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Optical Biology Core, Developmental Biology Center, University of California, Irvine
How to quantify the colocalization in your images?
Two or more fluorescence emission signals can often overlap in digital images recorded by
confocal or multiphoton microscopy due to their close proximity within the specimen. This effect is
known as colocalization and usually occurs when fluorescently labeled molecules bind to targets
that lie in very close or identical spatial positions.
This tutorial will show you how to conduct quantitative analysis of colocalization in your images.
Step1
Open your selected image from database.
Prepared by Zifu Wang, Ph.D.
© Developmental Biology Center, University of California, Irvine (2006)
Optical Biology Core, Developmental Biology Center, University of California, Irvine
Step 2 (Optional)
To show each individual channels together with the merged image, click
Split xy button
Step 2
Step 3
Step 3
Now click Histo button.
Step 4
Then click buttons of Colocalisation, Area and Crosshair. You now
see a scatter diagram with divided sections and the image.
Scatter diagram
Prepared by Zifu Wang, Ph.D.
© Developmental Biology Center, University of California, Irvine (2006)
Step 4
Optical Biology Core, Developmental Biology Center, University of California, Irvine
How a scatter diagram is generated?
All pixels having the same positions in both images are considered a pair. Of every pair of pixels
(P1, P2) from the two source images, the intensity level of pixel P1 is interpreted as X coordinate,
and that of pixel P2 as Y coordinate of the scatter diagram. Each pixel of the scatter diagram is a
value that shows how often a particular pair of pixels has occurred. Identical images produce a
clean diagonal line running from bottom left to top right, because only pixel pairs (0,0), (1,1), (2,2)
with the same intensity can occur. Differences between the images cause an irregular distribution
in the scatter diagram
Prepared by Zifu Wang, Ph.D.
© Developmental Biology Center, University of California, Irvine (2006)
Optical Biology Core, Developmental Biology Center, University of California, Irvine
Step 5
Click button Show Table. A table will appear below the scatter diagram and the
image.
Step 5
Please note, only the pixels in the scatter region 3 are colocalizing pixels. Therefore, the
parameters in the 3rd row of the table provide you with the quantitative information of
colocalization in your image.
Step 6
Drag the scroll bar to the right for more colocalization parameters.
Step 6
Prepared by Zifu Wang, Ph.D.
© Developmental Biology Center, University of California, Irvine (2006)
Optical Biology Core, Developmental Biology Center, University of California, Irvine
Step 7 If you are interested in colocalization of proteins in a special region, say, in the nucleus,
select the button with a shape you want (circle in this example) and draw a circle at the specific
region in the image. You will notice the changes both in the scatter diagram and the values of the
parameters in the table, which are now just for the area defined by the circle.
Step 7
To learn more about colocalization analysis and its applications in research, please check the
following references:
1. http://www.olympusconfocal.com/java/colocalization/
2. http://www.mediacy.com/pdfs/colocfluorprobes.pdf
3. Manders, E.M.M., Verbeek, F.J., Aten, J.A. Measurement of co-localization of objects in
dualcolor confocal images. Journal of Microscopy 169: 375-382 (1993).
4. Martin, R.M., Leonhardt, H. and Cardoso, M.C. DNA Labeling in Living Cells. Cytometry A
67A:45–52 (2005)
Prepared by Zifu Wang, Ph.D.
© Developmental Biology Center, University of California, Irvine (2006)
Optical Biology Core, Developmental Biology Center, University of California, Irvine
Appendix 1: Functional Elements
Prepared by Zifu Wang, Ph.D.
© Developmental Biology Center, University of California, Irvine (2006)
Optical Biology Core, Developmental Biology Center, University of California, Irvine
Appendix 2: Parameters in the table
- 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)
Colocalization coefficients:
c1 =
c2 =
pixelsCh1coloc
pixelsCh1total
pixelsCh 2 coloc
pixelsCh 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 irrespective of their intensity.
Weighted colocalization coefficients:
∑ Ch1icoloc
M 1 = i Ch1
∑ itotal
∑ Ch 2icoloc
M 2 = i Ch 2
∑ itotal
i
i
- 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
Correlation coefficient, Pearson´s correlation coefficient:
Rp =
∑ ( Ch1i −Ch1over )*(Ch 2i −Ch 2over )
i
∑ Ch1i −Ch1over ) 2 *(Ch 2i −Ch 2over ) 2
i
-
Provides information on the intensity distribution within the colocalizing region
Value range -1 to +1
1: all pixels are found on straight line in the scatter diagram
-1: total lack of overlap between pixels from the images
0: pixels in scatter diagram distribute in a cloud with no preferential direction
Overlap coefficient, overlap coefficient after Manders:
(Manders, Verbeek and Aten, J. Microscopy 169:375-382, 1993)
r=
∑ Ch1i *Ch 2i
∑
i
Ch1i ) 2 *( Ch 2 i ) 2
i
-
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)
Prepared by Zifu Wang, Ph.D.
© Developmental Biology Center, University of California, Irvine (2006)