[New appliance for fluorescence microscopy in incident and transmitted light].
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
Conformational changes in F-actin, induced by glutaraldehyde or phalloidin, were found in glycerinated m. psoas rabbit fibres (ghost and reconstructed fibres). It is shown that the conformational changes of F-actin decreased the thin filament flexibility as well as weakened fibre's contractility. It is assumed that the stabilization in F-actin structure may be an important factor involved in the mechanism of muscle contraction regulation.
Quantitative fluorescence microscopy is becoming an increasingly important tool in the study of cell biology. Fluorescence microscopy has long been used for qualitative characterizations of subcellular distributions of proteins, lipids, nucleic acids, and ions, but quantifying these distributions is complicated by a variety of optical, biological, and physical factors. Many factors that complicate quantification of fluorescence in cells can be circumvented by analyzing fluorescence ratios derived from pairs of fluorescence images. In this review we will discuss the factors that affect fluorescence quantification, the advantages of quantifying fluorescence as a ratio, and give examples of how fluorescence ratio microscopy is being applied in studies of cell biology.
Single molecule fluorescence detection of Atto590 in poly(vinyl alcohol) was achieved by using a wide-field epifluorescence microscope with CCD-camera detection. Image sequences are obtained from which the time traces of the detected molecules are built. We find a distinctive difference between the time evolution of the fluorescence originating from the molecules detected in the first image of the sequence compared to the time evolution of the fluorescence of the molecules detected in each image of the sequence. Atto590 shows very long blinking times and photobleaching and photoblinking that are both quadratically dependent on the irradiation power density. Our approach allows kinetic separation of photobleaching from blinking. The possibility of choosing different ensembles of molecules is demonstrated and taken advantage of for this aim. Initially dark molecules or low emitting ones that might be overlooked are important to describe the complete ensemble behavior.
Fluorescence microscopy may provide important information regarding interactions between nanoparticulate drugs carriers, such as liposomes and micelles, with target cells as well as their intracellular fate. Current paper describes various applications of fluorescence microscopy to investigate specific targeting of antibody-modified drug carriers to cancer cells. The enhanced antibody-mediated targeting of drug-loaded immunomicelles confirmed by fluorescence microscopy resulted in enhanced cancer cell killing compared to free drug or drug-loaded nontargeted micelles. Fluorescence microscopy was also used to prove the endosomal escape of properly assembled polymeric micelles (based on polyethylene glycol-phosphatidylethanolamine conjugate, PEG-PE) containing various additives destabilizing the endosomal membrane. When loaded with the anticancer drug (paclitaxel or vitamin K3), such micelles demonstrate increased cytotoxicity. Fluorescence microscopy was also applied to investigate the capture of cell-penetrating TAT peptide-modified liposomes by various cells and stability and intracellular trafficking of captured TAT-liposomes inside cells. It was also used to confirm the successful transfection of cells with TAT-liposomes bearing the plasmid encoding for the Green Fluorescent Protein (GFP).
By means of polarized ultraviolet fluorescence microscopy the conformational changes of F-actin occuring in glycerinated muscle fibers of rabbit and barnacle (Balanus rostratus Hock.) under the influence of adenosine triphosphate in the presence of ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid were discovered. These changes seem to be located near the surface of the globules thus hampering the penetration of univalent iones and neutral molecules into the F-actin macromolecule. It is suggested that similar changes of F-actin take place in thin myofilaments of living muscle fiber during the contraction-relaxation process.
In a novel application of two-photon scanning fluorescence microscopy, three-dimensional spatial distributions of the hydrophilic and hydrophobic fluorescent probes, sulforhodamine B and rhodamine B hexyl ester, in excised full-thickness human cadaver skin were visualized and quantified. Both sulforhodamine B and rhodamine B hexyl ester were observed to lie primarily in the lipid multilamellae region surrounding the corneocytes within the stratum corneum. From the two-photon scanning fluorescence microscopy scans, the changes in the concentration gradient and the vehicle to skin partition coefficient of each probe induced by the oleic acid enhancer action were calculated relative to the control sample (not exposed to oleic acid), and subsequently applied to theoretically derived mathematical expressions of transdermal transport to quantitatively characterize the oleic acid-induced relative changes in the skin diffusion coefficient and the skin barrier diffusion length of the permeant. For the hydrophobic probe rhodamine B hexyl ester, the permeability enhancement was primarily driven by an increase in the vehicle to skin partition coefficient, leading to an increase in the steepness of the concentration gradient across the skin. The primary oleic acid-induced changes in the transport properties of the hydrophilic probe sulforhodamine B included increases in the vehicle to skin partition coefficient and the skin diffusion coefficient. These findings utilizing the two-photon scanning fluorescence microscopy methodology and data analysis described here demonstrate that, in addition to providing three-dimensional images that clearly delineate probe distributions in the direction of increasing skin depth, the subsequent quantification of these images provides additional important insight into the mechanistic changes in transdermal transport underlying the visualized changes in probe distributions across the skin.
Non-isotopic (fluorescent) in situ hybridization has established itself as a useful technique for the localization of DNA sequences in both metaphase and interphase cells. The rapid development of digital fluorescence microscopy, especially confocal microscopy, has become a powerful aid for the evaluation of the hybridization results in cytogenetic and cell biological applications. In this review we will demonstrate the utility of these methodologies for the three-dimensional visualization and analysis of chromosome-specific (peri)centromeric repetitive DNA sequences within the intranuclear structure of human cells and cell lines.
A simple fluorescence technique is decribed to localise in situ the non-replicating alpha heterochromatin in the chromocentre region of Drosophila nasuta polytene nuclei. After incorporating 5-bromodeoxyuridine in larval salivary gland cells for one or two cycles of replication, the polytene nuclei are examined for Hoechst 33258 flourescence at pH 7.0. The non-replicating alpha heterochromatin remains brightly fluorescing as it does not incorporate any 5-bromodeoxyuridine while the rest of the replicating chromatin shows dull fluorescence due to the quenching of Hoechst 33258 fluorescence by the bromodeoxyuridine substituted DNA.