a confocal laser scanning microscope using a high numerical aperture objective lens. By comparison of the labeled sequence length to the domain size, compaction ratios were estimated to 1:129 (p53), 1:235 (p58), and 1:396 (c-myc). The measurements demonstrate the advantage of the SMI technique for the analysis of gene domain nano-architecture in cell nuclei. The data indicate that chromatin compaction is subjected to a large variability which may be due to different states of genetic activity or reflect the cell cycle state.