Hybrid Detectors Improved Time-Lapse Confocal Microscopy of PML and 53BP1 Nuclear Body Colocalization in DNA Lesions

2013 ◽  
Vol 19 (2) ◽  
pp. 360-369 ◽  
Author(s):  
Veronika Foltánková ◽  
Pavel Matula ◽  
Dmitry Sorokin ◽  
Stanislav Kozubek ◽  
Eva Bártová
Keyword(s):  
Confocal Microscopy ◽  
Protein Interactions ◽  
Laser Scanning ◽  
Nuclear Body ◽  
Dna Lesions ◽  
Nuclear Bodies ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Γ Irradiation ◽  
Pml Nuclear Bodies

AbstractWe used hybrid detectors (HyDs) to monitor the trajectories and interactions of promyelocytic leukemia (GFP-PML) nuclear bodies (NBs) and mCherry-53BP1-positive DNA lesions. 53BP1 protein accumulates in NBs that occur spontaneously in the genome or in γ-irradiation-induced foci. When we induced local DNA damage by ultraviolet irradiation, we also observed accumulation of 53BP1 proteins into discrete bodies, instead of the expected dispersed pattern. In comparison with photomultiplier tubes, which are used for standard analysis by confocal laser scanning microscopy, HyDs significantly eliminated photobleaching of GFP and mCherry fluorochromes during image acquisition. The low laser intensities used for HyD-based confocal analysis enabled us to observe NBs for the longer time periods, necessary for studies of the trajectories and interactions of PML and 53BP1 NBs. To further characterize protein interactions, we used resonance scanning and a novel bioinformatics approach to register and analyze the movements of individual PML and 53BP1 NBs. The combination of improved HyD-based confocal microscopy with a tailored bioinformatics approach enabled us to reveal damage-specific properties of PML and 53BP1 NBs.

scholarly journals Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

2003 ◽  
Vol 77 (9) ◽  
pp. 5401-5414 ◽  
Author(s):  
Maria Dimitrova ◽  
Isabelle Imbert ◽  
Marie Paule Kieny ◽  
Catherine Schuster
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interactions ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nonstructural Proteins

ABSTRACT Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

Contribution of Electron and Confocal Microscopy in the Study ofLeishmania–Macrophage Interactions

2004 ◽  
Vol 10 (5) ◽  
pp. 656-661 ◽  
Author(s):  
Birgitta Rasmusson ◽  
Albert Descoteaux
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Protozoan Parasite ◽  
Laser Scanning Microscopy ◽  
Sand Fly ◽  
Confocal Laser ◽  
Mammalian Host ◽  
Dependent Manner ◽  
Phagosome Maturation ◽  
Physical Barrier

Promastigotes of the protozoan parasite genusLeishmaniaare inoculated into a mammalian host when an infected sand fly takes a bloodmeal. Following their opsonization by complement, promastigotes are phagocytosed by macrophages. There, promastigotes differentiate into amastigotes, the form of the parasite that replicates in the phagolysosomal compartments of host macrophages. Although the mechanisms by which promastigotes survive the microbicidal consequence of phagocytosis remain, for the most part, to be elucidated, evidence indicates that glycoconjugates play a role in this process. One such glycoconjugate is lipophosphoglycan, an abundant promastigote surface glycolipid. Using quantitative electron and confocal laser scanning microscopy approaches, evidence was provided thatL. donovanipromastigotes inhibit phagolysosome biogenesis in a lipophosphoglycan-dependent manner. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that preventsL. donovanipromastigote-containing phagosomes from interacting with endocytic vacuoles. Inhibition of phagosome maturation may constitute a strategy to provide an environment propitious to the promastigote-to-amastigote differentiation.

scholarly journals CONFOCAL LASER SCANNING MICROSCOPY FOR DIAGNOSES OF MELANOCYTIC SKIN NEOPLASMS

2017 ◽  
Vol 20 (6) ◽  
pp. 324-331
Author(s):  
Anna V. Sokolova ◽  
N. P Malishevskaya
Keyword(s):  
Differential Diagnosis ◽  
Confocal Microscopy ◽  
Histological Examination ◽  
Laser Scanning ◽  
Diagnostic Algorithm ◽  
Surgical Excision ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Skin Melanoma ◽  
Main Criterion

