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Research and applications
Registration of whole immunohistochemical slide
images: an efficient way to characterize biomarker
colocalization
Xavier Moles Lopez,1,2 Paul Barbot,2 Yves-Rémi Van Eycke,2 Laurine Verset,3
Anne-Laure Trépant,3 Lionel Larbanoix,2 Isabelle Salmon,2,3 Christine Decaestecker1,2
▸ Additional material is
published online only. To view
please visit the journal online
(http://dx.doi.org/10.1136/
amiajnl-2014-002710).
1
Laboratories of Image, Signal
Processing and Acoustics,
Ecole Polytechnique de
Bruxelles, Université Libre de
Bruxelles, Bruxelles, Belgium
2
DIAPath, Center for
Microscopy and Molecular
Imaging (CMMI), Gosselies,
Belgium
3
Department of Pathology,
Erasme Hospital, Bruxelles,
Belgium
Correspondence to
Professor Christine
Decaestecker, Laboratories of
Image, Signal Processing and
Acoustics, École polytechnique
de Bruxelles, Université Libre
de Bruxelles CP 165/57, 50,
av. Franklin Roosevelt,
Brussels 1050, Belgium;
[email protected]
XML and PB contributed
equally.
LL’s contribution occurred
during his affiliation to
DIAPath (CMMI).
Received 8 February 2014
Revised 1 July 2014
Accepted 22 July 2014
ABSTRACT
Background and objective Extracting accurate
information from complex biological processes involved
in diseases, such as cancers, requires the simultaneous
targeting of multiple proteins and locating their
respective expression in tissue samples. This information
can be collected by imaging and registering adjacent
sections from the same tissue sample and stained by
immunohistochemistry (IHC). Registration accuracy should
be on the scale of a few cells to enable protein
colocalization to be assessed.
Methods We propose a simple and efficient method
based on the open-source elastix framework to register
virtual slides of adjacent sections from the same tissue
sample. We characterize registration accuracies for
different types of tissue and IHC staining.
Results Our results indicate that this technique is
suitable for the evaluation of the colocalization of
biomarkers on the scale of a few cells. We also show
that using this technique in conjunction with a
sequential IHC labeling and erasing technique offers
improved registration accuracies.
Discussion Brightfield IHC enables to address the
problem of large series of tissue samples, which are
usually required in clinical research. However, this
approach, which is simple at the tissue processing level,
requires challenging image analysis processes, such as
accurate registration, to view and extract the protein
colocalization information.
Conclusions The method proposed in this work
enables accurate registration (on the scale of a few cells)
of virtual slides of adjacent tissue sections on which the
expression of different proteins is evidenced by standard
IHC. Furthermore, combining our method with a
sequential labeling and erasing technique enables cellscale colocalization.
INTRODUCTION
To cite: Moles Lopez X,
Barbot P, Van Eycke Y-R,
et al. J Am Med Inform
Assoc Published Online First:
[please include Day Month
Year] doi:10.1136/amiajnl2014-002710
Whole-slide imaging is an essential tool for digital
pathology in which glass slides are digitized into
virtual slides (VSs). Digital pathology permits the
gathering and sharing of information for medical
education, diagnosis, and research.1 One particular
research field in digital pathology is concerned with
tissue-based biomarkers that are indicative of a
normal or abnormal process (or of a condition or
disease) and can be used for diagnostic, prognostic,
and therapeutic purposes. These biomarkers can
target the morphology and architecture of cells or
tissues as well as gene and protein expression,
which can be evidenced in tissue samples by using
Moles Lopez X, et al. J Am Med Inform Assoc 2014;0:1–11. doi:10.1136/amiajnl-2014-002710
certain staining techniques, such as in-situ hybridization (ISH) and immunohistochemistry (IHC).1–3
Multiple proteins must be simultaneously examined to extract relevant information from the
complex biological processes involved in serious
pathologies such as cancers. The colocalization of
protein expression that can be revealed by IHC in
formalin-fixed paraffin-embedded (FFPE) tissue
samples yields detailed information related to the
potential protein interaction to be extracted. This
type of information produces both morphologic
information and molecular and functional information, potentially increasing the specificity and/or
sensitivity of the resulting indicators in terms of
their diagnostic, prognostic, or theragnostic value.
Different approaches were developed to allow the
colocalization of two or more biomarkers in FFPE
or frozen tissue sections. Most of the approaches
use fluorescence labeling with frozen sections,4–8
and comparatively few use brightfield IHC with
FFPE tissue sections.9–12 However, the latter
approach is easier to implement and offers
better preservation of the tissue morphology.
Fluorescence-based labeling does have certain
advantages in colocalizing antigens (eg, its ability to
both multilabel and detect individual labels), but it
features several disadvantages in practice (eg, the
need for frozen tissue slices, labeling fading, tissue
autofluorescence, cost of the imaging equipment,
etc.). Consequently, brightfield IHC staining on
FFPE tissue sections is more suited for the highthroughput tissue sample processing involved both
in clinical research and in the daily routine of hospital pathology departments.
