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Expanding the multicolor capabilities of basic confocal microscopes by employing red and near-infrared quantum dot conjugates
Lara M Kingeter, Brian C Schaefer
BMC Biotechnology , 2009, DOI: 10.1186/1472-6750-9-49
Abstract: We modified a three-laser Zeiss Pascal confocal microscope by the addition of two band-pass filters and one long-pass filter for the detection of three different red to near-infrared quantum dot conjugates. We then performed direct comparisons between organic dye- and quantum dot-labeled detection reagents for the detection of subcellular structures. We found that the quality of staining was generally indistinguishable, although quantum dot reagents do have certain limitations, relative to organic dye conjugates. Using the modified Pascal system, three quantum dot conjugates, two organic dye conjugates, and one fluorescent protein, we demonstrated clean discrimination of six distinct fluorescent labels in a single sample.Our data demonstrate that nearly any basic confocal microscope can be modified by the simple addition of appropriate emission filters, allowing the detection of red and near-infrared quantum dot conjugates. Additionally, quantum dot- and organic dye-based secondary reagents can be successfully combined in complex intracellular staining experiments. Substantial expansion of the multi-parameter capabilities of basic confocal instruments can be achieved with a financial investment that is minimal in comparison to instrument replacement or upgrade with additional lasers.Over the past 20 years, confocal microscopy has become a centrally important technique for the analysis of biological samples. By using a pinhole to exclude scattered light, confocal instruments can be used to optically section biological samples, producing 2- and 3-dimensional images with spatially resolved details at the sub-micron level. Beyond simply visualizing fluorescently labeled specimens, confocal microscopy has become a powerful tool for biologists in many disciplines for diverse applications, including establishing structure-function relationships at the cellular and tissue level, defining dynamic processes in living specimens, and for detection of close interactions between
Real-Time Measurements of the Redox States of c-Type Cytochromes in Electroactive Biofilms: A Confocal Resonance Raman Microscopy Study  [PDF]
Bernardino Virdis, Diego Millo, Bogdan C. Donose, Damien J. Batstone
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0089918
Abstract: Confocal Resonance Raman Microscopy (CRRM) was used to probe variations of redox state of c-type cytochromes embedded in living mixed-culture electroactive biofilms exposed to different electrode polarizations, under potentiostatic and potentiodynamic conditions. In the absence of the metabolic substrate acetate, the redox state of cytochromes followed the application of reducing and oxidizing electrode potentials. Real-time monitoring of the redox state of cytochromes during cyclic voltammetry (CV) in a potential window where cytochromes reduction occurs, evidenced a measurable time delay between the oxidation of redox cofactors probed by CV at the electrode interface, and oxidation of distal cytochromes probed by CRRM. This delay was used to tentatively estimate the diffusivity of electrons through the biofilm. In the presence of acetate, the resonance Raman spectra of young (10 days, j = 208±49 μA cm?2) and mature (57 days, j = 267±73 μA cm?2) biofilms show that cytochromes remained oxidized homogeneously even at layers as far as 70 μm from the electrode, implying the existence of slow metabolic kinetics that do not result in the formation of a redox gradient inside the biofilm during anode respiration. However, old biofilms (80 days, j = 190±37 μA cm?2) with thickness above 100 μm were characterized by reduced catalytic activity compared to the previous developing stages. The cytochromes in these biofilm were mainly in the reduced redox state, showing that only aged mixed-culture biofilms accumulate electrons during anode respiration. These results differ substantially from recent observations in pure Geobacter sulfurreducens electroactive biofilms, in which accumulation of reduced cytochromes is already observed in thinner biofilms, thus suggesting different bottlenecks in current production for mixed-culture and G. sulfurreducens biofilms.
Simple Method for Sub-Diffraction Resolution Imaging of Cellular Structures on Standard Confocal Microscopes by Three-Photon Absorption of Quantum Dots  [PDF]
Anje Sporbert, Zoltan Cseresnyes, Meike Heidbreder, Petra Domaing, Stefan Hauser, Barbara Kaltschmidt, Christian Kaltschmidt, Mike Heilemann, Darius Widera
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0064023
Abstract: This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.
