Journal of Microscopy, Vol. 185, Pt 1, January 1997, pp. 2136.

Received 21 June 1996; accepted 16 September 1996

Multicolour analysis and local image correlation in

confocal microscopy*
D. DEMANDOLX & J. DAVOUST
Centre dImmunologie CNRS-INSERM de Marseille-Luminy, Case 906, 13288 Marseille
Cedex 9, France

Key words. Co-localization, confocal microscopy, uorescence microscopy,

uorogram, image analysis, image correlation, multicolour analysis, pixel
histogram.

Summary
Multiparameter uorescence microscopy is often used to
identify cell types and subcellular organelles according to
their differential labelling. For thick objects, the quantitative
comparison of different multiply labelled specimens requires
the three-dimensional (3-D) sampling capacity of confocal
laser scanning microscopy, which can be used to generate
pseudocolour images. To analyse such 3-D data sets, we
have created pixel uorogram representations, which are
estimates of the joint probability densities linking multiple
uorescence distributions. Such pixel uorograms also
provide a powerful means of analysing image acquisition
noise, uorescence cross-talk, uorescence photobleaching
and cell movements. To identify true uorescence colocalization, we have developed a novel approach based on
local image correlation maps. These maps discriminate the
coincident uorescence distributions from the superimposition of noncorrelated uorescence proles on a local basis,
by correcting for contrast and local variations in background intensity in each uorescence channel. We believe
that the pixel uorograms are best suited to the quality
control of multiuorescence image acquisition. The local
image correlation methods are more appropriate for
identifying co-localized structures at the cellular or
subcellular level. The thresholding of these correlation
maps can further be used to recognize and classify biological
structures according to multiuorescence attributes.

Introduction
In uorescence microscopy, the simultaneous or sequential
detection of several channels is of great importance in
biological applications where several markers have to be
superimposed and compared (Brelje et al., 1993). Protein
immunouorescence cytochemistry and uorescence in situ
Correspondence to: Denis Demandolx. Tel: (+33) 4 91 26 94 36; Fax: (+33) 491
26 94 30; E-mail: demandol@ciml.univ-mrs.fr
* Paper presented at MICRO 96, London, 24 July 1996.
1997 The Royal Microscopical Society

hybridization (FISH) of nucleic acids now permit the

detection of a wide variety of macromolecules within cells
and tissues. In conventional epiuorescence microscopy of
thin specimens, the multiple detection of nucleic acids by
FISH can be achieved by combining one or several
uorophores for each hybridization (Nederlof et al., 1990);
the resulting diversity allows the identication of more than
10 sites with three distinct labels mixed in multiple ratios
(Dauwerse et al., 1992; Nederlof et al., 1992a,b).
For thick specimens, the images acquired with conventional microscopes suffer from the out-of-focus blur of the
uorescence emissions, impairing the estimation of colocalization between several markers. Confocal microscopy
has extended the capacity of optical microscopy by allowing
3-D sampling of specimens while excluding out-of-focus
blur (Wijnaendts-van-Resandt et al., 1985; White et al.,
1987; Brakenhoff et al., 1989; Shotton, 1989). The
improved axial resolution allows the 3-D localization of
uorescence markers (Sheppard, 1989) and 3-D image
reconstruction (Van der Voort et al., 1989, 1993; White,
1995). Moreover, the lateral resolution may, under ideal
conditions, be improved compared with nonconfocal optical
microscopy (Sheppard & Cogswell, 1990; Wilson, 1990).
The confocal out-of-focus uorescence rejection laid the
foundations for the quantitative comparison of multiuorescence signals (Akner et al., 1991; Sandison & Webb,
1994; Sandison et al., 1995). In multiuorescence confocal
microscopy, the specimens are scanned with one or several
excitation laser lines, allowing sequential or simultaneous
detection of several uorescent markers through multiple
acquisition channels (Mossberg et al., 1990; Brelje et al.,
1993). Compared with ow cytometry, cells are analysed at
a slower rate, yielding a confocal image of high spatial
resolution of a limited number of cells. Multiuorescence
confocal microscopy can also yield valuable information for
the identication of multiply labelled structures, or to
monitor the time evolution of intracellular Ca2+, H+ ion or
cyclic AMP concentrations to be followed by uorescence
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D. D E M A N D O L X A N D J. DAVO U S T

ratio imaging of dedicated probes (Poenie et al., 1986;

Bright et al., 1989; Adams et al., 1991).
Double and triple immunouorescence labelling permits
one to estimate the co-localization between uorescent
markers in the same eld (Schubert, 1991; Brelje et al.,
1993; Paddock et al., 1993; Paddock, 1995). Several
methods based on the superimposition of colour channels
have been explored to reveal coincident immunouorescence labelling (Arndt-Jovin et al., 1990; Fox et al., 1991;
Humbert et al., 1992, 1993; Dutartre et al., 1996) or to
compare FISH vs. immunouorescence labelling (Leger et
al., 1994). The local subtraction of appropriately scaled
uorescence channels (Akner et al., 1991) and the double
thresholding of uorescence values (Mossberg et al., 1990)
have also been used to compare confocal images. Global
correlation coefcients have been explored to estimate the
degree of correspondence between two related uorescence
micrographs (Manders et al., 1992, 1993). In addition,
several authors have identied uorescence cross-talk and
image registration defects due to chromatic aberrations or
misalignment of confocal optics as fundamental problems in
multiuorescence acquisitions (Akinyemi et al., 1992;
Sandison et al., 1995; White et al., 1996). A judicious choice
of uorophores, laser types, objective lenses, uorescence
lter sets, detection pinhole settings and photon detectors
often reduces these limitations (Mossberg & Ericsson, 1990;
Sheppard et al., 1992; Brelje et al., 1993; Keller, 1995; Tsien
& Waggoner, 1995), which can further be compensated for
by digital image processing (Carlsson & Mossberg, 1992;
Brelje et al., 1993). Multicolour image analysis software is
highly desirable to control the acquisition conditions, to
evaluate the problems of registration and aberrations and to
reveal the co-localized uorescence distributions.
To check the quality of image acquisition procedures and
to study the correspondence between multiuorescence
labelled structures, we dene here several levels of image
analysis. We rst propose the use of an improved dualchannel look-up table (LUT), second, we create statistical
representations of multiple uorescence data, and third we
develop local image correlation methods. The enhanced
dual-channel LUT allows a higher colour discrimination of
double uorescence labelling, the pixel uorograms provide
a statistical representation of multiuorescence distributions,
and the local correlation maps reveal co-localized structures. Use of the improved LUT, pixel uorogram analysis
and local image correlation have to be combined for the
global and local analysis of uorescence co-localization.

