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Cell-based screening and assay development are challenges requiring extremely flexible solutions; no two experiments are the same. Therefore, Olympus has designed the scan^R Screening Station as a highly flexible “optical bench” enabling advanced microscope-based image acquisition and analysis. scan^R was developed into a powerful screening platform with an extremely broad application range on the basis of an experimental screening system designed by the European Molecular Biology Laboratory (EMBL).
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Capturing Mitosis in Live Cells
The Mitocheck project group at EMBL, is performing a live-cell RNAi screen on three scan^R Screening Stations to identify novel human genes involved in mitosis. A special screening protocol has been developed which analyses the frequency and dynamics of mitosis in siRNA transfected HeLa (Kyoto) H2B-GFP reporter cells. To accelerate acquisition and to reduce bleaching and photo-toxicity, a 3Dfocus map of the siRNA arrays is taken, saved and applied repeatedly for 48 hour during long term time-lapse screening.
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 Fig. 1: Image screen shots following data acquisition using scan^R, demonstrating the detection and separation of labels. The blue DAPI-stained nuclei are circled in cyan; the detected CFP tagged VSVG in the Golgi are circled in red and the detected Cy-5 VSVG antibodies at the cell surface are circled in blue.
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Protein Transportation in Fixed Cells
With 20–30 thousand open reading frames (ORFs) in the human genome, many potential genes are yet to be identified and characterised. One approach is genome-wide RNAi screening. At the EMBL, Dr Rainer Pepperkok [1] and coworkers use the scan^R Screening Station to perform genome-wide screens with the aim of detecting human genes involved in intracellular transport. Utilising 384 spot RNAi arrays on glass slides scan^R is used to observe deviations in distribution and localisation of the secretory marker protein VSVG.
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Acquisition and Analysis
Once an assay, consisting of an acquisition protocol and successive image and data analysis, has been configured on the scan^R, the whole screening process is performed fully automated. High resolution images are captured by the motorised Olympus IX81 inverted microscope fitted with new Olympus UIS2 optical components, an ultra precise motorised stage, a high sensitivity CCD camera and the innovative MT20 Fluorescence Illumination System. This unique device incorporates an 8 position filter wheel, a 14 position attenuator, a very fast shutter with an opening/closing time of less that a millisecond and an intensity stabilized Xe- or Xe/Hg burner, obligatory for quantitative measurements and reproducible automated analysis. The novel Olympus real-time controller allows precise synchronisation and parallel operation of the system components. Optimised devices and perfect system control guarantee maximum throughput and performance as well as minimised bleaching and photo-toxicity.
The advanced scan^R image and data analysis module can be used separately from the acquisition module to perform multi-parameter data extraction “online” during or “off-line” after image acquisition. Data are plotted, analysed, gated and classified in histograms and 2D scatter-grams, a concept successfully applied in flow cytometry and adapted by Olympus for analysis of image data. Each data point is linked to the respective image and detected object, which allows immediate quality control of the data analysis and gating schemes.
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Options
scan^R can be equipped with a plate loading robot and an IR-laser based autofocus to increase throughput. To enhance long term live cell imaging, the cell^cubator environmental control chamber can be fitted to manage CO2, temperature, humidity and pH.
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Conclusion
Olympus scan^R is a microscope-based screening platform for efficient acquisition of high quality imagery and importantly, the automated analysis of image derived data and information. The open platform concept allows it to be customised to the experimental design and specific requirements of the individual researcher.
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Acknowledgements
We thank the Pepperkok laboratory and the Mitocheck project team for providing data and comments on the text.
Reference
[1] Starkuviene V. et al.: Genome Res. 14, 1948–1956 (2004)
Author
Dr. Konstantin Joanidopoulos,
Olympus BioSystems GmbH, Germany
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