Confocal laser scanning microscopy analysis of S. epidermidis biofilms exposed to farnesol, vancomycin and rifampicin
Nuno Cerca, Fernanda Gomes, Sofia Pereira, Pilar Teixeira, Rosário Oliveira
BMC Research Notes , 2012, DOI: 10.1186/1756-0500-5-244
Abstract: 24 h biofilms were exposed to farnesol, vancomycin or rifampicin and were analysed by CLSM, after stained with a Live/Dead stain, a known indicator of cell viability, related with cell membrane integrity. Biofilms were also disrupted by sonication and viable and cultivable cells were quantified by colony forming units (CFU) plating. Farnesol showed a similar effect as vancomycin, both causing little reduction of cell viability but at the same time inducing significant changes in the biofilm structure. On the other hand, rifampicin showed a distinct action in S. epidermidis biofilms, by killing a significant proportion of biofilm bacteria.While farnesol is not very efficient at killing biofilm bacteria, it damages cell membrane, as determined by the live/dead staining, in a similar way as vancomycin. Furthermore, farnesol might induce biofilm detachment, as determined by the reduced biofilm biomass, which can partially explain the previous findings regarding its role as a possible chemotherapy adjuvant.Staphylococcus epidermidis, a normal inhabitant of human skin and mucosa, has recently emerged as a leading cause of biofilm-related infections, particularly, in patients with indwelling medical devices [1,2] due to its ability to adhere to abiotic surfaces and to form biofilms [2,3]. Biofilms are often defined as three-dimensional communities of microorganisms that are attached to a surface and encased in an extracellular matrix composed mainly of polysaccharides, proteins and extracellular DNA [4,5]. The major virulence factor of S. epidermidis is biofilm formation [6] and cells in biofilms are normally more tolerant to antibiotics than planktonic cells [7-9], making drug resistance in a S. epidermidis biofilm-related infection a serious problem, especially in nosocomial infections [10]. S. epidermidis are also inherently resistant to host defense mechanisms [11,12]. The increased resistance to conventional antibiotic therapy led to the search for new therapeutical a
Scanning optical and electron microscopes with computer image acquisition*
K. Konstankiewicz,A. Pukos
International Agrophysics , 1995,
Abstract: A Tandem Scanning Reflected Light Microscope (TSRLM) with computer image analyser and with a structure quantimeter (hardware, software and rotary microtome) are presented and compared to other microscopic methods with respect to new possibilities in visualisation of biological material. Scanning optical microscopes are the most recent constructions in optical microscopy. They offer the rejection of out-of-focus noise and higher contrast than the conventional imaging. The only allowed to reach a detector is the light emitted from the objective focal plane. This cuts off any out-of-focus image blurring. A short history of confocal microscopy from Marvin Minsky to Tandem Scanning Reflected Light Microscope (TSRLM) and Confocal Laser Scanning Microscope (CLSM) has been presented. The use of scanning optical and electron microscope method for the investigation of biological materials is estimated and compared.
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]
Biofilms’ Role in Planktonic Cell Proliferation  [PDF]
Elanna Bester,Gideon M. Wolfaardt,Nahid B. Aznaveh,Jesse Greener
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms141121965
Abstract: The detachment of single cells from biofilms is an intrinsic part of this surface-associated mode of bacterial existence. Pseudomonas sp. strain CT07 gfp biofilms, cultivated in microfluidic channels under continuous flow conditions, were subjected to a range of liquid shear stresses (9.42 mPa to 320 mPa). The number of detached planktonic cells was quantified from the effluent at 24-h intervals, while average biofilm thickness and biofilm surface area were determined by confocal laser scanning microscopy and image analysis. Biofilm accumulation proceeded at the highest applied shear stress, while similar rates of planktonic cell detachment was maintained for biofilms of the same age subjected to the range of average shear rates. The conventional view of liquid-mediated shear leading to the passive erosion of single cells from the biofilm surface, disregards the active contribution of attached cell metabolism and growth to the observed detachment rates. As a complement to the conventional conceptual biofilm models, the existence of a biofilm surface-associated zone of planktonic cell proliferation is proposed to highlight the need to expand the traditional perception of biofilms as promoting microbial survival, to include the potential of biofilms to contribute to microbial proliferation.
Research on improving performance to metallographic microscopes
Teodor Socaciu,Mihai ?imon
Scientific Bulletin of the ''Petru Maior" University of T?rgu Mure? , 2011,
Abstract: Precision optical components from an old optical microscope can be improved and emphasized with a dedicated digital microscope camera. This is an affordable way to obtain a high performance metallographic or biological microscope, with minimum of spending. This paper study those ways and adapts a camera to existing microscopes for researchers use, Optimizing visualization by projecting the image and improving the microscope use by different options of capture and image processing.
Disruption of Yarrowia lipolytica biofilms by rhamnolipid biosurfactant  [cached]
Dusane Devendra H,Dam Sushovan,Nancharaiah Yarlagadda V,Kumar Ameeta
Aquatic Biosystems , 2012, DOI: 10.1186/2046-9063-8-17
Abstract: Background Yarrowia lipolytica is an ascomycetous dimorphic fungus that exhibits biofilm mode of growth. Earlier work has shown that biosurfactants such as rhamnolipids are efficient dispersants of bacterial biofilms. However, their effectiveness against fungal biofilms (particularly Y. lipolytica) has not been investigated. The aim of this study was to determine the effect of rhamnolipid on a biofilm forming strain of Y. lipolytica. Two chemical surfactants, cetyl-trimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS) were used as controls for comparison. Results The methylene blue dye exclusion assay indicated an increase in fungal cell permeability after rhamnolipid treatment. Microtiter plate assay showed that the surfactant coating decreased Y. lipolytica biofilm formation by 50%. Rhamnolipid treatment disrupted pre-formed biofilms in a more effective manner than the other two surfactants. Confocal laser scanning microscopic studies showed that biofilm formation onto glass surfaces was decreased by 67% after sub-minimum inhibitory concentration (sub-MIC) treatment with rhamnolipids. The disruption of biofilms after rhamnolipid treatment was significant (P<0.05) when compared to SDS and CTAB. Conclusion The results indicate a potential application of the biological surfactant to disrupt Y. lipolytica biofilms.
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
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