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Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

ABSTRACT: Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

SUBMITTER: Yamagata K

PROVIDER: S-EPMC3275630 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

Yamagata Kazuo K Iwamoto Daisaku D Terashita Yukari Y Li Chong C Wakayama Sayaka S Hayashi-Takanaka Yoko Y Kimura Hiroshi H Saeki Kazuhiro K Wakayama Teruhiko T

PloS one 20120208 2

Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, ...[more]

PMID: 22347500

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Project description:BackgroundFrustrated phagocytosis occurs when an asbestos fiber >?10??m in length is engulfed imperfectly by a macrophage, and it is believed to be associated with chromosomal instability. Few studies have focused on dynamic cellular imaging to assess the toxicity of hazardous inorganic materials such as asbestos. One reason for this is the relative lack of fluorescent probes available to facilitate experimental visualization of inorganic materials. We recently developed asbestos-specific fluorescent probes based on asbestos-binding proteins, and achieved efficient fluorescent labeling of asbestos.ResultsLive-cell imaging with fluorescent asbestos probes was successfully utilized to dynamically analyze asbestos phagocytosis. The fluorescently labeled asbestos fibers were phagocytosed by RAW 264.7 macrophages. Internalized fibers of <?5??m in length were visualized clearly via overlaid phase contrast and fluorescence microscopy images, but they were not clearly depicted using phase contrast images alone. Approximately 60% of the cells had phagocytosed asbestos fibers after 2?h, but over 96% of cells remained alive even 24?h after the addition of asbestos fibers. Immediate cell death was only observed when an asbestos fiber was physically pulled from a cell by an external force. Notably, at 24?h after the addition of asbestos fibers an approximately 4-fold increase in the number of binucleated cells was observed. Monitoring of individual cell divisions of cells that had phagocytosed asbestos suggested that binucleated cells were formed via the inhibition of cell separation, by asbestos fibers of >?10??m in length that were localized in the proximity of the intercellular bridge.ConclusionsFluorescently labeled asbestos facilitated visualization of the dynamic biological processes that occur during and after the internalization of asbestos fibers, and indicated that (i) frustrated phagocytosis itself does not lead to immediate cell death unless the asbestos fiber is physically pulled from the cell by an external force, and (ii) macrophages that have phagocytosed asbestos can divide but sometimes the resulting daughter cells fuse, leading to the formation of a binucleated cell. This fusion only seemed to occur when a comparatively long asbestos fiber (>?10??m) was shared by two daughter cells.

Dataset Information

Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

Publications

Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

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