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Metaphase PtK1 cell microinjected with X-rhodamine-labeled tubulin and Alexa 488-labeled CENP-F antibodies to fluorescently label kinetochore fibers (red) and kinetochores/spindle poles (green), respectively. The movie shows that sister kinetochore pairs oscillate between the two spindle poles as their respective kinetochore fibers lengthen and shorten.
Digital images were collected with an Orca ER cooled CCD camera (Hamamatsu Photonics) coupled to a Yokogawa spinning disk confocal CS10 unit (Perkin Elmer Life Sciences Wallac), which was attached to a Nikon TE300 inverted microscope. Illumination was achieved via an Argon/Krypton laser and 488 nm and 568 nm filters in an excitation filter wheel (Sutter Instruments) through a 60 x 1.4 NA Plan-Apochromatic DIC objective lens. Nearly simultaneous fluorescence images were acquired at 488 and 568 nm at a single focal plane every 21 seconds. The movie is played at 10 frames per second. Elapsed time is shown in min:sec.
Lisa A Cameron
Edward D Salmon
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