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Enhanced Colocalization

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

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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

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