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A HeLa cell is shown over a period of 18 hours, which expresses both a sgGFP as well as an mRFP tagged version of Histone 2B labelling chromatin in two colours. Following the chromatin distribution over time shows, that it is relatively stable even during and after mitosis, indicating formation of chromosomal territories.
Image kindly provided by Dr. Horst Wolff, GSF-Institute for Molecular Virology, Retroviral Regulation group (Dr. R. Brack), Neuherberg, Germany and Manja Ziegler, Institute for Human Genetics (Prof. T. Cremer), University of Munich, Germany
The movies are provided as Video for Windows (avi) or QuickTime format (mov):
H2B-Bleaching.mov (340 KB)
H2B-Bleaching.avi (249 KB)
Description: A part of the nucleus of the cell was bleached using a regular HBO lamp by closing the field aperture diaphragm first in the GFP channel to provide a small illumination region; the bleaching was repeated for another region of the nucleus using the RFP filter. This procedure produced a three colour painting of the nucleus (Red/yellow/green, yellow resulting from the red/green overlay). The color merge view of the Nomarski (DIC) channel with the GFP and the RFP channels is shown. First, the bleaching was not followed by any observable recovery after photo bleaching ("FRAP") effect, indicating stable territorial organization of the chromatin. Even after cell division the resulting daughter nuclei do show distinct green and red colouring indicating that the territorial organization even survives mitosis.
For further reading on the subject see the article by Cremer et al.
http://www.ncbi.nlm.nih.gov
Technical details
HeLa cells were grown on glass bottom dishes and transfected with the appropriate expression plasmids. Image acquisition was started 24 hours post transfection in a fully automated Cell Observer System with a Plan Neofluar 40x/1.30 Oil Ph3 objective, incubator XL, heating insert P and CO2 cover HP for best pH and humidity control. The HBO light source was attenuated to 6% using neutral density filters in an excitation filterwheel featuring a fast incident light shutter for both fluorescence channels to reduce photo damage. The following filtersets were used: GFP with filterset #44; DsRed with filter set #15). Imaging was performed using an AxioCam HRm CCD camera using binning 2x2 to increase sensitivity and reduce exposure time). Microscope and camera were controlled by AxioVision 4 using the modules Multichannel, Time-lapse and Z-Stack. A 3- channel experiment including a phase contrast channel was set up with 3 Z-slices, each 4.25 µm apart covering an axial range of 8.5 µm to guarantee the capture of an in-focus plane even during cell division where typically cells round up and leave the focal plane. Images were acquired every 25 minutes for a total time of 18 hours. The finished time lapse image was processed to only contain in focus planes, annotated and exported using AxioVision’s export functionality. | |
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