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Genetics / Screens - Expression Strategies

Automated High Throughput Image Screening

Introduction

The Consortium's automated high throughput screening processes are based on 2 automated microscopes, the hardware and software of which was designed and installed in collaboration between Prof. B. Geiger and Prof. Z. Kam, in the Weizmann Institute. These systems were particularly “tailored" for migration, adhesion and cytoskeletal assays.

The Hardware

The basic systems consist of inverted fluorescent microscopes (Olympus) with high-NA air objectives with correction for plate-bottom thickness. It includes automated stage and focus control [XYZ], objective change for multiresolution imaging, computer-controlled filters, and illumination shutters (Transmission/fluorescence), fast, sample-independent laser Auto Focus system, high quantum efficiency back-illuminated CCD, 16 bit camera, environmental control for live cell imaging, chambers preventing evaporation without limiting gas exchange.

To obtain detailed information concerning the microscope optical and mechanical design, computer interface, data acquisition system, automated, autofocusing system, filter settings etc., please contact us . See related papers:

Liron Y, Paran Y, Zatorsky NG, Geiger B, Kam Z. Laser autofocusing system for high-resolution cell biological imaging. J Microsc. 2006 Feb;221(Pt2):145-51.PubMed

Paran Y, Lavelin I, Naffar-Abu-Amara S, Winograd-Katz S, Liron Y, Geiger B, Kam Z. Development and Application of Automatic HighResolution Light Microscopy for CellBased Screens. Methods in Enzymology. 2006 414:228-247 PubMed

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The Software

The automated screening software includes: Linux RedHat computer control, modular command-line experimental design controlling all microscope automation including looping on ZWT and positions [6D data]. Control of multiple-fluorescent and transmitted light exposures, exchange between 3D and fast 2.5D imaging modes, saving data on files, sufficient for accumulation of 40GBytes image data per day. Client-server interface for fast on-line analysis was used. The systems are extensively used by multiple research groups.

Quantitative analysis (on Linux RedHat computer farm):

  • Normalize inhomogeneous illumination.
  • Background subtraction [global and local].
  • Various novel segmentation algorithms

    Binary, WaterShed, Fiber Analysis, multi-resolution schemes.

  • Connected component (objects), selective featuring:

    size, shape, total intensity, length, branching (fibers).

  • Quantify parameters for segmented “Objects”
  • Background subtracted total intensity for all colors, morphological parameters:

    area, perimeter, shape factors, counting, density, length (fibers).

  • User-interactive analysis platform.
  • Multiparameter statistics tailored for each essay:

    Histograms, Scatter plots, Multidimensional clustering, Neural nets.

  • Automated statistical scoring for the whole plate.

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Image Processing

For information concerning the system requirements for downloading and using these software packages please contact us.

"Water" Segmentation - for quantification of "patches" (eg., Focal adhesions, nuclei, Vesicles) Zamir et al.,1999

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Ratio imaging - for comparing 2 molecules or 2 time points. Zamir et al.,1999

Ratio imaging between two colors

  • measurement of pixel intensity of Cy3 and FITC
  • normaliztion
  • calculation of the ratio value for each pixel

Temporal Ratio Imaging

  • measurement of pixel intensity on one antigen at 2 time points
  • calculation of the intensity ration for each pixel
  • color display of the ratio value for each pixel
 

Example: Superposition (red-green) and ratio images (blue-to-red) of different focal adhesion-associated molecules

 

In this example, the effect of inhibitors of actin polymerization and contratility on the dynamic properties of focal adhesion was visualized (red/blue structured and dynamic, yellow - static)
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"FiberScore" Segmentation

- for quantification of fibers (e.g. actin cables, microtubules) Lichtenstein et al., 2003

 

References

Zamir E, Katz BZ, Aota S, Yamada KM, Geiger B, Kam Z. Molecular diversity of cell-matrix adhesions. J Cell Sci. 1999 Jun;112 ( Pt 11):1655-69. PubMed

Lichtenstein N, Geiger B, Kam Z. Quantitative analysis of cytoskeletal organization by digital fluorescent microscopy. Cytometry. 2003 Jul;54A(1):8-18. PubMed

Liron Y, Paran Y, Zatorsky NG, Geiger B, Kam Z. Laser autofocusing system for high-resolution cell biological imaging. J Microsc. 2006 Feb;221(Pt2):145-51.PubMed

Paran Y, Lavelin I, Naffar-Abu-Amara S, Winograd-Katz S, Liron Y, Geiger B, Kam Z. Development and Application of Automatic HighResolution Light Microscopy for CellBased Screens. Methods in Enzymology. 2006 414:228-247 PubMed

 

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