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- ItemBright fluorescent silica-nanoparticle probes for high-resolution STED and confocal microscopy(Frankfurt am Main : Beilstein-Institut, 2017) Tavernaro, Isabella; Cavelius, Christian; Peuschel, Henrike; Kraegeloh, AnnetteIn recent years, fluorescent nanomaterials have gained high relevance in biological applications as probes for various fluorescencebased spectroscopy and imaging techniques. Among these materials, dye-doped silica nanoparticles have demonstrated a high potential to overcome the limitations presented by conventional organic dyes such as high photobleaching, low stability and limited fluorescence intensity. In the present work we describe an effective approach for the preparation of fluorescent silica nanoparticles in the size range between 15 and 80 nm based on L-arginine-controlled hydrolysis of tetraethoxysilane in a biphasic cyclohexane–water system. Commercially available far-red fluorescent dyes (Atto647N, Abberior STAR 635, Dy-647, Dy-648 and Dy-649) were embedded covalently into the particle matrix, which was achieved by aminosilane coupling. The physical particle attributes (particle size, dispersion, degree of agglomeration and stability) and the fluorescence properties of the obtained particles were compared to particles from commonly known synthesis methods. As a result, the spectroscopic characteristics of the presented monodisperse dye-doped silica nanoparticles were similar to those of the free uncoupled dyes, but indicate a much higher photostability and brightness. As revealed by dynamic light scattering and ζ-potential measurements, all particle suspensions were stable in water and cell culture medium. In addition, uptake studies on A549 cells were performed, using confocal and stimulated emission depletion (STED) microscopy. Our approach allows for a step-by-step formation of dye-doped silica nanoparticles in the form of dye-incorporated spheres, which can be used as versatile fluorescent probes in confocal and STED imaging.
- ItemIMAGE-IN: Interactive web-based multidimensional 3D visualizer for multi-modal microscopy images(San Francisco, California, US : PLOS, 2022) Gupta, Yubraj; Costa, Carlos; Pinho, Eduardo; A. Bastião Silva, Luís; Heintzmann, RainerAdvances in microscopy hardware and storage capabilities lead to increasingly larger multidimensional datasets. The multiple dimensions are commonly associated with space, time, and color channels. Since “seeing is believing”, it is important to have easy access to user-friendly visualization software. Here we present IMAGE-IN, an interactive web-based multidimensional (N-D) viewer designed specifically for confocal laser scanning microscopy (CLSM) and focused ion beam scanning electron microscopy (FIB-SEM) data, with the goal of assisting biologists in their visualization and analysis tasks and promoting digital work-flows. This new visualization platform includes intuitive multidimensional opacity fine-tuning, shading on/off, multiple blending modes for volume viewers, and the ability to handle multichannel volumetric data in volume and surface views. The software accepts a sequence of image files or stacked 3D images as input and offers a variety of viewing options ranging from 3D volume/surface rendering to multiplanar reconstruction approaches. We evaluate the performance by comparing the loading and rendering timings of a heterogeneous dataset of multichannel CLSM and FIB-SEM images on two devices with installed graphic cards, as well as comparing rendered image quality between ClearVolume (the ImageJ open-source desktop viewer), Napari (the Python desktop viewer), Imaris (the closed-source desktop viewer), and our proposed IMAGE-IN web viewer.
- ItemsimpleISM—A straight forward guide to upgrade from confocal to ISM(San Francisco, California, US : PLOS, 2022) Goswami, Monalisa; Lachmann, René; Kretschmer, Robert; Heintzmann, RainerResolution in a confocal laser scanning microscopes (CLSM) can be improved if the pinhole is closed. But closing the pinhole will deteriorate the signal to noise ratio (SNR). A simple technique to improve the SNR while keeping the resolution same by upgrading the system to an image scanning microscope. In this paper, we explain in detail, based on an Olympus Fluoview 300 system, how a scanning microscope can be upgraded into an image scanning microscope (ISM) using a simple camera-based detector and an Arduino Due providing a galvo driving and camera synchronization signals. We could confirm a resolution improvement as well as superconcentration and made the interesting observation of a reduced influence of laser fluctuations.
- ItemAnalyses and localization of pectin-like carbohydrates in cell wall and mucilage of the green alga Netrium digitus(Wien [u.a.] : Springer, 2010) Eder, M.; Lütz-Meindl, U.The unicellular, simply shaped desmid Netrium digitus inhabiting acid bog ponds grows in two phases. Prior to division, the cell elongates at its central zone, whereas in a second phase, polar tip growth occurs. Electron microscopy demonstrates that Netrium is surrounded by a morphologically homogeneous cell wall, which lacks pores. Immunocytochemical and biochemical analyses give insight into physical wall properties and, thus, into adaptation to the extreme environment. The monoclonal antibodies JIM5 and JIM7 directed against pectic epitopes with different degrees of esterification label preferentially growing wall zones in Netrium. In contrast, 2F4 marks the cell wall only after experimental de-esterification. Electron energy loss spectroscopy reveals Ca-binding capacities of pectins and gives indirect evidence for the degree of their esterification. An antibody raised against Netrium mucilage is not only specific to mucilage but also recognizes wall components in transmission electron microscopy and dot blots. These results indicate a smooth transition between mucilage and the cell wall in Netrium. © 2009 The Author(s).
- ItemSingle cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells([London] : Macmillan Publishers Limited, part of Springer Nature, 2016) Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, UteHepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.