In cases of complicated differential diagnosis of benign melanocytic neoplasms and skin melanoma, clinical visual examination and superficial dermatoscopy do not always allow to make an accurate diagnosis, which is especially important in cases of suspected skin melanoma. In recent years the method of confocal microscopy (CM) has been used for non-invasive skin examination. In the present study 15 patients with suspected skin melanoma were examined by confocal laser microscopy in cases of complex differential diagnosis (18 melanocytic neoplasms in total). Ten patients were diagnosed with skin melanoma, including two amelanotic melanomas, which were subsequently confirmed by histological examination of the material obtained after surgical excision. The results of the study to determine the effectiveness of differential diagnosis of skin melanoma by laser confocal microscopy showed that differential diagnosis of skin melanoma is possible in the presence of the main criteria of malignancy: cellular atypia at the site of the dermo-epidermal junction (the first main criterion) and abnormality of papillary architecture (the second main criterion). At detection at least one of these criteria, a diagnosis of skin melanoma was made, which in all cases was confirmed by the results of histological examination. Inclusion of CM in the diagnostic algorithm of examination of patients with melanocytic neoplasms, suspicious of melanoma, makes it possible to improve the diagnosis of melanoma at early stages.

scholarly journals MICROSCOPY TECHNIQUES ARE EFFECTIVE TOOLS FOR IMPLEMENTING STUDIES IN DAIRY SCIENCE AND TECHNOLOGY

2018 ◽  
Author(s):  
Paolo D'Incecco ◽  
Luisa Pellegrino
Keyword(s):  
Protein Interactions ◽  
Milk Fat ◽  
Laser Scanning ◽  
Biological Membrane ◽  
Immunogold Labelling ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Storage Lipids ◽  
Fat Globules ◽  
Microscopy Techniques

Milk is a complex system where lipids, proteins, sugars and salts are present in different phases, and thus shows a characteristic behaviour during either technological treatments or storage. Lipids are organized as globules, small drops of triglycerides surrounded by a biological membrane that ensures stability of their emulsion. Casein is the main milk protein and is organized as micelles containing salts and strongly hydrated. This last feature has an important effect on the micelle stability. Furthermore, micelle stability is ensured by glycosylated k-casein fragments. Interaction between fat globules and casein micelles are likely to occur since ~1010 globules e 1014 micelles are present in 1 mL of milk and their reactive surface is approximately 0.07 and 4 m2 respectively. Different milk processes, i.e. mechanic or thermal, are responsible for interactions which may vary in number and chemical nature. Microscopy techniques represent an indispensable tool to study milk microstructure during milk processing or even on finished products upon storage. Confocal laser scanning microscopy, through specific probes, is suitable to study phenomena of coalescence among fat globules and fat-protein interactions in fluid milk, gel (clotted milk) or cheese. Transmission electron microscopy and immunogold labelling are used to more deeply investigate either milk components ultrastructure or specific interactions established between the milk fat globule membrane and the casein fractions. Food products are matrices where a multidisciplinary approach is necessary for their study, and microscopy certainly plays a key role.

scholarly journals Clinical Applications of In Vivo and Ex Vivo Confocal Microscopy

2021 ◽  
Vol 11 (5) ◽  
pp. 1979
Author(s):  
Stefania Guida ◽  
Federica Arginelli ◽  
Francesca Farnetani ◽  
Silvana Ciardo ◽  
Laura Bertoni ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Real Life ◽  
Laser Scanning Microscopy ◽  
Pathological Examination ◽  
Confocal Laser ◽  
Fluorescence Confocal Microscopy ◽  
Skin Cancers

