|The 4D workstation includes a Zeiss Axioplan 2 microscope equipped with a motor-driven stage, differential interference contrast (DIC) optics and a Zeiss AxioCam MRc5 IEEE 1394 color CCD camera housed within an incubator set at 37°C and 5% CO2. The light is controlled by a timer that is synchronized with the AxioVision image acquisition software and the motorized stage.|
3 2D workstations housed in a 37°C, 5% CO2 incubators with image acquisition controlled by Fire-I software.
We developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.2, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 µm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. J3D-DIAS 4.2 converts images to mathematical representations from which quantitative parameters are then derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. Our data on breast cancer cell lines suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.