Materials and methods

Materials, cells and antibodies
All chemicals and proteins were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Major histocompatibility

complex (MHC) class II IAk and invariant (Ii) chain positive

broblast cells were obtained after transfection of IAka, IAkb
and Ii chains using the calcium phosphate precipitation
method as described (Salamero et al., 1990; Humbert et al.,
1993). Anti-MHC class II IAk 10.2.16 monoclonal antibodies and rabbit polyclonal anti-cathepsin D antibodies
were obtained and used as described (Humbert et al., 1993).
Rabbit polyclonal anti-Ii chain antibodies were generously
provided by Nicolas Barois (Centre dImmunologie CNRSINSERM de Marseille-Luminy, France). Texas-Red-coupled
wheat germ agglutinin (WGA) was obtained from Molecular
Probes Inc. (Eugene, OR, U.S.A.).

Intracellular indirect immunouorescence

Mouse broblast and B lymphoma cells were cultured for
48 h on glass coverslips. After washing three times with
PBS, cells were xed for 15 min with a 4% paraformaldehyde solution in phosphate-buffered saline (PBS) and
permeabilized with 0.1% Triton X100 solution. Cells were
subsequently washed and blocked with PBS containing
0.2% gelatin. Antibodies were diluted to a 10 mg mL 1
concentration in PBSgelatin. To perform indirect immunouorescence labelling, cells were incubated for 20 min with
primary antibodies at room temperature, and then washed
three times, before incubation with either FITC- or TexasRed-coupled anti-IgG antiserum (1/200 dilution in PBS
gelatin). The labelled cells adherent to the glass coverslips
were mounted on glass slides with Mowiol embedding
medium (Humbert et al., 1993). To stain the surface of
living cells, we incubated cells grown on coverslips with
1 mg mL 1 of Texas-Red-coupled WGA for 20 min at 20 C.
The cells were mounted in optical chambers containing
400 mL of medium as described (Pollerberg et al., 1986).

Conguration of the confocal laser scanning microscope

Confocal laser scanning microscopy was carried out using a
Leica TCS 4D instrument (Leica Lasertechnik, Heidelberg,
Germany), based on a Leitz DMRBE microscope interfaced
with an argonkrypton laser specied to have a maximum
power of 25 mW in each line (488, 568 and 647 nm). The
total laser output power was set to 25 mW. Selecting the
488- and 568-nm lines simultaneously, we have 8 mW for
each line. Owing to an initial attenuation with lters, to
optical bre coupling, to the confocal excitation aperture
and to an over-illumination of the 1.4 numerical aperture
(NA) of the microscope 100 PL APO objective, the input
power is reduced 25-fold. The total illumination power
thus lies around 0.3 mW for each laser line on the specimen
under these conditions. To minimize the noise and to keep
the photobleaching rate below 1% for FITC, we selected an
acquisition time of 1 s per scan and averaged 16 scans to
produce each 1024 1024-pixel image, as a standard
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M ULT IC OLOU R A NA LYSIS I N C ON FO CA L M IC ROSC O P Y

procedure for all the applications. (For the application of

Fig. 2, where we wanted to accentuate the effects of
photobleaching, we tripled the laser power.) For most of the
applications, we selected a eld of interest of 512 512
pixels from the raw micrographs. The Nyquists criterion
indicates that to digitize a signal whose cut-off frequency is
fc, it is necessary to sample at a frequency of at least 2fc. This
principle can be applied in confocal microscopy to select the
appropriate sampling rate of the detected signal (Young,
1988). With the 100 PL APO objective, the lateral optical
resolution is about 0.18 mm and the axial resolution along
the z-axis is 0.5 mm. We used a sampling step of 0.095 mm in
the plane of section and 0.25 mm in the axial direction.
In multicolour analysis, the image registration between
channels is of great importance. We checked that
wavelength-dependent distortions were small compared
with the optical resolution for objects close to the coverslip.
The possible chromatic aberrations (Akinyemi et al., 1992)
are considerably reduced by using plan apochromatic
objective lenses, provided that for an oil-immersion lens
imaging an aqueous specimen, the plane of focus is adjacent
to the coverslip (Keller, 1995). Actual measurements
provided by Leica Lasertechnik indicate that the relative
axial shifts between 488 and 568 nm lines are never greater
than 50 nm using 100 1.4-NA PL APO oil-immersion
objective. Experimental verications using the detection of
double-labelled subcellular endosomal structures present in
the cells close to the coverslip/medium interface showed no
visible shift between green and red uorescence emissions
even at the periphery of the eld. The use of a single argon
krypton laser feeding a monomode optical bre guarantees
the alignment of laser lines. Focal series of four horizontal
planes of section spaced at 0.25 mm have been monitored
simultaneously for FITC and Texas Red. As we recorded
series of optical sections close to the coverslips, we have
minimized the optical aberrations due to differences of
refractive index inside the specimen. Moreover, the Mowiol
mounting medium has a refraction index close to the glass
coverslip (n = 1.52). For standard acquisitions, we used a
double dichroic mirror for the excitation beam, a band pass
520560 nm barrier lter for FITC, a long pass barrier lter
above 580 nm for Texas Red detection and two photomultiplier tubes (PMT). Depending on the relative amount
of FITC and Texas Red, and the gain settings of the two
detectors, simultaneous dual-channel acquisition can generate a small amount of uorescence cross-talk between
FITC and Texas Red channels (Mossberg & Ericsson, 1990;
Carlsson & Mossberg, 1992; Brelje et al., 1993).

Implementation of image processing programs

The individual 8-bit-encoded 1024 1024- or 512 512pixel images, one from each channel for each plane of section,
were combined and visualized with a 16-million-colour
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23

display screen and printed using a Codonics NP-1600 photographic network colour printer utilizing dye-sublimation
technique (Codonics Inc., Middleburg Heights, OH, U.S.A.).
Image processing, false-colour mapping, pixel uorogram
and local image correlation algorithms were implemented
in C programming language using Microwares C Language
Compiler System (Microware, Des Moines, IA, U.S.A.) and
then linked with Leicas image processing library. Our
current programs run on a Motorola 68040 microprocessor
system (Motorola, U.S.A.) with Microwares OS-9 operating
system. To display dual-colour uorescence images, we have
used both classical and extended LUT as described in the
Results. Pixel uorogram and local image correlation
techniques are extensively described in the Results and the
Appendix.