Confocal laser scanning microscopy (CLSM) has been introduced in clinical settings as a tool enabling a quasi-histologic view of a given tissue, without performing a biopsy. It has been applied to many fields of medicine mainly to the skin and to the analysis of skin cancers for both in vivo and ex vivo CLSM. In vivo CLSM involves reflectance mode, which is based on refractive index of cell structures serving as endogenous chromophores, reaching a depth of exploration of 200 μm. It has been proven to increase the diagnostic accuracy of skin cancers, both melanoma and non-melanoma. While histopathologic examination is the gold standard for diagnosis, in vivo CLSM alone and in addition to dermoscopy, contributes to the reduction of the number of excised lesions to exclude a melanoma, and to improve margin recognition in lentigo maligna, enabling tissue sparing for excisions. Ex vivo CLSM can be performed in reflectance and fluorescent mode. Fluorescence confocal microscopy is applied for “real-time” pathological examination of freshly excised specimens for diagnostic purposes and for the evaluation of margin clearance after excision in Mohs surgery. Further prospective interventional studies using CLSM might contribute to increase the knowledge about its application, reproducing real-life settings.

Confocal microscopy of the fertilization-induced calcium wave in sea urchins

1991 ◽  
Vol 49 ◽  
pp. 392-393 ◽  
Author(s):  
Stephen A. Strieker ◽  
Stephen W. Paddock ◽  
Gerald Schatten
Keyword(s):  
Confocal Microscopy ◽  
Sea Urchin ◽  
Laser Scanning ◽  
Sea Urchins ◽  
Metabolic Activation ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cortical Granule ◽  
Free Calcium

At fertilization, the egg undergoes a rapid rise in intracellular free calcium (=[Ca2+]i), which in turn triggers several important events, including cortical granule release and metabolic activation. In sea urchins, the fertilization induced increase in [Ca2+]i begins at the site of sperm fusion and sweeps through the egg in a wavelike fashion. Whether fertilization affects calcium levels only in the peripheral ooplasm or throughout the interior of the sea urchin egg is difficult to ascertain by conventional microscopic techniques, because out-offocus rays obscure subcellular organization in these relatively bulky cells. Recently, however, confocal microscopy has been introduced as a means of producing thin optical sections that reveal details not normally discernable by conventional light microscopy. In this paper, we use confocal laser scanning microscopy (CLSM) and time-lapse reconstructions to image calcium dynamics during fertilization in living sea urchin eggs labeled with the calcium-sensitive fluorescent dye fluo-3.

CONFOCAL MICROSCOPY APPLIED TO PALEONTOLOGICAL SPECIMENS

2016 ◽  
Vol 22 ◽  
pp. 39-55 ◽  
Author(s):  
Alexander D. Ball ◽  
Tomasz Goral ◽  
Seyit A. Kamanli
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Fluorescent Dyes ◽  
Three Dimensional ◽  
Sample Surface ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Image Stack ◽  
Selective Staining ◽  
Wide Range

AbstractConfocal laser scanning microscopy is a well-established optical technique allowing for three-dimensional (3-D) visualization of fluorescent specimens with a resolution close to the diffraction limit of light. Thanks to the availability of a wide range of fluorescent dyes and selective staining using antibodies, the technique is commonly used in life sciences as a powerful tool for studying different biological processes, often at the level of single molecules. However, this type of approach is often not applicable for specimens that are preserved in historical slide collections, embedded in amber, or are fossilized, and cannot be exposed to any form of selective staining or other form of destructive treatment. This usually narrows the number of microscopic techniques that can be used to study such specimens to traditional light microscopy or scanning electron microscopy. However, these techniques have their own limitations and cannot fully reveal 3-D structures within such barely accessible samples. Can confocal microscopy be of any help? The answer is positive, and it is due to the fact that many paleontological specimens exhibit a strong inherent autofluorescence that can serve as an excellent source of emitted photons for confocal microscopy visualizations either through reconstruction of the induced autoflourescent signal from the sample, or through reconstruction of the reflected signal from the sample surface. Here, we describe the workflow and methodology involved in acquiring confocal data from a sample and reprocessing the resulting image stack using the image-processing program imageJ before reconstructing the data using the open-source 3-D rendering program, Drishti. This approach opens new possibilities for using confocal microscopy in a nondestructive manner for visualizing complex paleontological material that has never previously been considered as suitable for this type of microscopic technique.

