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Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
Publication Name:
Current Protocols in Cell Biology
Unit Number:
UNIT 4.19
DOI:
10.1002/0471143030.cb0419s39
Online Posting Date:
June, 2008 Abstract
Colocalization is an important finding in many cell biological studies. This unit describes a protocol for quantitative evaluation of images with colocalization based on the calculation of a number of specialized coefficients. First, images of double-stained sections are subjected to background correction. Then, various coefficients are calculated. Meanings of the coefficients and a guide to interpretation of their results indicating either presence or absence of colocalization are given. Success in colocalization studies depends on the quality of analyzed images, proper preparation of them for coefficients calculations, and correct interpretation of obtained results. This protocol helps to ensure reliability of colocalization coefficients calculations. Curr. Protoc. Cell Biol. 39:4.19.1-4.19.16. © 2008 by John Wiley & Sons, Inc.
Keywords: quantitative colocalization; confocal fluorescence microscopy; image analysis
Materials
Basic Protocol: Quantiative Colocalization Analysis
Materials
- Cryostat-cut 6- to 8-µm thick sections of rat liver
- Acetone for tissue fixation
- Blocking solution: 10% (v/v) goat serum in 0.1 M Tris-buffered saline (TBS) containing 0.1% Triton X-100
- Primary antibodies against Bsep and Mrp2 proteins [Santa Cruz Biotechnology; anti-Bsep antibody was donated by Dr. Bruno Stieger (Department of Medicine, University of Zurich, Switzerland)] or other proteins of interest (primary antibodies should be raised in different species)
- Non-immune IgG to control the specificity of immunostaining
- 0.1 M Tris-buffered saline (TBS; appendix 2A)
- Corresponding secondary antibodies with different excitation spectra, e.g., Alexa 488 and Alexa 594 from Molecular Probes (antibodies should not be cross-reacting)
- Glycerol
- Poly-l-lysine-coated glass slides
- Cover glass for mounting sections
- Confocal microscope for image acquisition (any brand)
- Argon-krypton laser (Siemens)
- Software: either CoLocalizer Pro or CoLocalizer Express (CoLocalization Research Software; http://homepage.mac.com/colocalizerpro/) for QCA
- Additional reagents and equipment for immunofluorescence staining (unit 4.3) and fluorescence microscopy (unit 4.2)
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Figures
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Figure 4.19.1Main and Colocalization windows of CoLocalizer Pro software used for quantitative colocalization analysis. The image to be examined opens in the main window (1) and can be magnified to view areas with colocalization in greater detail (2). The ROI selection tool can be used for background correction in Manual mode and for selecting image area for coefficients calculations. After background correction procedure, image opens in a Colocalization window (3), where all the necessary calculations according to the selected pair of channels can be performed. -
Figure 4.19.2Confocal immunofluorescence micrograph of colocalized Bsep (red fluorescence) and Mrp2 (green fluorescence) proteins in the rat liver (A) alongside with its corresponding scatter gram (B). Note a significant number of pixels along the diagonal of Scatter Gram, a typical pixel representation of the image with colocalization. The scale bar indicates 10 µm. -
Figure 4.19.3Background correction in Auto mode is straightforward and done by selecting one of the presets reflecting the most common image patterns. Image of colocalization of Bsep and Mrp2 proteins before (A) and after (C) correction. Background correction window (B) and the respective scatter gram following background correction using the Average Contrast and Fluorescence preset (D) are shown. Black areas at the right and the bottom of scatter gram indicate removed pixels (D). The areas are relatively narrow, which means that the number of removed pixels is acceptable. The scale bar indicates 10 µm. -
Figure 4.19.4Background correction in Manual mode using selected ROI on the same test image before (A) and after (C) correction. Area at image background used for selecting pixel values for background correction is indicated by an arrow (A). Background correction window (B) and the respective scatter gram after background was corrected (D) are presented. Remaining pixels (yellow) tend to concentrate along the diagonal of the Scatter Gram, while the majority of pixels with Red (right portion) and Green (bottom portion) values are removed (D). The scale bar indicates 10 µm. -
Figure 4.19.5Colocalization window of the software shows scatter gram (1) of the selected ROI, the ROI itself (2), a pair of channels according to which the coefficients can be calculated (3), pixel information of the analyzed ROI (4), and the options to perform calculations of the coefficients (5). The important option to view exclusively colocalized pixels in the image is also given (6). All calculations results can be exported as Text and Microsoft Excel files. All data used for calculations can be saved in PDF and HTML formats and presented as session reports.
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| Key References | |
| Manders et al., 1993. See above. | |
| First description of the correlation coefficient and examples of its use. | |
| Smallcombe, 2003. See above. | |
| Practical look at colocalization, some critical views and advice for performing colocalization experiments. | |
| North, 2006. See above. | |
| Review with focus on proper interpretation of the results of fluorescence microscopy studies, drawbacks and limitations of biological imagery. | |
| Internet Resources | |
| http://homepage.mac.com/colocalizerpro/ | |
| Web site of CoLocalization Research Software, creators of CoLocalizer Pro and CoLocalizer Express software applications used for quantitative estimation of colocalization. | |
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pl get me a good protocol for colocalization to avoid back ground flourescence and mounting
Thank you very much for the protocol. Surprisingly, this technique is not as difficult as I expected!
I found this protocol after my supervisor told me to perform analysis of my samples with colocalisation. The protocol is well written and easy to follow. The guidelines are very useful. I was surprised to learn that mounting media can influence the results of measurements. The table with interpretation of results was especially useful. Thanks a lot.
We would like to attract attention of protocol users to a recent review with information about advances of the use of quantitative colocalization analysis in the field of neuroscience. It is appearing in Progress in Histochemistry and Cytochemistry:
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B7GJ3-4W7RJTN-1...
The review provides excellent examples of the practical use of the described protocol and illustrates its advantages when it is applied in combination with other cell and molecular biological techniques.
In addition, we would also like to share news that protocol is poised to evolve further, since the developers of software are looking at the ways to improve the use of background correction tools. The idea is to offer the option to correct background depending on the properties of analyzed images, i.e. have separate, but compatible algorithms for correcting images with homogenous (cells in culture) and heterogenous (tissue sections) backgrounds.
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