Results
Improved look-up tables
Through all this work, we have used double uorescence
confocal micrographs obtained from broblast tissue culture
cells expressing the IAkab MHC class II molecules and the
associated Ii chain (Humbert et al., 1993). Figure 1a shows
a typical plane of section recorded 1 mm above the solid
coverglass support used for cell culture and simultaneously
observed with FITC and Texas Red acquisition channels.
MHC class II and Ii chain are represented in green and red,
respectively. This type of double uorescence micrograph
can be visualized with a standard redgreen LUT producing
a limited range of shades from green for FITC to red for
Texas Red. Note that the plasma membrane is only labelled
by indirect FITC immunouorescence revealing the surface
expression of MHC class II molecules. Internal vesicles are
labelled with both uorophores but with different ratios,
indicating a partial segregation of MHC class II molecules
and Ii chains (Humbert et al., 1993).
As shown above, multiuorescence confocal micrographs
are usually represented by the superimposition of their red,
green and blue colour-coded channels. However, this direct
representation leads to interpretation problems linked with
a differential sensitivity of our perception of red, green and
blue. To circumvent these difculties, we have extended this
dual-channel LUT from cyan for FITC to magenta for Texas
Red through green, yellow and red for halfway combinations, improving the discrimination of the uorescence
ratios between FITC and Texas Red (Fig. 1b). The concept
behind this extended dual-channel LUT is inspired by
consideration of the classical huesaturationluminance
(HSL) colour space. Here, a specic hue value is dened for
each ratio between the pure FITC and Texas Red
uorescence components x and y (see LUT bar in Fig. 1a).
The corresponding luminance is here calculated by the or
fuzzy logical operator x + y xy/255 varying from 0 to 255

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M ULT IC OLOU R A NA LYSIS I N C ON FO CA L M IC ROSC O P Y

for initial 8-bit-encoded uorescence values. Other luminance operators are conceivable, such as the maximum
value of x and y. The nal choice and the usefulness of such
colour correction depends on accepted conventions. If the
hues of the extended LUT are too far from conventional red
green LUT, their interpretation can require an adaptation
time.

Statistical representations of the uorescence micrographs

To provide a statistical representation of the image data sets
for each channel, we can calculate the pixel histogram
distribution, showing the number n(k) of each pixel value
within the image:
nk cardinalfxi; j kg;

where n(k) denotes the number of pixels whose uorescence

value x(i,j) equals k for all possible coordinates (i,j).
A large contribution of weakly uorescent areas leads to a
sharp peak at the origin of the graphs for each channel
(Fig. 1c,d). To classify the occurrences of double-labelled
pixels in the micrographs, we decided to use second-order
histograms to estimate the joint probability density linking
two uorescence distributions (Pratt, 1991). Second-order
histograms show the number n(k,l) of pixels for which the
values of each channel x(i,j) and y(i,j) equal, respectively k
and l:
nk; l cardinalfxi; j k; yi; j lg:

As an application, second-order semilogarithmic histograms

have been calculated for each cell circled on the micrograph
of Fig. 1b (Fig. 1eh). By analogy with the well-known
cytouorogram representation from ow cytometry, we are
proposing to call this representation a pixel uorogram.
Pixel values of FITC and Texas Red components (k,l) are
placed, respectively, along the x- and y-axes. These graphs
can also be represented by means of contour lines
(Demandolx & Davoust, 1995). The sharp peak at the
origin of these pixel uorograms corresponds to the low
luminance levels of the double uorescence labelling. It
should be noticed that these pixel uorograms are composed

25

of a multitude of radial streaks showing the local correlations between uorescence distributions. Each co-localized
structure corresponds to a radial streak. On the other hand,
horizontal or vertical clouds of dots lying along the x- and yaxes are related to single labelled structures in the
micrographs. Usually, each combination of pixel values
(k,l) corresponds to a specic colour on the dual-channel
micrographs. It is therefore possible to derive colour-coded
pixel uorograms from the actual semilogarithmic representation, as shown in Fig. 1i,k. Dots on such pixel
uorograms are represented with the same colour as in
the raw dual-colour image. A straightforward visualization
of double-labelled pixels in the micrograph can be achieved
by selecting the pixels above a threshold for each
uorescence channel in the pixel uorogram (Fig. 1j,l).
This is equivalent to a double thresholding using the image
amplitudes as attributes (Mossberg et al., 1990; Pratt, 1991;
Pal & Pal, 1993). We preferred to limit ourselves to each
elliptical area of interest because it is often difcult to use a
global threshold for a whole eld containing several cells.
Provided that the nonspecic uorescence is uniformly
distributed within the object, this method allows us to
distinguish double positive, single positive and background
areas according to the selected portion of the pixel
uorogram.

Application of the pixel uorograms

The pixel uorograms can be used to identify doublelabelled pixels of interest and are helpful in the analysis of
small variations between closely related images. Signal-tonoise ratio (SNR) and basic movements can be analysed in
this way. As an example, a confocal uorescence micrograph of B lymphoma cells is shown in Fig. 2a. Cells are
labelled indirectly with primary anti-IAk MHC class II
molecules and FITC-coupled secondary antibodies. We have
represented the pixel uorograms obtained between two
sequentially single scanned images (Fig. 2b), four-timeaveraged scanned images (Fig. 2c) and 16-time-averaged
scanned images (Fig. 2d). The diagonal cloud of dots on the
pixel uorogram gets thinner as the uorescence signal and

