RESUMEN
In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells.
Asunto(s)
Células Dendríticas/ultraestructura , Imagenología Tridimensional/métodos , Proteínas Luminiscentes/análisis , Microscopía Confocal/métodos , Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/análisis , Células Cultivadas , Células Dendríticas/virología , Fluorescencia , Substitución por Congelación , Congelación , VIH , Humanos , Microscopía Electrónica de Rastreo/métodos , Microtomía , Proteínas Recombinantes/análisis , Adhesión del Tejido , Virión/ultraestructura , Proteína Fluorescente RojaRESUMEN
Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a approximately 45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (approximately 3-10 microm) and microtubules (approximately 1-8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing. Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 microm and 1 microm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be approximately 1 microm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support. Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.
Asunto(s)
Filamentos Intermedios/química , Filamentos Intermedios/ultraestructura , Microscopía de Fuerza Atómica , Proteínas Recombinantes/química , Vimentina/química , Citoesqueleto de Actina/química , Actinas/química , Humanos , Microscopía Electrónica , Modelos Moleculares , DocilidadRESUMEN
The tiron-hydrogen peroxide reaction used for catalytic determination of trace metals is studied with respect to the indicator substance-the oxidation product of tiron absorbing at 44Onm. Contrary to assumptions in the literature, it is shown by electrochemical techniqus EPR spectroscopy and visible spectrophotometry that the indicator substance is not the o-quinone derivative of tiron (lambda(max) = 372 nm) but the tiron semiquinone radical with a g-value of g(o) = 2.0049 and molar absorptivity = (3.5 +/- 0.5) x 10(3) 1.mole(-1).cm(-1) at 440 nm.
RESUMEN
Morphologically, glutaraldehyde-fixed and -dried intermediate filaments (IFs) appear flexible, and with a width of 8-12 nm when observed by electron microscopy. Sometimes, the filaments are even unraveled on the carbon-coated grid and reveal a protofilamentous architecture. In this study, we have used atomic force microscopy to further investigate the morphology of IFs in a more physiological environment. First, we have imaged hydrated glutaraldehyde-fixed IFs adsorbed to a graphite support. In such conditions, human vimentin and desmin IFs appeared compact with a height of 5-8 nm and revealed either a beading repeat or a helical morphology. Second, we have analyzed the architecture of hydrated vimentin, desmin, and neurofilament IFs adsorbed to mica, graphite, and hydrophilic glass without the presence of fixative. On mica, vimentin IFs had a height of only 3-5 nm, whereas desmin IFs appeared as 8-10 nm height filaments with a helical twist. Neurofilaments were 10-12 nm in height with a pronounced 30-50 nm beading along their length. On graphite, the different IFs were either not adsorbing properly or their architecture was modified yielding, for example, broad, flattened filaments. Finally, hydrophilic glass was the surface which seemed to best preserve the architecture of the three IFs, even if, in some cases, unraveled vimentin filaments were observed on this support. These results are straightening the idea that mature IFs are dynamic polymers in vitro and that IFs can be distinguished from each others by their physicochemical properties.