| |
HeLa cells are shown which express a GFP tagged version of the small regulatory Rev-protein of HIV-1 as well as an mRFP tagged version of Histone-2B, labelling the chromatin.
The sample was kindly provided by Dr. Horst Wolff, GSF-Institute for Molecular Virology, Retroviral Regulation Group (Dr. R. Brack), Neuherberg, Germany. Image acquisition was done during a training course for Carl Zeiss Imaging specialists.
The movies are provided as Video for Windows (avi) or QuickTime format (mov):
Dividing_HeLa.mov (947 KB)
Dividing_HeLa.avi (610 KB)
Description: The localization of the green Rev-protein during cell division is followed over time in three dimensions. The Rev protein is excluded from the nuclei/nucleoli during mitosis but starts to relocate to the nucleoli (see yellow arrows appearing in the last time points) about 30 minutes after the nuclear envelope has been reformed. The technology which makes this kind of time lapse imaging possible, involves a collection of measure which minimizes the deleterious effects of fluorescence imaging on living cells.
Technical details: Cells were grown in glass bottom dishes, transfected with expression plasmids for Rev-sgGFP and H2B-mRFP and observed 24 hours later. Image acquisition was done in a fully automated CellObserver System with an EC Plan Neofluar 63x/1.25 Oil objective, incubator XL, heating insert P and CO2 cover HP for best pH and humidity control. The HBO light source was dimmed to 1.5% for both fluorescence channels using neutral density filters in an excitation filterwheel featuring a fast incident light shutter to avoid photo damage. Imaging was performed using an AxioCam HRm CCD camera with binning 2x2 to increase sensitivity and decrease needed exposure time. The incident light shutter was under direct trigger control of the camera to minimize light exposure. Z-stacks were acquired for both fluorescent channels (20 slices, 1.2 µm slice distance), a DIC image was acquired for the central Z-slice only (“Center plane only mode”) to provide a morphological reference. Image stacks were acquired every 7 minutes for a total time of about 75 minutes.
Stacks were processed as follows: 3D Deconvolution was performed on the fluorescent channels using the Regularized Inverse Filter method. 6 Z-planes containing the desired information were extracted from the result image and processed using the Extended Focus module to generate a single plane time lapse image containing all spatially relevant information. Annotations were performed using AxioVision’s annotation features and exported as movies.
The 3D rendering of one time point was created using the Inside4D rendering module.
The following AxioVision modules were used in addition to the basic software functions: Multichannel, Time Lapse, Z-Stack, Extended Focus and 3D Deconvolution. | |
|