Quantitative Colocalization Analysis of UNIT 4.

19
Confocal Fluorescence Microscopy
Images
Vadim Zinchuk1 and Olga Zinchuk2
1
Department of Anatomy and Cell Biology, Kochi University, Faculty of Medicine, Japan
2
Institute of Anatomy, University of Berne, CH-3000, Berne, Switzerland

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 calcula-
tion 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 pres-
ence or absence of colocalization are given. Success in colocalization studies depends
on the quality of analyzed images, proper preparation of them for coefficients calcu-
lations, 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.  C 2008 by John Wiley & Sons, Inc.

Keywords: quantitative colocalization r confocal fluorescence microscopy r

image analysis

INTRODUCTION
Quantitative colocalization analysis (QCA) is a method to estimate the degree of colo-
calization of antigens in multicolor confocal immunofluorescence microscopy images.
Colocalization is observed when staining of two or more antigens in the same section,
labeled by corresponding antibodies with different excitation spectra and therefore vi-
sualized in different colors, overlaps. Although colocalization is a mere coexistence of
molecules in a very close physical location, it can provide valuable clues clarifying
their common characteristics. Scientific applicability of colocalization observed per se
is, however, limited because it is perceived differently by the eye and thus can frequently
be misleading. Thus, the method described here evaluates colocalization objectively by
estimating its quantitative characteristics.

The method is applicable to dual-color confocal images of sections stained with anti-
bodies with well-separated excitation spectra, acquired using proper set up of confocal
microscopes, and stored in the image file format that preserves image data necessary
for quantification. It relies on the use of specialized software that calculates a number
of colocalization coefficients to understand the observed colocalization in greater detail.
Prior to calculating the coefficients, the software performs background correction of the
images. This important step is required to avoid obtaining false-positive results and is
not available with other tools.

The protocol described in this unit demonstrates the use of QCA for studying functionally
synergetic proteins. As an example, the use of the method is illustrated by quantifying
colocalization of bile salt export pump (Bsep) and multidrug resistance protein 2 (Mrp2)
in the liver. The background correction procedure is performed using different methods
and is accompanied by the respective scatter grams. The meanings of coefficients and
Microscopy

Current Protocols in Cell Biology 4.19.1-4.19.16, June 2008 4.19.1

Published online June 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471143030.cb0419s39 Supplement 39
Copyright C 2008 John Wiley & Sons, Inc.
 their importance is explained. Finally, the interpretation of the results of coefficients
calculations in terms of their biological significance is given.

STRATEGIC PLANNING
General
Successful QCA requires good knowledge and experience in basic immunohistochemi-
cal techniques (UNIT 4.3), confocal fluorescence microscopy (UNIT 4.5), and handling and
processing of digital images. This unit provides practical information about quantitative
colocalization and coefficients used to estimate it, gives tips for success in background
correction procedure, and describes applicability of calculations results. Please refer to
UNITS 4.2 and 4.3 for basic information on basic fluorescence microscopy and immunoflu-
orescence staining, respectively.

Steps of QCA
1. Ensure that image is properly prepared, acquired, and processed.

2. Open image in colocalization analysis software and perform background correction.

3. Calculate colocalization coefficients.

4. Save obtained results for further reference.

See also Table 4.19.1 for comments and tips.

Importance of Using Properly Prepared Images

Although QCA is the most important step in colocalization studies, it is just the final step.
Its results are fully dependent on all previous steps of sample preparation, microscope set
up, and image acquisition and handling. To emphasize the importance of image suitability
to ensure reliable results of coefficients calculations, it is presented as a first step of QCA.
The most important rules which should be followed to obtain confocal images useable
for quantification are as follows:

Table 4.19.1 Steps of QCA

Step Comments and tips

1. Ensure suitability of Make sure to use proper microscope set up and keep images unedited
images to prevent the loss of original information
2. Open image and Correct background in either Auto or Manual mode. Try several
correct background background correction settings to find out the best set up for your
images. Once determined, keep them the same for all images in a
study to ensure compatibility of calculations results.
3. Calculate coefficients As a rule, calculate at least Pearson’s correlation coefficient (Rr ) and
overlap coefficient according to Manders (R). Determine other
coefficients as well if these two do not provide conclusive information
and/or if you are interested in individual contribution of the antigens
to the observed colocalization. Make sure that results of coefficients
calculations fall within their values and pay attention to their
dynamical changes. Use the opportunity provided by the software to
Quantitative view actually colocalized pixels in their image location.
Colocalization
Analysis of 4. Save obtained results Save as much information about the analyzed image as is allowed by
Confocal for further reference the software. You may find valuable information when examining
Fluorescent
Microscopy image data in relation with coefficients calculations later on.
Images

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Supplement 39 Current Protocols in Cell Biology
Sample preparation
1. Ensure that the antibodies you are using are specific and do not cross react.

