Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells.

• MeSH
• buněčné jádro metabolismus   MeSH
• fluorescenční mikroskopie metody   MeSH
• lidé MeSH
• struktury buněčného jádra * MeSH
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• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Extracellularly distributed collagen and chondrocytes seeded in gelatine and poly-ɛ-caprolactone scaffolds are visualized by two-photon excitation microscopy (TPEM) and second-harmonic generation (SHG) imaging in both forward and backward nondescanned modes. Joint application of TPEM and SHG imaging in combination with stereological measurements of collagen enables us not only to take high-resolution 3-D images, but also to quantitatively analyze the collagen volume and a spatial arrangement of cell-collagen-scaffold systems, which was previously impossible. This novel approach represents a powerful tool for the analysis of collagen-containing scaffolds with applications in cartilage tissue engineering.

• MeSH
• chondrocyty cytologie   metabolismus   transplantace   MeSH
• fluorescenční mikroskopie metody   MeSH
• interpretace obrazu počítačem metody   MeSH
• kolagen metabolismus   ultrastruktura   MeSH
• králíci MeSH
• kultivované buňky MeSH
• nelineární dynamika MeSH
• tkáňové podpůrné struktury MeSH
• zobrazování trojrozměrné metody   MeSH
• zvířata MeSH
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• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We investigated possibilities of the combination of the one- and two-photon excitation microscopy for examination of the experimental melanoma tissue in vivo, in mice under general anesthesia, and ex vivo on freshly harvested specimens. Our aim was to obtain sufficiently informative images of unstained tumor tissues and their modifications after hyperthermia treatment. The mouse experimental melanoma structure was studied and compared with normal tissue from the same animal by using confocal and nonlinear microscopy techniques based on (i) one-photon excitation (1PE) fluorescence, (ii) 1PE reflectance, (iii) second harmonic generation imaging, and (iv) two-photon excitation autofluorescence. We checked different spectral conditions and other settings of image acquisition, as well as combinations of the above imaging modalities, to fully exploit the potential of these techniques in the evaluation of treated and untreated cancer tissue morphology. Our approach enabled to reveal the collagen fiber network in relation with the other tissues, and to identify invasive tumor cells. It also proved to be useful for the examination of interrelationships between functional and morphological aspects based on optical properties of the tissues, especially in studies of changes between the tumor and control tissue, as well as changes induced by physical treatments, e.g., delivery of microwave hyperthermia treatment. These differences were also evaluated quantitatively, when we found out that the maximum Euler-Poincare characteristic reflects well the melanoma morphological structure. The results showed that the proposed investigative approach could be suitable also for a direct evaluation of tissue modifications induced by clinical interventions. (c) 2009 Wiley-Liss, Inc.

• MeSH
• indukovaná hypertermie MeSH
• melanom experimentální patologie   radioterapie   MeSH
• mikroskopie MeSH
• myši MeSH
• zvířata MeSH
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• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH