Enhanced Colocalization - PowerPoint PPT Presentation

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Enhanced Colocalization

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  1. Enhanced Colocalization Enhanced Colocalication Tool

  2. Enhanced Colocalization • The new colocalization Dialog • Scattergram, image display and data table are interactively linked Image display NEW Scattergram Tools NEW Data table

  3. Enhanced Colocalization • Interactive link between scattergram and image display • Select ROIs in scattergram and view corresponding pixels in image display Color assignment for pixels in ROI(RGB or overlay)

  4. Enhanced Colocalization • Interactive link between scattergram and image display • Select ROIs in image display and view corresponding pixels in scattergram

  5. Enhanced Colocalization • Scattergram and thresholding with crosshair Crosshair Scatterregion 2: Pixels in channel 1 only Scatterregion 3: Colocalizing pixels Scatterregion 1: Pixels in channel 1 only Scatterregion 4: Sub-threshold pixels (background intensities)


  6. Enhanced Colocalization • Thresholding with crosshair • Drag crosshair in scattergram • Use Intensity Threshold dialog • move slider or • type in intensity values or • define ROI in image display and use „Set from Image ROIs“ • (system will calculate mean intensities + 2x standard deviation and set crosshair accordingly)

  7. Enhanced Colocalization • Thresholding with crosshair • Colors may be assigned to pixels in scatter regions 1-3 and displayed in the image display as „RGB“ or „overlay“

  8. Enhanced Colocalization • Data Table • Quantitative Parameters: • No. of pixels in image ROI or scatter region • Area / relative area of image ROI or scatter region • Mean intensities / SD within image ROI or scatter region • Colocalization coefficients • Weighted colocalization coefficients • Overlap coefficient after Manders • Correlation coefficients (R and R2)

  9. Enhanced Colocalization • Data Table • Quantitative Parameters: • Colocalization coefficients • Relative number of colocalizing pixels in channel 1 or 2, respectively, as compared to the total number of pixels above threshold. • Value range 0 – 1 (0: no colocalization, 1:all pixels colocalize) • All pixels above background count irrespective of their intensity.

  10. Enhanced Colocalization • Data Table • Quantitative Parameters: • Weighted colocalization coefficients • Sum of intensities of colocalizing pixels in channel 1 or 2, respectively, as compared to the overall sum of pixel intensities above threshold and in this channel. • Value range 0 – 1 (0: no colocalization, 1: all pixels colocalize) • Bright pixels contribute more than faint pixels

  11. Enhanced Colocalization • Data Table • Quantitative Parameters: • Overlap coefficient, overlap coefficient after Manders (Manders, Verbeek and Aten, J. Microscopy 169:375-382, 1993, PDF on this CD) • Another parameter used to quantify colocalization in image pairs • Insensitive to differences in signal intensities between the two channels, photo-bleaching or amplifier settings • Value range 0 – 1 (0: no colocalization, 1:all pixels colocalize)

  12. Enhanced Colocalization • Data Table • Quantitative Parameters: • Correlation coefficient, Pearson´s correlation coefficient • Provides information on the intensity distribution within the colocalizing region • Value range -1 to +1 (-1,+1: all pixels are found on straight line in the scattergram 0: pixels in scattergram distribute in a cloud with no preferential direction

  13. Enhanced Colocalization • Data Table • Quantitative data display for individual scatter regions and image regions