2. Make the proper choice of fluorophores by selecting ones with well-separated excita-
tion and emission spectra.

3. Use the same mounting medium for all of the samples that you will be performing
calculations on.

4. As antifading reagents tend to increase background fluorescence, try to avoid using

them.

5. Always use unstained control samples to check for autofluorescence.

Microscope set up
1. Reduce chromatic shift by using plan apochromatic lenses.

2. Maximize emission collection while avoiding bleed-through effect by using optimized

emission filters.

Figure 4.19.1 Main and Colocalization windows of CoLocalizer Pro software used for quantitative colocal-
ization 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. For color version of this figure see http://www.currentprotocols.com.
Microscopy

4.19.3
Current Protocols in Cell Biology Supplement 39
 3. Make sure you are using the same objective lens for obtaining all images you are
planning to compare.

4. Ensure proper set up of the size of microscope pinhole.

Image acquisition and handling

1. Remember to acquire images sequentially to minimize bleed-through effect.

2. Avoid acquiring images that are too bright and images with too much contrast, as it
may result in image saturation.

3. Never resave image files in any other graphic format than TIFF, as it may result in the
loss of original data necessary for quantification.

4. Remember that any adjustments of brightness, contrast, etc. of your images using
graphics-editing software may prevent further use of them for quantification purposes.

5. After making sure your images are suitable for colocalization studies, open them in
QCA software (Fig. 4.19.1) and perform quantitative analysis according to the protocol
below.

As an example of colocalized proteins, colocalization of bile salt export pump (Bsep)

and multidrug resistance protein 2 (Mrp2) in the liver is shown (Fig. 4.19.2A). These
proteins work in concert to regulate transport of bile salt and organic anions, respectively
(Gerloff et al., 1998; Konig et al., 1999), but they are regulated differently and change
their location and colocalization in response to various stimuli (Zinchuk et al., 2002,
2005b; Dombrowski et al., 2006). Bsep is stained in red, while Mrp2 is stained in green.
The areas of location of both proteins are seen in yellow color. A respective scatter gram
(Fig. 4.19.2B) reveals distribution of pixels according to the selected pair of channels.

A B

Figure 4.19.2 Confocal 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 signif-
icant number of pixels along the diagonal of Scatter Gram, a typical pixel representation of the image with colo-
calization. The scale bar indicates 10 μm. For color version of this figure see http://www.currentprotocols.com.

4.19.4
Supplement 39 Current Protocols in Cell Biology
In the scatter gram, colocalized pixels are located along the diagonal, while those with
no colocalization occupy left and bottom portions. As can be seen in the figure, the
scatter gram represents unique information about an image file and is useful for viewing
colocalized pixels in the image.

QUANTIATIVE COLOCALIZATION ANALYSIS BASIC

PROTOCOL
The present protocol is used to quantify colocalization in dual-color confocal microscopy
images of sections stained with fluorescence-labeled antibodies with well-separated ex-
citation spectra, obtained using correct microscope set up, and saved in the image file
format that ensures preservation of image data required for quantification. It relies on
the use of specialized software that enables determination of colocalization coefficients
within a single session of image analysis, making calculation results not only highly
relevant and comparable, but also easily reproducible. The validation of the results of
quantitative colocalization experiments and their applicability are presented.

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)
Perform immunostaining
1. Pick up a 6- to 8-μm thick section of liver on poly-L-lysine-coated glass slides, air
dry for 1 hr, and fix in acetone for 30 min at −20◦ C.
2. Apply 500 μl blocking solution for 1 hr at room temperature to block nonspecific
binding.
3. Incubate with 500 μl anti-Bsep antibody diluted 1:100 for 1 hr at room temperature.
In parallel, perform the same procedure using non-immune IgG instead of primary anti-
bodies to control specificity of immunostaining.