Fig. 1. Double-labelling analysis with pixel uorograms. (a) Double immunouorescence labelling of broblast transfectants monitored by
confocal microscopy. IAkab MHC class II molecules are represented in green after FITC labelling and the Ii chain is represented in red
after Texas Red labelling. The additive superimposition of both channels gives a range of yellow shades as shown in the LUT bar in the bottom
right corner. Field of view 50 50 mm. (b) Same eld as in (a) but the combination between FITC and Texas Red is visualized using an
extended LUT with more shades for different FITC and Texas Red ratios. Four elliptical areas of interest 14 have been dened around
the cells to compute the pixel uorograms. (c,d) Semilogarithmic pixel histograms of the FITC and Texas Red components, respectively.
(eh) Second-order pixel uorograms from the four elliptical areas of interest 14 displayed in (b). The grey level is inversely proportional
to the logarithm of pixel numbers. To distinguish isolated dots, we have used a black background. As a standard procedure, FITC and Texas
Red components are represented along the x- and y-axes, respectively. (i) Colour-coded pixel uorograms of the same elliptical areas of interest
1 from panel (b). Pairs of pixel values present in the area of interest are represented by a dot of the same colour in the pixel uorogram and in
the image. (j) Double-labelled pixels selected and displayed in white in the colour-coded pixel uorogram (i) are visualized on the micrograph
(j). (k,l) This is the same as (i,j) but for the area of interest 3 of (b).
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D. D E M A N D O L X A N D J. DAVO U S T

Fig. 2. Pixel uorograms for noise and movement analysis. (a) Confocal immunouorescence micrograph of IAk MHC class II molecules in B
lymphoma cells indirectly labelled with FITC. Field of view 70 70 mm. (b-d) Pixel uorograms of sequential acquisitions from the section (a)
for 1-, 4- and 16-scan averaged images, respectively. Components from the rst and second acquisition are represented along the x- and yaxis, respectively. The tilt of the cloud-of-dots (d) is due to the photobleaching of FITC after 16 scans with triple laser power. (e) Time-lapse
confocal image of living broblasts labelled on their surface with Texas Red-coupled WGA. Two time lapse acquisition with a 1-min interval
are superimposed. Field of view 50 50 mm. (f) Colour-coded pixel uorogram of the two superimposed components of (e). (g,h) Same as (e,f),
but with a time interval of 15 min.

the number of scans increase, indicating an improved SNR

(Fig. 2bd). The width of the cloud-of-dots is proportional to
the standard deviation of the acquisition noise and
decreases as 1/ n where n denotes the number of scans
(Mossberg et al., 1990). In confocal uorescence microscopy, the noise originates mostly from the photon number
which follows Poisson statistics (Young, 1996) characterized by a standard deviation proportional to the mean of the
corresponding signal. This explains the parabolic distribution of the cloud-of-dots of Fig. 2. The photobleaching of
FITC between two acquisitions is also detected as a tilt of the
diagonal as shown in Fig. 2d (Brakenhoff et al., 1994). If
parts of the specimen undergo limited movement during
time lapse recording of a living specimen, off-diagonal dots
will appear on the pixel uorogram. Such an example is
shown in the second part of Fig. 2, where we have presented
the superimposition of the surface labelling of living cells
recorded at time 0 and time 1 min (Fig. 2e) and at time 0
and time 15 min (Fig. 2g). The corresponding colour-coded
pixel uorograms become more spread as a function of time
(Fig. 2f,h, respectively). For a clear detection of the
movement, good SNR and limited uorescence photobleaching are obviously required. More sophisticated methods,
optimized to track the cell movements, can also be

considered. However, the pixel uorograms developed here

are very helpful in the analysis of SNR, photobleaching and
can reveal partial displacements in the specimens.

Application of intensity-weighted pixel uorograms

To further quantify the uorescence values for each class of
pixels identied on the pixel uorograms, we have dened a
uorescence intensity-weighted pixel uorogram from the
double confocal immunouorescence micrograph of Fig. 3a.
In such a pixel uorogram, the number of pixels n(k,l) is
multiplied by the FITC or the Texas Red uorescence
intensities k and l leading to the integrated FITC and Texas
Red uorescence values k.n(k,l) and l.n(k,l). Colour-coded
intensity-weighted pixel uorograms are then obtained by
merging FITC and Texas Red uorescence integrated values
using dual-colour LUT (see Fig. 3be). As illustrated below,
uorescence intensity-weighted pixel uorograms can be
used to analyse uorescence cross-talk and background
uorescence.
The uorescence cross-talk occurs when part of a
uorophore emission is detected in the wrong uorescence
channel. In particular, FITC and Texas Red uorophores can
emit photons of longer wavelength which are detected and
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27

Fig. 3. Application of intensity-weighted pixel uorograms. (a) Double confocal immunouorescence micrograph of broblasts. FITC-labelled
antibodies directed against the lysosomal enzyme cathepsin D labels intracellular vesicles whereas Texas Red-coupled WGA binds mostly at
the cell surface. Field of view 50 50 mm. (b) Intensity-weighted pixel uorogram of (a). The white line corresponds to the slant induced by
the 12% cross-talk of FITC to the Texas Red channel. (c) Same as (b) but with a cross-talk compensation. (d) Same as (c) but FITC and Texas
Red uorescence have been offset to correct for background uorescence before the computation of the uorogram. Offset values of 16 for
FITC and 16 for Texas Red are shown by vertical and horizontal lines, respectively. (e) Same as (d) but using a preprocessing 13 13-pixel
low-pass lter.

added in another uorescence channel (Carlsson & Mossberg, 1992). FITC emission can leak into the Texas Red
acquisition channel and Texas Red can leak into the
Cyanine 5 channel. Fluorescence cross-talk results in very
characteristic pixel uorograms. Everything happens as if
the uorophore x,y-base was no longer orthogonal. Since
FITC-stained regions of the specimen emitting green
photons are also emitting a smaller number of red photons
detected in the Texas Red channel, the FITC x-axis makes an
acute angle as shown in Fig. 3b. Pixel uorogram
representations are thus important to evaluate the crosstalk between uorescence channels and to perform the
appropriate corrections. Different approaches have been
proposed for the compensation of colour images (Carlsson et
al., 1994). A practical approach implies the specication of
a matrix operator that will remove cross-talk contributions
as described by Brelje et al. (1993). Matrix coefcients can
be determined by estimating the cross-talk percentage from
the slant in the contours of the intensity-weighted pixel
uorogram. The tangent of the angle made with the FITC
axis denes the ratio of the FITC intensity detected in the
Texas Red channel to the FITC intensity detected in the FITC
channel (Fig. 3b) and consequently the matrix coefcients
for its correction (Fig. 3ce).
1997 The Royal Microscopical Society, Journal of Microscopy, 185, 2136

