enhanced colocalization

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

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)

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

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)

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