4. Rinse three times with 10 ml of 0.1 M TBS.

5. Incubate with 500 μl anti-Mrp2 antibody diluted 1:100 for another 1 hour at room
temperature.
Microscopy

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Current Protocols in Cell Biology Supplement 39
 6. Rinse three times with 10 ml 0.1 M TBS.
7. Incubate with a mixture of corresponding secondary antibodies conjugated with
Alexa 488 and Alexa 594, respectively, for 1 hr at room temperature. Dilute both
antibodies 1:400.
8. Wash three times with 10 ml 0.1 M TBS.
9. Mount sections with glycerol and apply a coverglass.
Perform microscopy
10. Use immersion lens (e.g., Plan-Neofluar 40 /0.75 mm) of confocal microscope to
examine sections.
11. Check samples for autofluorescence. If autofluorescence is detected, discard sam-
ples. If not, continue examining them.
12. Use an argon-krypton laser at the wavelength of 488 and 543 nm to visualize double
fluorescence for green and red channels, respectively.
13. Keep the pinhole of the microscope at the same position when examining samples.
14. After finding satisfactory staining (not too bright and not too contrasty), perform
sequential scanning for each channel to minimize the crosstalk of fluorophores.
15. Save images according to each channel (in this case you will need to merge them
into one image for quantification purposes later on) or overlayed in TIFF format
with maximum possible image resolution.
Transfer images from microscope and perform quantitative colocalization analysis
16. Transfer obtained images to a computer for analysis.
We use a Mac Pro computer operated under Mac OS X 10.4.10, but even a less powerful
computer is fully satisfactory. The minimum system requirement for the software is Mac
OS X 10.2.8.

17. Import image to CoLocalizer Pro software for analysis and perform background
correction on the imported image. Choose suitable background correction settings
for your images by trying Auto mode first.
Correct background
Note the following: (1) The software will issue a warning if user attempts to perform
coefficients calculations with no background corrected. (2) Low Contrast and Weak Flu-
orescence presets (Auto mode) may be too rough in some cases as the number of removed
pixels can be excessive. (3) Successful background correction of the image with colocal-
ization will show pixels concentrating along the diagonal of the scatter gram, while the
majority of pixels at the right and bottom portions of it will be removed. The areas at right
and bottom should however not be too wide. (4) For beginners, we recommend resetting
the image to its original state every time when trying another background correction mode.
With some experience, this may become unnecessary. (5) Once determined, background
correction settings should be used consistently for all images you intend to compare.
(6) Background correction will have an impact on coefficients calculations. Unsatisfac-
tory corrected background can result in erroneous coefficients readings.

Quantitative For automatic correction of background

Colocalization
Analysis of 18a. Correct background in Auto mode. After opening image (Fig. 4.19.3A) in the main
Confocal window of the software, choose Background Correction under the Tools in the
Fluorescent
Microscopy application menu bar or click Background icon.
Images

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Supplement 39 Current Protocols in Cell Biology
 A B

C D

Figure 4.19.3 Background 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. For color version of
this figure see http://www.currentprotocols.com.

19a. Select Auto in the Background Correction window (Fig. 4.19.3B) and click Apply.
Then, dismiss the Background Correction window by clicking Done.
Following background correction the image appears slightly changed due to the absence
of the portion of pixels (Fig. 4.19.3C). The corresponding scatter gram reveals the extent
of background correction (Fig. 4.19.3D).

20a. If the scatter gram shows a relatively small number of removed pixels (Fig. 4.19.3D),
process the image for coefficients calculations.
If the background is more than shown, reset image and try correcting background in
manual mode using Selected ROI.

Microscopy

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Current Protocols in Cell Biology Supplement 39
 B
A

C D

Figure 4.19.4 Background 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 Scat-
ter Gram, while the majority of pixels with Red (right portion) and Green (bottom portion) values are removed
(D). The scale bar indicates 10 μm. For color version of this figure see http://www.currentprotocols.com.

For manual correction of background

18b. Correct background in manual mode (this is the mode we recommend to use)
using Selected ROI. After opening image (Fig. 4.19.4A) in the main window of the
software, choose Background Correction under the Tools in the application menu
bar or click Background icon. Select Manual in the Background Correction window
(Fig. 4.19.4B).
19b. Using rectangular or oval ROI selection tool, select a small area, e.g., 10 × 10 pixel
Quantitative size, in the image background.
Colocalization
Analysis of If you are examining cells in culture, it is best to select any area outside of the cells.
Confocal If you are examining tissue sections, select an area in the tissue background which is
Fluorescent
Microscopy dark, but not completely black, i.e., that absorbed some fluorescence. Such areas can be
Images considered as the areas containing only pixels of the background level.

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Supplement 39 Current Protocols in Cell Biology
20b. Then, click Apply and dismiss Background Correction window by clicking Done.
Following background correction the image will appear changed depending on the
number of removed pixels (Fig. 4.19.4C). The corresponding scatter gram reveals the
extent of background correction (Fig. 4.19.4D). Figure 4.19.4D shows what the scatter
gram of a properly corrected image should look like: remaining pixels concentrate
mainly along the diagonal of the scatter gram and the areas of removed red (at right)
and green (at left) pixels are not too wide.
You may also try correcting background in Manual mode using Threshold Value. We
find it useful for images that are more bright and contrasty than the average. It works
similarly to Auto mode, with the exception that you are free to choose the exact number
of pixels to be removed. It can provide good correction results, but, in comparison to
the selected ROI option, does not give the important advantage of being able to tailor
correction to the unique pixel profile of analyzed images.

21. After background correction step, perform coefficients calculations.

Calculate coefficients and view colocalized pixels
22. Using the whole image as a ROI, open Colocalization window, and proceed as
described below.
23. Depending on the shape appearance of areas with colocalization, use a suitable ROI
tool in the menu bar to select image areas containing predominantly colocalization.
In the test image used in this protocol, we used the Lasso tool.

24. Repeat calculations for at least three different areas with colocalization.
Thus, you will have at least four sets of data: one for the whole image and the other
three for each of the selected ROIs. The resulting coefficients values will be the average
of these four.

25. Select a pair of channels according to which coefficients will be calculated, for
example red-green.
After selection is made, respective pixel information will be shown in the Pixel Informa-
tion section of the Colocalization window (Fig. 4.19.5).
The total number of pixels shown in the Pixel Information section of the Colocali-
zation window may not necessarily be 100% and this is correct, because pixels for
colocalization analysis are counted according to channel pairs, taking into consideration
color components. If, for example, a red-green pair of channels is selected, pixels with
some levels of blue component may not be included to the count.

26. In the colocalization window, select coefficients you would like to calculate.
Pearson’s coefficient is a default coefficient.

27. Calculate all coefficients paying particular attention to the results of Pearson’s and
Manders’ coefficients.
28. After calculating coefficients for image ROI, perform calculations of coefficients
for scatter gram ROI (M1 and M2 coefficients) by clicking Scatter Gram ROI tab in
the Coefficients section of the Colocalization window.
29. Then, click Reveal Pixels tab in the same window section and view colocalized
pixels in the image by clicking Colocalized button.
Under this tab, you may also select pixels of interest in the scatter gram and then view
them in their image location by clicking Selected button. Highlight pixels by using any
color you like using color picker.
30. Export calculations results as Text and Microsoft Excel files for further statistical
analysis. Microscopy

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Current Protocols in Cell Biology Supplement 39
 Figure 4.19.5 Colocalization 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. For color version of
this figure see http://www.currentprotocols.com.

Table 4.19.2 Comparison of Coefficients Obtained without Background Correction and with Background Corrected in
Auto and Manual Modesa

Background corrected in Background corrected

Coefficients Background not corrected
Auto mode using Selected ROI

Pearson’s correlation 0.91±0.1 0.86±0.1 0.74±0.08

coefficient (Rr )
Overlap coefficient according 0.76±0.06 0.71±0.1 0.69±0.1
to Manders (R)
Overlap coefficients k1 and k2 0.99±0.1 and 0.83±0.1 0.97±0.08 and 0.73±0.07 0.91±0.07 and 0.70±0.1
Colocalization coefficients m1 1.0±0.1 and 1.0±0.1 0.99±0.1 and 0.98±0.09 0.99±0.09 and 0.94±0.08
and m2
a Calculation of coefficients with background intact results in obtaining ∼20% of false-positive results. Results obtained after background corrected
using Selected ROI appear to be the most applicable. An average of coefficients of five examined areas is shown. p <0.05.
Quantitative
Colocalization
Analysis of
Confocal
Fluorescent
Microscopy
Images

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Supplement 39 Current Protocols in Cell Biology
31. Make a report of the calculations session in either PDF or HTML format.
In addition to the analyzed image and calculations results, include in the report the image
with scatter gram inserted in it. This is a very handy way to present the analyzed image
together with its unique colocalization profile for publishing.
Results of coefficients calculations on the test image without and with background cor-
rection are presented in Table 4.19.2.
COMMENTARY
Background Information
The word “colocalization” is one of the
most frequently used words in modern cell and
molecular biological studies. It is used to de-
scribe the existence of two or more different
molecules in a very close spatial position in
the cell (Smallcombe, 2001). The molecules
are visualized when examining images ac-
cording to two or more fluorescence channels
generated by corresponding fluorophores and
observing the same specimen region. At the
same time, the phenomenon behind it is per-
haps one of the most misrepresented and mis-
understood. The most common misconception
is that colocalization of antigens equals shar-
ing of their functional characteristics (North,
2006).
The theoretical basis of quantitative colo-
calization has been available for some time,
but its adoption in practical studies has been
slow. As a result, even in the current publi-
cations proteins continue to be described as
“more” or “less” colocalized with no quantita-
tive justification of any kind. Importantly, the
lack of quantitative assessment does not al-
low researchers to extend their observations of
colocalization to analyzing important changes
of proteins in dynamics, as well as in associa-
tion with other proteins (Lippincott-Schwartz
et al., 2001).
In this protocol, we gathered in one unit
all the necessary information required for
performing reliable quantitative colocalization
studies.
Background correction
Although confocal fluorescence mi-
croscopy allows demonstration of the precise
location of fluorophores, confocal images
are not usable for quantification right away
because they have low signal-to-noise ratio.
This means that they have high levels of
background noise, which not only negatively
affects image resolution by hiding structural
details, but can also introduce false-positive
results. It was estimated that these levels of
noise can reach as high as 30% of maximum
intensity of fluorescence (Landmann and
Current Protocols in Cell Biology
Marbet, 2004). Thus, it can be approximately
said that if QCA is performed on “raw”
(with background intact) confocal images, the
results will be 30% wrong. Therefore, prior
to performing calculations of coefficients,
background noise in confocal images must
be removed, i.e., the background needs to
be corrected. The background correction
procedure, as well as all other steps, including
coefficients calculations described in this
protocol, are performed with the help of
CoLocalizer Pro 2.0 software. The software
is now widely used in various colocalization
studies (Zinchuk et al., 2004, 2005a,b;
Criscuoli et al., 2005; Kato et al., 2005; Cario
et al., 2006; Head et al., 2006; Patel et al.,
2006; Rey et al., 2006; Swaney et al., 2006;
Tsutsumi et al., 2006; Berg et al., 2007;
Clizbe et al., 2007; Desplanques et al., 2007;
Mutch et al., 2007; Rocker et al., 2007;
Van Acker et al., 2007; Watanabe et al.,
2007). Although other software options to
estimate colocalization exist as well, an
important advantage of CoLocalizer Pro is
that it is a stand-alone application not bundled
with the microscopes and not tied to any
proprietary image file format. The software
has background correction functionality
built-in, so that moving from the background
correction step to calculation of colocalization
coefficients is done in just a single button
click. It should be mentioned that several
image restoration techniques were recently
introduced to improve the signal-to-noise
ratio in confocal images by applying complex
image restoration algorithms, but using them
results in a significant image transformation.
The approach based on the mentioned
software, on the contrary, is quick, simple,
and uses original image data. Importantly,
background correction settings can be reused,
thus ensuring that all images in a study
are prepared in the same way, and thus the
results of coefficients calculations are easily
compatible and comparable. There are several
options to perform the background correction
procedure.
Microscopy
4.19.11
Supplement 39
 Background correction in Auto mode
(option 1)
A simple and efficient way to correct back-
ground is by using presets options according
to the most common image patterns. Software
then uses special formulas to remove a pre-
defined number of pixels according to these
patterns, such as: (1) Average Contrast and
Fluorescence, (2) Low Contrast, and (3) Weak
Fluorescence.
According to their names, Average Contrast
and Fluorescence, the default preset, should
be used for images with average contrast and
fluorescence intensity and should be suitable
in the most cases. Low-contrast images should
be corrected using the Low Contrast preset. If
the image has weak fluorescence, the Weak
Fluorescence preset should be chosen.
Background correction in Manual mode
(option 2)
However, Auto mode may be too straight-
forward in some cases and remove too many
pixels. Manual mode is much more sensitive
and customizable. More importantly, it allows
performance of background correction using
the unique pixel profile of analyzed images.
In this mode, background can be corrected us-
ing either: (1) Selected ROI or (2) Threshold
Value.
Using these options, background can be
corrected either for one or for all channels.
Correcting for all channels is advantageous,
because it is often difficult to predict what
particular channel needs to be corrected. Se-
lecting All channels guarantees that no “back-
ground noise” pixels will be left in the im-
age. Viewing the corresponding scatter gram
in the software colocalization window helps
to determine the extent of pixel removal. If
too many pixels are removed, the image can
be reset and background correction repeated
again by selecting a different area. Although
this procedure requires some experience, you
will become an expert in the background cor-
rection fairly quickly. When using Threshold
Value, it is possible to select the exact extent
of pixel removal.
Colocalization coefficients
Colocalization is determined by calculat-
Quantitative ing a number of specialized values represent-
Colocalization ing the proportion of colocalized pixels. These
Analysis of
Confocal values are estimated according to colocaliza-
Fluorescent tion coefficients (Fig. 4.19.5). The following
Microscopy coefficients are used.
Images
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Supplement 39
Pearson‘s correlation coefficient (Rr )
∑ (S1 − S1 ) ⋅ (S 2
i aver i − S 2 aver )
Rr = i
∑ (S1 − S1 ) ⋅ ∑ (S 2 − S 2aver )
2 2
i
i aver
i
i
where S1 represents signal intensity of pixels
in channel 1 and S2 represents signal inten-
sity of pixels in channel 2; S1aver and S2aver
shows the average intensities of these respec-
tive channels. This coefficient, being one of
standard measures in pattern recognition, was
first employed to estimate colocalization and
is used for describing the correlation of the
intensity distributions between channels. It
takes into consideration only similarity be-
tween shapes, while ignoring the intensities
of signals. Its values range between −1.0 and
1.0, where 0 indicates no significant correla-
tion and −1.0 indicates complete negative cor-
relation.
Overlap coefficient according to Manders (R)
∑ S1 ⋅ S 2 i 1
R= i
∑ (S1 ) ⋅ ∑ (S 2 )
2 2
i 1
i i
where S1 represents signal intensity of pixels
in channel 1 and S2 represents signal intensity
of pixels in channel 2. This coefficient was
developed specifically for estimating colocal-
ization (Manders et al., 1993). Its advantage is
that it is insensitive to the limitations of typ-
ical fluorescence imaging, such as efficiency
of hybridization, sample photobleaching, and
camera quantum efficiency. The values of this
coefficient are in the range from 0 to 1.0. If
the image has an overlap coefficient 0.5, it im-
plies that 50% of both its objects, i.e., pixels,
overlap. A value of zero means that there are
no overlapping pixels.
Overlap coefficients k1 and k2
∑ S1 ⋅ S 2 i i
k1 = i
∑ (S1 )
2
i
i
and
∑ S1 ⋅ S 2 i i
k2 = i
∑ (S 2 )
2
i
i
where S1 represents signal intensity of pixels
in channel 1 and S2 represents signal inten-
sity of pixels in channel 2. These coefficients
Current Protocols in Cell Biology
split the value of colocalization into a pair of Colocalization coefficients m1 and m2
separate parameters. They depend on the sum
of the products of the intensities of two chan- ∑ S1 i , coloc

nels and are sensitive to the differences in the m1 = i

intensities of signals.
∑ S1
i
i

Table 4.19.3 Coefficients Used to Estimate Colocalization with Their Meanings, Values, and Use

Coefficient Meaning Values Usability

Pearson’s correlation Describes the correlation From −1.0 to 1.0; 0 indicates no Useable in any colocalization
coefficient (Rr ) of the intensity significant correlation and −1.0 experiment
distribution between indicates complete negative
channels correlation
Overlap coefficient Indicates an actual From 0 to 1.0; 0.4 implies that Can be used in any
according to Manders (R) overlap of the signals, is 40% of both selected channels colocalization experiment,
considered to represent colocalize especially applicable when
the true degree of the intensities of fluorescence
colocalization of detected antigens differ
Overlap coefficients k1 Split the value of Vary Useable in any colocalization
and k2 colocalization into the experiment
two separate parameters,
allows determination of
the contribution of each
antigen to the areas with
colocalization
Colocalization Describe the contribution From 0 to 1.0; m1 and m2 of 1.0 Useable in any colocalization
coefficients m1 and m2 of each one from two and 0.3 for red-green pair imply experiment
selected channels to the that all red pixels colocalize with
pixels of interest green, but only 30% of green
pixels colocalize with red
Colocalization Identical to m1 and m2 , From 0 to 1.0; M1 and M2 of 1.0 Useable in any colocalization
coefficients M1 and M2 but applied to analyzing and 0.3 for red-green pair imply experiment
scatter gram ROI that all red pixels colocalize with
green, but only 30% of green
pixels

Table 4.19.4 Troubleshooting Guide to QCA

Problem Possible cause Solution

Scatter gram shows too many pixels
removed following background
correction
Scatter gram shows too few pixels
removed following background
correction
Results of coefficients calculations do Mistake(s) in image acquisition
not fall within their standard values
Wrong choice of background If background was corrected in Auto mode,
correction method try using Manual mode. In Manual mode,
use the Selected ROI preset and try selecting
different image areas.
Background was not corrected If background was corrected using Selected
enough ROI, select different ROI. If Threshold Value
was employed, use slider to increase the
number of pixels to be removed.
Obtain another image
and handling
Microscopy

4.19.13
Current Protocols in Cell Biology Supplement 39
Table 4.19.5 Summary of the Values of Coefficients Calculations Indicating Ranges When Antigens Can Be Inter-
preted as Colocalized and When Nota

Values indicating absence of

Coefficient Values indicating colocalization
colocalization

Pearson’s correlation coefficient (Rr ) From 0.5 to 1.0 From −1.0 to 0.5
Overlap coefficient according to From 0.6 to 1.0 From 0 to 0.6
Manders (R)
Overlap coefficients k1 and k2 Any close values, like 0.5 and 0.6 Any distant values, like 0.5 and 0.9 or
or 0.8 and 0.9 0.2 and 0.7
Colocalization coefficients m1 and m2 More than 0.5 Less than 0.5
Colocalization coefficients M1 and M2 More than 0.5 Less than 0.5
a The table is based on a large pool of statistically significant coefficients results obtained when analyzing colocalization of various antigens in
brain, heart, liver, kidney, muscle, and blood labeled by FITC, Texas Red, and various Alexa dyes.

and antigens can be considered as colocalized and

when not.
∑ S2 i , coloc
m2 = i Limitations
∑S2i
i Reliable QCA is limited only by the suit-
ability of images to be analyzed and their pixel
where S1i,coloc = S1i if S2i >0 and S2i,coloc = resolution. Requirements for the suitability of
S2i if S1i >0. For example, if the red-green pair images are described in the protocol in de-
of channels is selected and m1 and m2 are 1.0 tail. Microscope-dependent suitability is con-
and 0.5, respectively, this means that all red stantly improving, as is the pixel resolution,
pixels colocalize with green pixels, but only together with the advancements of the quality
half of green pixels colocalize with red ones. of confocal microscopes.
The value of 1.0 for both channels indicates
perfect colocalization. Time Considerations
In comparison with performing immuno-
Colocalization coefficients M1 and M2 histochemical staining and confocal micro-
scope observations, QCA requires very little
∑ S1 i , coloc time and is dependent solely on the results of
m1 = i
immunofluorescence staining. The most time-
∑ S1
i
i
consuming part can be the background cor-
rection step, but only until the proper setup
and is determined. Calculations of coefficients are
usually done in a matter of minutes.
∑S2 i , coloc
m2 = i

∑S2i
i
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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 experi-
ments. Microscopy
4.19.15
Supplement 39
 North, 2006. See above.
Review with focus on proper interpretation of the
results of fluorescence microscopy studies, draw-
backs and limitations of biological imagery.

Internet Resources
http://homepage.mac.com/colocalizerpro/
Web site of CoLocalization Research Software, cre-
ators of CoLocalizer Pro and CoLocalizer Express
software applications used for quantitative estima-
tion of colocalization.

Quantitative
Colocalization
Analysis of
Confocal
Fluorescent
Microscopy
Images

4.19.16
Supplement 39 Current Protocols in Cell Biology