Most indirect immunouorescence micrographs contain

nonspecic uorescence levels referred to as background
uorescence. This background uorescence may be due to
autouorescence of the specimen, or to nonspecic absorption of uorescence coupled secondary antibodies usually
present all over cellular specimens. This gives a signicant
contribution to the integrated uorescence, as shown in the
intensity-weighted pixel uorogram close to the origin of the
graph. Therefore, we recalculated the intensity-weighted
pixel uorograms after appropriate uorescence offsetting
(Fig. 3d,e). In these representations, dots on the pixel
uorograms are weighted with offset-corrected uorescence
intensities. Mossberg et al. (1990) proposed a method for the
choice offsets from each channel histogram. Ideally the
offsets should correspond to the averaged FITC and Texas
Red background uorescence recorded under the same
optical conditions for a negative control specimen. By
calculating intensity-weighted pixel uorograms using offset corrected uorescence values, we can now resolve
differently the bins of integrated uorescence. The joint
distribution of uorescence integrated values now reveals
the contribution of specic uorescence measurements in a
better way. To minimize the local dispersion of the pixel
uorogram distributions, linear low-pass or nonlinear

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median ltering is usually advantageous. To remove from

the pixel uorograms the contribution of the high spatial
frequency components due to the acquisition noise, we have
applied a 3 3-pixel low-pass convolution lter (Pratt,
1991) throughout this paper. Using as a test a more drastic
Gaussian convolution lter whose standard deviation is set
to 3 pixels on a 13 13-pixel window, all clouds-of-dots are
smoothed (Fig. 3e).
The intensity-weighted pixel uorograms can reveal
several bins of pixels corresponding to statistically signicant areas of interest, such as different levels of background,
and single or double-labelled areas. However, very large or
complex images contain many overlapping bins of pixels
which leads to confused situations for the interpretation of
pixel uorograms. Background uorescence correction and
local averaging are needed to extract more information on
multiuorescence images. The evaluation of specic vs.
background uorescence is always more accurate on a local
basis.

Local image correlation

In order to detect co-localization of uorescence distributions on a local basis, we have to extract more information
from the neighbouring pixels. In the strict sense, a colocalization means a correspondence between two uorescence distributions varying locally in the same way.
Provided that the co-localized area stretches over relatively
few pixels and that we do not reach uorescence saturation
level (Visscher et al., 1994), the cloud-of-dots of a very local
pixel uorogram is clustered along a straight line (Fig. 4a,b).
The slope of such a cloud-of-dots is related to the
uorescence ratio between the markers used. The shape
and the width of the cloud depend on acquisition noises.
The global pixel uorograms are indeed composed of a
multitude of radial streaks describing each individual double

positive structure. In the following, we have developed a

local approach using a sliding window to characterize the
dispersion of the cloud-of-dots in second-order histograms.
To nd out the correspondences between local uorescence
distributions, we have compared two methods based on
local image correlation. The rst is based on the calculation
on the local correlation coefcient of raw images (CCRI) and
the second on the local joint moment of standardized
images (JMSI). This second-order joint moment, also called
the noncentral covariance or correlation function (Svedlow
et al., 1978; Anderson, 1984), is computed between the
images using a sliding window after a statistical differencing
lter to standardize the two image data sets (Pratt, 1991).
The local CCRI method consists of computing the correlation coefcient for a sliding window between the two raw
images. In the two methods, detailed in the Appendix, we
obtain a complete correlation map expressing a local colocalization coefcient for each pixel. Both methods are
suited for registered image pairs.
To illustrate the principle of the JMSI method, we have
chosen a small area of interest of about 100 pixels around a
vesicle in which both uorophores are co-localized, zoomed
and circled in Fig. 4a. If we form a pixel uorogram
representation, the corresponding binary cloud-of-dots is
elongated, showing a linear dependence between intensities
and thus a signicant correlation (Fig. 4b). To perform a
statistical differencing, we have, respectively, subtracted
from each pixel x(i,j) and y(i,j) of the raw images, the

averaged values x(i,j) and y(i,j) of the surrounding pixels

computed with a 9 9-pixel Gaussian convolution lter
whose standard deviation is set to 1.33 pixel (Fig. 4c) (Pratt,
1991). We have next divided the pixel values of x and y
images by their modied standard deviation jx(i,j) and
jy(i,j) calculated over the same window at the coordinate
(i,j) as dened in Eqs. (4)(7) in the Appendix. The local
means and standard deviations of the resulting images are

Fig. 4. Standardization of image data sets. (a) A subregion of Fig. 1b is selected to illustrate the principle of the local joint moment of standardized images (JMSI). The circle identies a small co-localized vesicle of about 100 pixel area. Field of view 12.5 12.5 mm. (b) Pixel uorogram from the circled area of interest in the raw micrograph (a). (c) First step of statistical differencing showing the subtraction of the lowpass component of each raw image. (d) Second step of statistical differencing showing the normalization of the cloud-of-dots. By dividing each
central image by their modied local standard deviation, the cloud-of-dots has been straightened along the diagonal of the graph.
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M ULT IC OLOU R A NA LYSIS I N C ON FO CA L M IC ROSC O P Y

normalized (Fig. 4d). As a result of this standardization, the

cloud-of-dots is now straightened along the bisecting line of
the pixel uorogram. The elongated shape along the
diagonal shows the linear dependence between correlated
intensities in the source image of Fig. 4a. Noncorrelated

29

structures give rise to cloud-of-dots dispersed in all

directions. To visualize the resulting joint moment, we
have multiplied and locally averaged the two standardized
images. Considering each pixel neighbourhood, this locally
averaged joint moment is sensitive to the shape of the

Fig. 5. Application of local image correlation methods. (a) Correlation maps derived from the local correlation coefcient of raw images
(CCRI), applied to the micrograph shown in Fig. 1a, using a black and white LUT between values 0 and 1 of the CCRI result. (b) Same
as (a) but for the correlation map of the local JMSI method displayed with the same LUT. (c,d) Same micrographs as in Fig. 1b, but the contours of the thresholded correlation maps (a) and (b), respectively, are shown as black lines to reveal co-localized vesicles.
1997 The Royal Microscopical Society, Journal of Microscopy, 185, 2136

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D. D E M A N D O L X A N D J. DAVO U S T

standardized cloud-of-dots. Highly diagonal clouds give rise

to maximum values of the joint moment.

