We developed a light microscope-based image acquisition and analysis platform to perform high resolution screening (HRS). By optimizing the system performance we achieved high throughput at large scale of in-plane resolution (from 10x/0.3 to 60x/0.9, max resolution of 0.3 mm), accepting variety of multi-well plate types. To match the throughput in the interpretation to that of the acquisition we built an automated analysis pipeline. Currently we have two screening systems.
The systems specifications:
Microscope: model IX71 or IX81 [Olympus, Japan]. IX81 has computer-controlled exchange of fluorescence filter cubes, precise focus positioning and motorized nosepiece. Both microscopes have computer control on filter wheels, focus and stage. The microscope automation is provided by ProScan [Prior, Cambridge, UK].
Digital camera: ANDOR camera [ANDOR technology, Ireland] equipped with EEV back illuminated CCD chip with very high quantum efficiency in the visible and the near IR.
Automatic focus: The focusing during screening is achieved by a laser based autofocus, which employ an accessory red laser beam and confocality principle to adjust the objective into focus by detecting the reflections of the laser from the substrate surfaces. Allow the use of wide range of plate types. Sweeping focus projections are possible for fast imaging of specimens with thickness greater than the objectives focal depth. Environment for living
Cells: the microscopes are inserted in a plexiglass box heated to 370 by air circulator. CO2 is supplied into a small plexiglass chamber and exchanges into the wells via Luminex BioFolie foil [Hanover, Germany]. Image acquisition: Programmed optimal scanning path (wells, points within wells, number of images per well, time lapse movies). Acquisition rate 15 to 40 images/minute (for exposure time of 1 sec).
Image visualization and analysis modules: Interactive visualization; Image processing; Image segmentation and connected component analysis; Statistical analysis; Multi-parametric scoring of changes in treated versus control cells.
Sample preparation: A robotic system [Beckman Coulter, CA] in a sterile environment is used for sample preparation.
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Microscope |
Robot |
