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Confocal microscopy in the diagnosis of melanoma
Apostolovi?-Stojanovi? Milica,Dobrosavljevi?-Vukojevi? Danijela,La?kovi? Vesna,Stojanovi? A.
Archives of Biological Sciences , 2013, DOI: 10.2298/abs1301279s
Abstract: Melanoma is the most deadly form of skin cancer of melanocytic origin. The tumor has a high malignant potential and early metastasis. Prognosis is directly linked to the stage of the disease. Diagnosing thin melanoma at an early stage offers patients their best chance for survival. The crucial innovation in the early recognition of melanoma was the development of in vivo examination of the skin in high-resolution, by confocal microscopy. Confocal microscopy and its modifications provides a “virtual biopsy“, owing to melanosomes and melanin, which are a source of endogenous contrast. Confocal scanning laser microscopy (CSLM) provides visualization of microanatomical structures and cellular detail in real time (pigmented keratinocytes, melanocytes, melanosomes and melanophages) in the epidermis, dermoepidermal junction and superficial dermis at a resolution equivalent to the resolution of conventional microscopes. [Projekat Ministarstva nauke Republike Srbije, br. 41002]
Basic confocal microscopy  [cached]
Manuela Monti
European Journal of Histochemistry , 2012, DOI: 10.4081/ejh.2012.br3
Abstract: This is an eleven chapter’s effort done by a bunch of Authors coordinated by Prof. R.L. Price and W.G. Jerome (who have personally written almost half of the book) that with great skills are revealing us the secrets of confocal microscopy. Considering the significant progresses in different fields of biology, confocal microscopy is extremely important to dynamically see all the different molecules involved in the controlling networks build up by gene expressions in time and space. Necessary prerequisites to accomplish such goals are some fundamental microscopic technologies well and clearly presented in the first chapters....
Confocal Microscopy in Biopsy Proven Argyrosis  [PDF]
Melis Palamar,Suzan Guven Yilmaz,Taner Akalin,Sait Egrilmez,Ayse Yagci
Case Reports in Ophthalmological Medicine , 2013, DOI: 10.1155/2013/875989
Abstract: Purpose. To evaluate the confocal microscopy findings of a 46-year-old male with bilateral biopsy proven argyrosis. Materials and Methods. Besides routine ophthalmologic examination, anterior segment photography and confocal microscopy with cornea Rostoch module attached to HRT II (Heidelberg Engineering GmbH, Heidelberg, Germany) were performed. Findings. Squamous metaplastic changes on conjunctival epithelium and intense highly reflective extracellular punctiform deposits in conjunctival substantia propria were detected. Corneal epithelium was normal. Highly reflective punctiform deposits starting from anterior to mid-stroma and increasing through Descemet’s membrane were evident. Corneal endothelium could not be evaluated due to intense stromal deposits. Conclusion. Confocal microscopy not only supports diagnosis in ocular argyrosis, but also demonstrates the intensity of the deposition in these patients. 1. Introduction Prolonged exposure to silver might cause irreversible pigmentation of the skin (argyria) and/or the eyes (argyrosis) [1]. Hands, eyes, and mucous membranes are affected in most of the patients, and discoloration of the ocular surface is the main ocular evidence [1–3]. A direct relationship was shown between the amount of discoloration and the length of time worked [1]. Confocal microscopy provides high-resolution, high-contrast in vivo images and is a powerful tool for studying the ultrastructure of the cell, its molecular components, and their functions. The Rostock cornea module is an option of the Heidelberg Retina Tomograph (HRT II, Version 3.0; Heidelberg Engineering GMBH, Dossenheim, Germany) introduced as an improvement over older confocal microscopes. The module consists of a monochrome laser radiation source which avoids chromatic aberrations and provides extremely sharp images and a high-powered lens that allows the operator to change the confocal plane within the cornea to capture images at different depths without losing sharpness [4]. The aim of this study is to demonstrate the location of conjunctival and corneal silver deposits by confocal microscopy and to evaluate the correlation between conjunctival histopathology and confocal microscopy findings in a case of occupational argyrosis. To the best of our knowledge this is the first biopsy proven argyrosis case to be evaluated by confocal microscopy. 2. Report of a Case A 46-year-old long-standing silver worker who was diagnosed as ocular argyrosis 4 years earlier was evaluated with confocal microscopy. His visual acuity was 10/20 and intraocular pressures were normal
QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY  [cached]
Merete Krog Raarup,Jens Randel Nyengaard
Image Analysis and Stereology , 2006, DOI: 10.5566/ias.v25.p111-120
Abstract: This paper discusses recent advances in confocal laser scanning microscopy (CLSM) for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm) tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging Microscopy), FCS (Fluorescence Correlation Spectroscopy) and FRAP (Fluorescence Recovery After Photobleaching) are introduced and their applicability for quantitative imaging of biomolecular (co-)localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.
Twin-Photon Confocal Microscopy  [PDF]
D. S. Simon,A. V. Sergienko
Physics , 2010, DOI: 10.1364/OE.18.022147
Abstract: A recently introduced two-channel confocal microscope with correlated detection promises up to 50% improvement in transverse spatial resolution [Simon, Sergienko, Optics Express {\bf 18}, 9765 (2010)] via the use of photon correlations. Here we achieve similar results in a different manner, introducing a triple-confocal correlated microscope which exploits the correlations present in optical parametric amplifiers. It is based on tight focusing of pump radiation onto a thin sample positioned in front of a nonlinear crystal, followed by coincidence detection of signal and idler photons, each focused onto a pinhole. This approach offers further resolution enhancement in confocal microscopy.