Detection of co-localized areas with local image correlation

Image correlation maps contain positive and negative areas
depicting positive or inverse correlations between local
uorescence distributions. The local correlation maps of the
CCRI and of the JMSI methods have been calculated from
the original images of Fig. 1a (Fig. 5a and 5b, respectively).
Here, we have only displayed the positive part of correlation
maps since it is the most signicant one for our applications.
Both methods use local and standardized data sets, and both
correlation maps reveal the co-localized structures of
interest in the raw images. As shown in Fig. 5a, the local
CCRI correlates the edges better than the top areas whose
distributions are less stretched out than the edges. In local
JMSI method, the sliding estimation of local statistical
parameters allows a better discrimination of top areas and
an easier determination of the threshold needed for further
segmentation of co-localized structures (Fig. 5b). To compare the local image correlation maps with the corresponding multiuorescence micrographs, we have rst segmented
the co-localized areas from each correlation map (Pal & Pal,
1993). Practically, for each correlation map, we have
extracted regions above a global correlation threshold of
0.5. We have then superimposed in black the contours of
these segmented areas on the raw image of Fig. 1b for the
CCRI and JMSI correlation maps (Fig. 5c and 5d,
respectively). For sake of clarity on the images, we do not
show the contours of correlated objects smaller than 9
pixels. This segmentation allows the extraction of highly
correlated structures whatever their uorescence intensities. Some cyan and magenta structures are scored as colocalized indicating a linear dependence between uneven
labelling.
Since the JMSI method reveals the co-localized vesicles
better than the CCRI, it facilitates subsequent segmentation.
In our second example, we have applied the JMSI method to
confocal micrographs from other broblast cells having a
higher surface expression of MHC class II molecules labelled
indirectly with Texas Red-coupled antibodies. The second
indirect labelling was carried out with antibodies coupled
with FITC and directed against the cathepsin D lysosomal
enzyme. As in Fig. 5d, we have extracted and superimposed
in black the contours of correlated regions on the raw image
(Fig. 6a). Black-surrounded areas, thresholded from the
local JMSI map, reveal the presence of a collection of colocalized structures located in the cytoplasm of double
positive cells. Finally, to estimate the correspondences
between local correlation methods and pixel uorograms,
one can either classify pixels from individual segmented
objects as shown in Fig. 4a or pool the segmented pixels
surrounded in Fig. 6a in an intensity-weighted pixel

Fig. 6. Pixel uorograms and correlated areas. (a) Application of

the JMSI method to identify and surround co-localized structures
in a double immunouorescence labelling of broblasts. FITClabelled antibodies directed against the lysosomal enzyme cathepsin D reveal intracellular vesicles and Texas Red antibodies directed against mutated MHC class II molecules now label the cell
surface and some internal vesicles. The JMSI correlation map
reveals colocalized vesicles but excludes nonrelated uorescence
superimposition. Field of view 50 50 mm. (b,c) Intensity-weighted
pixel uorograms of (b) correlated and (c) noncorrelated areas
dened in (a). The local JMSI correlation map extracts both weakly
and highly labelled structures (b). Double positive pixels that have
not been selected in colocalized areas in (a) are displayed in the
intensity-weighted pixel uorograms (c) with single positive and
background pixels.

uorogram representation (Fig. 6b). On the other hand,

the pixel uorogram calculated on noncorrelated areas only
is highly dominated by the background uorescence
(Fig. 6c). As expected from the correlation method, superimpositions of nonrelated FITC and Texas Red labelling are
excluded from the correlated areas as shown in the
micrograph (Fig. 6a) and in the intensity-weighted pixel
uorogram (Fig. 6c).
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M ULT IC OLOU R A NA LYSIS I N C ON FO CA L M IC ROSC O P Y

31

Discussion

Statistical analysis of multichannel images

Optical microscope users usually face difculties during the

course of multiuorescence imaging experiments carried
out with confocal or nonconfocal equipment. Among these,
multicolour visualization, quality control of multiuorescence acquisition and nally how to evaluate the statistical
signicance of uorescence co-localization are the main
practical problems. For dual-channel imaging purposes, we
have proposed rst to enhance the standard redgreen
superimposition with an extended dual-channel LUT,
thereby allowing a higher discrimination between the two
labels. Second, we have dened pixel uorograms, which
provide a statistical representation of multiple uorescence
acquisitions, in a similar way to ow cytometric cytouorograms. Third, we have developed a local approach for the
detection of colocalization, by estimating the linear dependence between the uorescence distributions on a sliding
window assuming that intensity vs. binding follows the
same characteristics for the two probes.

Despite using an improved LUT, it is generally difcult to

estimate the occurrences of dual uorescence measurements on multicolour micrographs. To visualize the
uorescence joint distribution, we have created secondorder pixel uorograms showing the frequency of registered
pairs of pixels coming from both uorescence channels. By
dening a threshold for each channel on these graphs, we
can now classify pixels according to their intensities in four
categories: the background, the single-labelled areas, and the
double-labelled areas. To provide a user-friendly correspondence between images and pixel uorograms, we have
created a colour-coded pixel uorograms. As a complement
to the spatial representation of images, we think that
second-order pixel uorograms are appropriate to reveal
intrinsic properties of multiuorescence images that would
otherwise be difcult to infer from the micrographs directly.
The rst group of applications concerns the analysis of the
time-dependence of single labelling including the SNR,
uorescence photobleaching and cell movement detection.
A second group of applications deals with the multicolour
analysis of the channel registration, and measurements of
uorescence cross-talk and background uorescence. These
two groups of applications require either time-resolved or
multiuorescence images. Photobleaching and uorescence
cross-talk can be corrected sequentially depending on the
available data sets provided that single labelled structures
are present in the micrographs.
Owing to the low number of detected photons (Pawley,
1995), the photon statistics have been proven to be the
main limiting factor of the SNR (Young, 1996). As shown in
our results, the pixel uorograms allow one to visualize the
noise distribution for each grey level from two successively
scanned images. The intrinsic dispersion of the cloud-of-dots
due to the acquisition noise limits the precision with which
two images can be compared. Temporal and/or spatial
ltering techniques improve the SNR, as revealed by pixel
uorograms constructed from time-averaged images. On the
other hand, a too long or too intense laser excitation of
uorophores induces a uorescence attenuation due to
photobleaching, which can also be estimated from pixel
uorograms. The acquisition noise can be detected as the
width of the diagonal cloud-of-dots, and the photodamage
as a tilt of the pixel distribution. Finally, limited movements
of specimens generate random distributions of points on the
pixel uorograms.
In multiuorescence acquisitions, image registration and
uorescence cross-talk are the main experimental problems
for which a statistical analysis of multicolour pixels is highly
desirable. Relative distortions between multiuorescence
channels originate from chromatic aberrations in the
objective lenses, in the confocal optics, or from the
movement of the specimen between sequential acquisitions.

Visualization of multiple uorescence micrographs

For multicolour representations, when the emission spectrum of each uorophore produces a distinct colour which
is close to one of redgreenblue (RGB) primary colours, we
generally associate each uorescence channel to the RGB
colour plane that best matches the uorophore emission
colour. This straightforward method produces colour
combinations close to the ones observed through the
microscope oculars provided that the emission energy is
similarly distributed among different uorescence detectors.
In the other cases, an arbitrary RGB colour plane can be
attributed to each channel. For double-labelling images, it is
possible to improve upon the standard redgreen LUT by
using the third primary colour to create an extended dualchannel LUT, allowing a higher discrimination of uorescence ratios while conserving the luminance information.
Triple-channel acquisition allows less exibility. In the
extended dual-channel LUT presented here, ve hues dene
ve sectors of uorescence ratios, and the luminance is a
function of both uorescence intensities as dened in the
Results section. We think that our sequence from magenta,
red, yellow, green to cyan enhances our perception of colour
combination, as shown in Fig. 1. Other choices of hue and
luminance formulae can be applied to visualize uorescence
ratio imaging, such as obtained with Ca2+ or H+ probes
(Poenie et al., 1986; Bright et al., 1989). Anyhow, the
interpretation of colour superimposition remains linked to
subjective criteria. The extension of multicolour representation to 3-D uorescence data sets is not obvious. Still,
some interesting approaches have been proposed (Van der
Voort et al., 1989; Messerli et al., 1993; Messerli & Perriard,
1995).
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D. D E M A N D O L X A N D J. DAVO U S T

Defect in the colour registration is detected by off-diagonal

dots on pixel uorograms if the specimen contains sharp
and colocalized uorescence structures.
To reveal the cross-talk between uorescence channels,
we have designed intensity-weighted pixel uorograms.
Nevertheless, the conditions of acquisition should rst be
optimized to reduce cross-talk during the acquisition by
adequate selection of laser lines, uorophores and chromatic lters (Mossberg & Ericsson, 1990; Brelje et al.,
1993). For given acquisition settings, the cross-talk
coefcients can be evaluated by comparing the two
acquisition channels on second-order pixel uorograms for
each single-labelled specimen (Mossberg et al., 1990). As
carried out in uorescence ow cytometry, a real time crosssubtraction method can be implemented at the level of the
signal acquisition electronics. Furthermore, the autouorescence and/or the nonspecic absorption of uorescent
antibodies generate a non-negligible part of the signal, in
addition to any instrumental acquisition noise. Our
intensity-weighted pixel uorograms show that the low
luminance levels constitute the main part of the total image
uorescence. Special intensity-weighted pixel uorograms
were designed after offsetting the uorescence values to
remove a large part of the nonspecic background
uorescence from the integration. However, the statistical
properties of the noise vary within the observed areas, and it
is generally difcult to associate bins for each component of
the noise in wide-eld pixel uorograms. Therefore, we
recommend others to restrict the area of interest from
which the pixel uorogram is calculated. Within local areas,
colocalized structures are dened by an elongated cloud-ofdots, the slope of which is related to the ratio of uorescence
labelling. A single hue of the extended dual-channel LUT is
attributed to each co-localized distribution, provided that
the background uorescence is small enough compared
with the specic uorescence.
Pixel uorograms can easily be calculated on serial
optical sections, but again it is preferable to limit the volume
of interest in 3-D data sets. If needed, 3-D representation
techniques could be used to visualize third-order pixel
uorograms associated with triple uorescence labelling.
Pixel uorogram analysis beyond the third-order presents
serious problems of representation and interpretation
(Bonnet et al., 1995).

Detection of co-localization
To detect the local correlations of uorescence distributions,
we had to consider a collection of neighbouring pixels. A
standardization of the uorescence values was required to
obtain comparable data sets for each channel despite the
different acquisition conditions. Various advanced methods
have been proposed to compare images, either in pattern
recognition to match a template and a portion of image

(Pratt, 1991) or for the spatial registration of multispectral

or multitemporal satellite images (Svedlow et al., 1978).
Among these, the correlation-based methods prove to be
well-suited to compare registered digital images, or to
determine the spatial shift separating two acquisitions.
Manders et al. (1992, 1993) have applied correlation
methods to nd out the degree of correspondence on entire
confocal micrographs. However, this approach does not
provide local information on the sites of colocalization. To
enlighten highly correlated structures in the images, we
have developed two image correlation methods adapted on a
local scale for the detection of uorescence colocalization.
The methods use standardized data sets to estimate the
linear dependence between the uorescence distributions.
One is based on the local correlation coefcient between raw
images (CCRI) and the second on the local second-order
joint moment of standardized images (JMSI), also called
noncentral covariance or correlation function (Svedlow et
al., 1978; Anderson, 1984).
To compute the correlation measurements locally, we
have chosen a Gaussian sliding window whose standard
deviation appears to be the main parameter of the methods.
The standard deviation of the Gaussian window should be
large enough to estimate correctly the local statistical
parameters such as the mean and the standard deviation of
pixel values with regard to the optical resolution and the
sampling frequency of the micrograph. Too broad a window
lowers the resolution of the correlation map, hampering the
detection of the small objects. The standard deviation
dening the Gaussian window should be chosen according
to the size of objects to be colocalized, the optical resolution
and the sampling rate. We chose a 1.33-pixel standard
deviation on a 9 9-pixel window containing 99.9% of the
Gaussian distribution, which turned out to be large enough
to estimate local statistical parameters while conserving a
high resolution in the correlation map. A compromise
should be found between spatial resolution and validity of
statistical measurements. Using standardized data sets, the
correlation methods can reveal the correlation of very
weakly labelled areas. Indeed, we noted that the autouorescence of the specimen as well as a part of the nonspecic
absorption of antibodies give rise to highly correlated
structures due to the normalization by very small standard
deviations. To prevent these nonsignicant outputs when
the variance of very at areas is low, we have added an
additional constant to the variance of each channel, which
modies the denominator of the correlation formulae. In
statistical differencing a constant is added to the denominator (Pratt, 1991). This additional constant can be also
interpreted as the variance of a noncorrelated noise added
to each channel. This noise constant can be adjusted to
drown any type of weak correlated structures (autouorescence or correlated components of weak labelling). In our
256-grey-level images, we have added a uniform noise
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M ULT IC OLOU R A NA LYSIS I N C ON FO CA L M IC ROSC O P Y

