AIM Rel 3.2
Enhanced Colocalization
Zimmermann / Rel 3.2 Colocalization October 2002 1

AIM Rel 3.2
Enhanced Colocalization
 The new colocalization Dialog
 Scattergram, image display and data table are interactively linked
Image display
Scattergram
NEW
Tools
NEW
Data table
Zimmermann / Rel 3.2 Colocalization October 2002 2

AIM Rel 3.2
Enhanced Colocalization
 Interactive link between scattergram and image display
Select ROIs in scattergram and view corresponding pixels in image display
Zimmermann / Rel 3.2 Colocalization October 2002
Color assignment for pixels in ROI
(RGB or overlay)
3

AIM Rel 3.2
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 4

AIM Rel 3.2
Enhanced Colocalization
 Scattergram and thresholding with crosshair
Crosshair
Scatterregion 2:
Pixels in channel 1 only
Scatterregion 4:
Sub-threshold pixels
(background intensities)
Scatterregion 3:
Colocalizing pixels
Scatterregion 1:
Pixels in channel 1 only
Zimmermann / Rel 3.2 Colocalization October 2002 5

AIM Rel 3.2
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 6

AIM Rel 3.2
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 7

AIM Rel 3.2
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 R 2 )
Zimmermann / Rel 3.2 Colocalization October 2002 8

AIM Rel 3.2
Enhanced Colocalization
Data Table

Quantitative Parameters:
- Colocalization coefficients c
1
 pixels
Ch 1 , coloc pixels
Ch 1 , total c
2
 pixels
Ch 2 , coloc pixels
Ch 2 , total
 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
Zimmermann / Rel 3.2 Colocalization October 2002 9

AIM Rel 3.2
Enhanced Colocalization
Data Table

Quantitative Parameters:
- Weighted colocalization coefficients
M
1

 i i

Ch 1 i,coloc
Ch 1 i,total
M
2

 i
 i
Ch 2 i,coloc
Ch 2 i,total
 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
Zimmermann / Rel 3.2 Colocalization October 2002 10

AIM Rel 3.2
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) r

 i
Ch 1 i
 i
( Ch 1 i
)
2
* Ch 2 i
*
 i
( Ch 2 i
)
2
 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
Zimmermann / Rel 3.2 Colocalization October 2002 11

AIM Rel 3.2
Enhanced Colocalization
Data Table

Quantitative Parameters:
- Correlation coefficient, Pearson
´s correlation coefficient
R p

 i
( Ch 1 i
 i
( Ch 1 i

Ch 1 aver
) * ( Ch 2 i

Ch 1 aver
)
2
*
 i
( Ch 2 i

Ch 2 aver
)

Ch 2 aver
)
2
 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
Zimmermann / Rel 3.2 Colocalization October 2002 12

AIM Rel 3.2
Enhanced Colocalization
Data Table
 Quantitative data display for individual scatter regions and image regions
Zimmermann / Rel 3.2 Colocalization October 2002 13