Clinical applications of corneal confocal microscopy  [cached]
Mitra Tavakoli,Parwez Hossain,Rayaz A Malik
Clinical Ophthalmology , 2008,
Abstract: Mitra Tavakoli1, Parwez Hossain2, Rayaz A Malik11Division of Cardiovascular Medicine, University of Manchester and Manchester Royal Infirmary, Manchester, UK; 2University of Southampton, Southampton Eye Unit, Southampton General Hospital, Southampton, UKAbstract: Corneal confocal microscopy is a novel clinical technique for the study of corneal cellular structure. It provides images which are comparable to in-vitro histochemical techniques delineating corneal epithelium, Bowman’s layer, stroma, Descemet’s membrane and the corneal endothelium. Because, corneal confocal microscopy is a non invasive technique for in vivo imaging of the living cornea it has huge clinical potential to investigate numerous corneal diseases. Thus far it has been used in the detection and management of pathologic and infectious conditions, corneal dystrophies and ecstasies, monitoring contact lens induced corneal changes and for pre and post surgical evaluation (PRK, LASIK and LASEK, flap evaluations and Radial Keratotomy), and penetrating keratoplasty. Most recently it has been used as a surrogate for peripheral nerve damage in a variety of peripheral neuropathies and may have potential in acting as a surrogate marker for endothelial abnormalities.Keywords: corneal confocal microscopy, cornea, infective keratitis, corneal dystrophy, neuropathy
In vivo evaluation of DSAEK interface with scanning-laser confocal microscopy  [cached]
Ferrari Giulio,Reichegger Verena,Ludergnani Luca,Delfini Elisabetta
BMC Ophthalmology , 2012, DOI: 10.1186/1471-2415-12-32
Abstract: Background Descemet Stripping Automated Endothelial Keratoplasty (DSAEK) allows selective replacement of the endothelium. Post-operative haze and particles can affect the interface quality and, ultimately, visual outcome. In this study, we evaluated DSAEK interface with in vivo laser confocal microscopy (LCM) in order to: (i) correlate interface status with best corrected visual acuity, and (ii) with time from surgery; (iii) correlate interface particle number with best corrected visual acuity. Host-donor interface was imaged and graded using a published reflectivity scale. Particles at the interface were counted. Methods 18 eyes of 16 patients (6 males and 10 females); mean age: 74 ± 8.3 years which underwent DSAEK were examined by means of in vivo laser confocal microscopy between 1 and 24 months after surgery. Host-donor interface was imaged and graded using a published reflectivity scale. Particles present at the interface were counted. Results Interface reflectivity was 2.17 ± 1.2 and significantly correlated with visual acuity (Spearman correlation coefficient 0.83; P < 0.001), and with time after surgery (Spearman correlation coefficient 0.87; P < 0.001). Visual acuity was 0.67 ± 0.27. The number of particles was 205 ± 117.8; no correlation was found between this number and visual acuity (Spearman correlation coefficient 0.41; P = 0.15). Conclusion DSAEK interface imaged with LCM is helpful in diagnosing poor host-donor interface quality in DSAEK surgery. A good quality interface is related to a better visual acuity. Moreover, the quality of the interface appears to improve as time passes from the surgery. Interface quality is related with visual acuity and improves with time from surgery. LCM should be considered as an added tool in post-DSAEK follow-up of patients. Finally, our study shows that the presence of particles does not influence visual outcome.
Fluorescent ligands for studying neuropeptide receptors by confocal microscopy
Beaudet, A.;Nouel, D.;Stroh, T.;Vandenbulcke, F.;Dal-Farra, C.;Vincent, J.-P.;
Brazilian Journal of Medical and Biological Research , 1998, DOI: 10.1590/S0100-879X1998001100017
Abstract: this paper reviews the use of confocal microscopy as it pertains to the identification of g-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. principles that should guide the choice of suitable ligands and fluorophores are discussed. examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or bodipy-tagged bioactive peptides. the results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. in the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. these mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.
Fluorescent ligands for studying neuropeptide receptors by confocal microscopy  [cached]
Beaudet A.,Nouel D.,Stroh T.,Vandenbulcke F.
Brazilian Journal of Medical and Biological Research , 1998,
Abstract: This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.
The use of confocal microscopy in quantifying changes in membrane potential
D Elgar, F Verdonck, A Grobler, C Fourie, J Du Plessis
African Journal of Biotechnology , 2006,
Abstract: Monitoring the plasma membrane potential and its changes can be a time consuming and challenging task especially when conventional electrophysiological techniques are used. The use of potentiometric fluorophores, namely tetramethylrhodamine methylester (TMRM), and digital imaging devices (laser scanning confocal microscopy) provides reliable and time efficient method. Two scorpion pore-forming peptides, namely PP and OP1, were used as a tool to induce depolarization of the plasma membrane potential of neuroblastoma cell line and cardiac myocytes. Alternative methods for the neuroblastoma cells and cardiac myocytes were used. Depolarization of the neuroblastoma cells was calibrated with 140 mM KCl solution with 1 ìM valinomycin, after which intensity readers were substituted in the Nernst equation for quantification. Calibration of the alternative method used of the cardiac myocytes’ plasma membrane potential changes was calibrated with the use of 5, 20, 40, and 80 mM KCl solutions with 1 ìM valinomycin. A calibration curve was then constructed from which plasma membrane potential could be calculated
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