whose standard deviation was set to four quantization levels

for both channels. The photon statistics and the dark
current give typical noncorrelated noises, but these were
too small to mask nonsignicant structures in the low
uorescence areas of the micrographs. As expected, the
uorescence cross-talk increases the correlation measurements and should be compensated for before computing the
correlation maps. In addition to pixel uorograms, image
correlation maps can be used to adjust acquisition settings,
to eliminate uorescence cross-talk and to optimize image
registration locally. Among the two methods described in
the Results and the Appendix, the JMSI method better
denes the tops of vesicular structures. Since this method
uses an image-orientated way of standardizing the data sets
called statistical differencing (Pratt, 1991), it provides
intermediate images with normalized uorescence distributions. Except for very low signals, for which local
variance is covered by the noise constant, both methods
detect colocalization independently of the local mean of the
signal. Double positive pixels which do not correspond to a
local covariation of uorescence distributions are not
revealed by these methods (White et al., 1996), as shown
in Fig. 6. Local image correlation excludes the superimposition of at uorescent proles when a high spatial
resolution is required for the analysis. At low spatial
resolution, at areas can be considered if they are smaller
than the local scanning window. We think that the
identication of colocalized structures is meaningful only
for a specied scale. Accordingly, unknown structures of
any size can be compared with known uorescence
elements.

33

The extension of local correlation methods to serial

confocal sections requires the use of 3-D Gaussian windows.
The uorescence attenuation could then automatically be
corrected by the standardization of the data. Independent
methods for the correction of this attenuation have been
proposed (Rigaut & Vassy, 1991; Visser et al., 1991;
Strasters et al., 1994; Liljeborg et al., 1995). To apply
local image correlation methods to triple labelled specimens
in a basic way, we propose to consider the three channels
two-by-two, and then nally to superimpose the three maps
by associating each of them to a RGB colour plane.

Acknowledgments
This work was supported by the Centre National de la
Recherche Scientique (CNRS), the Institut National de la

Sante et de la Recherche Medicale (INSERM) and the

Association pour la Recherche contre le Cancer (ARC). D.D.
is the recipient of a predoctoral BDI fellowship from the

CNRS and the Conseil Regional Provence-Alpes-CotesdAzur. We wish to thank Dr Jacques Barbet (Centre
dImmunologie de Marseille-Luminy, France) and especially
Dr David Shotton (University of Oxford, U.K.) for critical
reading of the manuscript. We also thank Dr Graca Raposo

(Institut Curie, Paris, France) and Dr Patrizia Rovere (Centre

dImmunologie de Marseille-Luminy, France) for the preparation of various specimens and especially Mr Nicolas
Barois (Centre dImmunologie de Marseille-Luminy, France)
for providing us with rabbit polyclonal anti-Ii chain
antibodies.

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Appendix: the local image comparison methods

The correlation coefcient of raw images
Considering a local area of interest in the pair of
uorescence micrographs, the correlation coefcient estimates the linear dependencies between the two pixel value
distributions independently of background uorescence
levels. The correlation coefcient of raw images (CCRI),
given in the correlation formula (3), is computed on sliding
windows across the whole image eld, i.e. for each pixel
neighbourhood, to produce a complete image correlation
map. All means on local windows are computed using a
Gaussian low-pass lter.
CCRI

xy xy
;
jx jy

where x and y denote the digital uorescence images as

arrays of pixel values for both images. The image arrays j2x
and j2y denote the modied local variances as indicated in
Eqs. (4) and (5). All arithmetical operations are calculated
pixel-to-pixel between pairs of images. The bar ( ) indicates
an image operation where each pixel value is replaced by
the averaged value of its neighbourhood. For example, the
image xy is the result of the image product between x and y

convoluted by a Gaussian convolution lter, whereas xy is

the pixel-to-pixel product between the ltered images x and

y. In our example the experimental averages ( ) are all

calculated by smoothing the input images through a 9 9pixel Gaussian convolution lter whose standard deviation
is set to 1.33 pixel. The variances j2x and j2y have been
modied by adding the constants ax and ay (Eqs. 4 and 5) to
prevent the generation of nonsignicant correlation coefcient values due to very at areas with small variances.
The constants ax and ay can be also interpreted as
variances of noncorrelated noises added to the images. ax
and ay can be set to zero to obtain the exact variances and
the exact correlation coefcient varying from 1 to 1.

j 2 x 2 x 2 ax
x

j 2 y 2 y 2 ay :
y

36

D. D E M A N D O L X A N D J. DAVO U S T

The joint moment of standardized images

ys

yy
:
jy

In an alternative approach, we applied a statistical

differencing (Pratt, 1991) to locally standardize the mean
and the variance in the images. The standardized images
are then compared by the calculation of their local secondorder joint moment (Anderson, 1984). The statistical
differencing of images allows the generation of locally
standardized images successively by subtracting the locally

averaged image x from the raw image x and dividing the

result by the standard deviation image jx dened above. The
overall operator is dened by

The notations are the same as for Eqs (3), (4) and (5). All
the means () are computed using the Gaussian convolution
lter dened above.
Having dened two standardized image data sets xs and
ys, we can compute their joint moment between sliding
windows across the whole image eld. This second-order
joint moment between standardized images (JMSI) is the
averaged value of the product between images xs and ys:

xx
xs
jx

The JMSI values are not necessarily between 1 and 1 but

are usually in this range.

JMSI xs ys :

1997 The Royal Microscopical Society, Journal of Microscopy, 185, 2136