The three‐dimensional architecture of the mitotic spindle, analyzed by confocal fluorescence and electron microscopy

Abstract

Fluorescence microscopy techniques have become important tools in mitosis research. The well-known disadvantages of fluorescence microscopy, rapid bleaching, phototoxicity and out-of-focus contributions blurring the in-focus image are obstacles which still need to be overcome. Confocal fluorescence microscopy has the potential to improve our capabilities of analyzing cells, because of its excellent depth-discrimination and image processing power. We have been using a confocal fluorescence microscope for the study of the mechanism of poleward chromosome movement, and report here 1) a cell preparation technique, which allows labeling of fixation sensitive spindle antigens with acceptable microtubule preservation; 2) the use of image processing methods to represent the spatial distribution of various labeled elements in pseudocolour; 3) a novel immunoelectron microscopic labeling method for microtubules, which allows the visualization of their distribution in semithin sections at low magnification; and 4) a first attempt to study microtubule dynamics with a confocal fluorescence microscope in living cells, microinjected with rhodamine labeled tubulin. Our experience indicates that confocal fluorescence microscopy provides real advantages for the study of spatial colocalization of antigens in the mitotic spindle. It does not, however, overcome the basic limits of resolution of the light microscope. Therefore, it has been necessary to use an electron microscopic method. Our preliminary results with living cells show that it is possible to visualize the entire microtubule network in stereo, but that the sensitivity of the instrument is still too low to perform dynamic time studies. It will be worthwhile to further develop this new type of optical instrumentation and explore its usefulness on both fixed and living cells.

Measurement of co‐localization of objects in dual‐colour confocal images

“…Due to the recent development of dual-colour confocal microscopy, co-localization studies of objects or processes in doubly labelled specimens have become feasible (Akner et al, 1990;Draeger et al, 1990;Kitamura et al, 1990;Mossberg et al, 1990;Sato et al, 1990;Stamatoglou et al, 1990;Fox et al, 1991;Merdes et al, 1991). Akner et al (1991) presented a method to visualize the total overlap of two patterns by subtracting one component of an image from the other.…”

The Three-dimensional Study of Chromosomes and Upstream Binding Factor-immunolabeled Nucleolar Organizer Regions Demonstrates Their Nonrandom Spatial Arrangement during Mitosis

“…These chromosomes limit a circular cavity, which corresponds to the presence of a half spindle with a conic shape (Merdes et al, 1991).…”

“…On the other hand, the depression of the concave polar face, which was a half sphere during early anaphase, acquires a more profound and larger cylindrical shape during early telophase, and adopts a less profound but wider cylindrical form limited by a regular ledge during late telophase. Such modifications could be linked not only to the shortening and spreading of the kinetokorial fibers but also to other proteic complexes during the migration of the chromosomes toward the pole (Merdes et al, 1991;Paddy and Chelsky, 1991;Gaglio et al, 1997).…”

Transmission X‐ray microscopy of intact hydrated PtK2 cells during the cell cycle

“…7c). These X-ray micrographs differed markedly from the images of identical structures obtained with different types of high-resolution light microscopy, such as polarization microscopy, differential interference microscopy and fluorescence microscopy (Inoué, 1986;Cassimeris et al, 1988;Lacey, 1989;Merdes et al, 1991). None of the above-mentioned optical methods allows completely unstained, fully hydrated individual microtubules to be visualized in the intact cell, but it should be noted that it is possible to visualize the individual native microtubules in specimens of extruded squid axoplasm by video-enhanced microscopy although not reflecting their real size (see Allen et al, 1985).…”