scholarly journals Confocal microscopy applied to water mite taxonomy with the description of a new genus of Axonopsinae (Acari, Parasitengona, Hydrachnidia) from Central America

Zootaxa ◽  
2008 ◽  
Vol 1820 (1) ◽  
pp. 41 ◽  
Author(s):  
ANTONIO G. VALDECASAS
Keyword(s):  
New Species ◽  
Confocal Microscopy ◽  
Central America ◽  
Science Citation Index ◽  
Laser Scanning ◽  
New Genus ◽  
Citation Index ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Water Mite

Vagabundia sci n. gen. n. sp. of the subfamily Axonopsinae is proposed and described. Confocal Laser Scanning Microscopy, not previously applied to water mite taxonomy, allowed the acquisition and posterior processing of clean optical slices. The new species is compared to other mites that have been described as ‘Axonopsella-like’. Vagabundia sci n. sp. is named after the Science Citation Index, a sociological tool that, as explained in the text, has done more harm than good to the population of taxonomists.

scholarly journals Murine Salmonellosis Studied by Confocal Microscopy: Salmonella typhimurium Resides Intracellularly Inside Macrophages and Exerts a Cytotoxic Effect on Phagocytes In Vivo

1997 ◽  
Vol 186 (4) ◽  
pp. 569-580 ◽  
Author(s):  
Agneta Richter-Dahlfors ◽  
Alison M.J. Buchan ◽  
B. Brett Finlay
Keyword(s):  
Confocal Microscopy ◽  
Salmonella Typhimurium ◽  
Mouse Liver ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Phagocytic Cells ◽  
Intracellular Location ◽  
Subcellular Level

Salmonella typhimurium is considered a facultative intracellular pathogen, but its intracellular location in vivo has not been demonstrated conclusively. Here we describe the development of a new method to study the course of the histopathological processes associated with murine salmonellosis using confocal laser scanning microscopy of immunostained sections of mouse liver. Confocal microscopy of 30-μm-thick sections was used to detect bacteria after injection of ∼100 CFU of S. typhimurium SL1344 intravenously into BALB/c mice, allowing salmonellosis to be studied in the murine model using more realistic small infectious doses. The appearance of bacteria in the mouse liver coincided in time and location with the infiltration of neutrophils in inflammatory foci. At later stages of disease the bacteria colocalized with macrophages and resided intracellularly inside these macrophages. Bacteria were cytotoxic for phagocytic cells, and apoptotic nuclei were detected immunofluorescently, whether phagocytes harbored intracellular bacteria or not. These data argue that Salmonella resides intracellularly inside macrophages in the liver and triggers cell death of phagocytes, processes which are involved in disease. This method is also applicable to other virulence models to examine infections at a cellular and subcellular level in vivo.

scholarly journals Meibomian Glands or Not? Identification of In Vivo and Ex Vivo Confocal Microscopy Features and Histological Correlates in the Eyelid Margin

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Yu-jing Wang ◽  
Min Ke
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Meibomian Gland ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Eyelid Margin ◽  
Healthy Humans ◽  
Skin Appendages

Purpose. In vivo confocal laser scanning microscopy (CLSM) is an emerging diagnostic tool allowing fast and easy microscopic tissue examination. For the diagnostics of pathological eyelid margin lesions, the knowledge of the normal eyelid margin is essential. Methods. We examined 18 eyelid margins of healthy humans using the in vivo CLSM device and 10 samples of healthy eyelid margins from donor sites with ex vivo CLSM and compared the findings to the corresponding histological sections of donor sites. Cross-section images of different depths and depths of different skin appendages were measured. Results. The depth observed by in vivo CLSM is less than 150 μm into the eyelid. Images of the epidermis and superficial dermis skin, appendages including hair follicle, and sebaceous catheters can be captured associated with histopathology and ex vivo confocal microscopy. In correlation with histopathology, we identified different layers of the eyelid margin, different layers of the epidermis, and skin appendages by ex vivo confocal microscopy. Conclusions. The study offers an overview of the in vivo confocal microscopy human eyelid margin characteristics in comparison to the standard histological examination and confirms that in vivo CLSM could not observe the meibomian gland acini structure.

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