enhanced colocalization
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
1
Enhanced Colocalication Tool
Enhanced Colocalization
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
2
Enhanced Colocalization
The new colocalization Dialog
Scattergram, image display and data table are interactively linked
Scattergram
Data table
Image display
Tools
NEW
NEW
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
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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)
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
4
Enhanced Colocalization
Interactive link between scattergram and image display
- Select ROIs in image display and view corresponding pixels in scattergram
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
5
Enhanced Colocalization
Scatterregion 3:Colocalizing pixels
Scatterregion 1:Pixels in channel 1 only
Scatterregion 2:Pixels in channel 1 only
Scatterregion 4:Sub-threshold pixels(background intensities)
Scattergram and thresholding with crosshair
Crosshair
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
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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)
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
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“
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
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)
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
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.
totalCh
colocCh
pixels
pixelsc
,1
,11
totalCh
colocCh
pixels
pixelsc
,2
,22
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
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
ii,total
ii,coloc
Ch
ChM
1
1
1
ii,total
ii,coloc
Ch
ChM
2
2
2
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
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)
i iii
iii
ChCh
ChChr
22 )2(*)1(
2*1
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
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 scattergram0: pixels in scattergram distribute in a cloud with no preferentialdirection
i iaveriaveri
iaveriaveri
p
ChChChCh
ChChChChR
22 )22(*)11(
)22(*)11(
Zimmermann / Rel 3.2 Colocalization October 2002
AIM Rel 3.2
13
Enhanced Colocalization
Data Table
Quantitative data display for individual scatter regions and image regions