Brightfield multichromogenic methods for multiple antigen labeling, in which each biomarker is
evidenced by a different color, are limited and have
had many challenges over the years.13–16 The essential problems result from antibody cross-reactions
and interpretation difficulties. These latter occur if
different targeted proteins are expressed in the same
cellular compartment (eg, the cell nuclei), then
color merging or masking arises. As an alternative
method, biomarker colocalization can be determined by cutting adjacent (or serial) tissue sections
ranging in thickness from 3 to 5 μm, submitting
each section to standard IHC, digitizing the slides,
and finally registering the adjacent VSs. Because the
slides are thinner than the average size of human
cells, pluricellular structures such as glands or epithelium are often well preserved across several
slides. Thus, analyzing adjacent slides enables the
colocalization of protein expression at a histological
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Research and applications
level; however, it also requires a more complicated image analysis
processes, such as the accurate registration of VSs for viewing
and extracting the colocalization information.12 17 18
An interesting alternative to multichromogenic IHC was
recently proposed by Glass et al,19 who described a method to
label the same tissue slide multiple times using brightfield IHC.
This ‘sequential immunoperoxidase labeling and erasing’
(SIMPLE) method requires the creation of a VS after each labeling. After digitization, the staining is erased (or washed) and a
new cycle of staining–digitization–erasing can be performed.
Staining erasing is a difficult technique that uses an antibody
elution technique intended to preserve tissue and antigen epitopes for the next staining. By correcting certain parameters and
including a new elution methodology, we recently improved the
SIMPLE methodology and successfully utilized it with antigens
expressed in the same cellular compartment (unpublished data).
Figure 1A compares the workflows of our SIMPLE-like method
to the standard IHC procedure applied to serial slides.
The registration of VSs thus becomes an essential step for the
colocalization of IHC biomarkers with the serial slides or the
SIMPLE-like method. It is a relatively new problem that presents different challenges than those traditionally encountered
in the registration of digital radiology images.17 The most
important of these challenges is the size of the images: VSs are
typically 30 000 pixels in height by 60 000 pixels in width,
which is approximately 2×109 pixels in total. In addition, this
method differs from multimodal digital radiology images where
different modalities applied to the same region are registered; in
the case of serial slides, we need to register different but adjacent
slides that were independently processed to evidence different
biomarkers by IHC. The IHC staining pattern depends on the
targeted antigen and its cellular location and can possibly yield
large variations in pixel intensity between adjacent VSs. It
should also be noted that the tissue process may introduce nonlinear deformations such as tearing, shearing, folding, and
shrinking.
Although VS registration is a relatively new problem, the
registration of small histological images (HIs) is a well-studied
field of research. These images show either the entire tissue with
a lower resolution or a small section of the tissue with a higher
resolution. Three-dimensional (3D) volume reconstruction is
also often addressed in this context,20–28 as it requires sequentially registering pairs of two-dimensional (2D) HIs and propagating the registrations to realign all the images of the
stack.25 26 28 To avoid error propagation, some authors proposed the introduction of constraints related to the 3D nature
of their dataset.21 23 27 29
In contrast, the problem of registering multiple HIs to measure
the overlap or colocalization of multiple IHC biomarkers is
addressed much less in the literature (and even less for complete
VSs). Often, colocalization was manually and qualitatively evaluated by viewing the same histological region in different slides
and showing the different pictures side by side.10 30 31
Rademakers et al18 used an interactive ‘register’ function in
IPLab (Scanalytics) that could rotate and shift images to manually
register VS pairs to assess the colocalization of two IHC biomarkers on adjacent tissue sections from biopsies.
To the best of our knowledge, all of the methods studying
full-sized VSs used a pyramidal registration technique that first
registered a downsampled version of the image and then refined
the registration result by initializing new registration steps with
incrementally increasing resolutions. For example, Cooper
et al32 applied an automatic two-step procedure to register
H&E- and IHC-stained slides of mouse mammary glands and
2
of follicular lymphomas.33 The first step in the procedure was a
rigid registration that was guided by matching pairs of histological structures. The second step was a non-rigid transformation that was guided by matching either image patches32 or a
subset of histological structures.33
In Mueller et al,17 the authors tackle the problem of visualizing two or more VSs, which enables the visualization of an
H&E slide together with an IHC-stained slide that shows a
prognostic biomarker. The challenge was to provide an
almost-real-time registration so that two slides could be easily
viewed at the same time. They also proposed a two-step
approach based on the open-source program elastix, originally
developed to register in vivo images.34 The first step in the
approach was a global pre-registration step that was performed
offline prior to viewing. A second, online transformation was
then instantly applied to each field of view (FOV) requested by
the pathologist during his/her navigation throughout the VSs.
The registration error was computed on five regions of interest
(ROIs), only for the offline step, each ROI containing 10 pointbased landmarks. The results had a maximal registration error
of 130 μm after the offline registration stage.
Finally, Metzger et al12 also used a two-step registration
method to analyze H&E- and IHC-stained slides scanned at 20×
magnification. This method combines a coarse manual alignment
with an automatic fine registration using a software module
called TurboReg, which uses a pyramid approach initially proposed for the registration of positron emission tomography and
fMRI images.35 During the first step, manual manipulations were
used to flip and rotate three adjacent IHC VSs so that they were
in rough alignment with a reference H&E VS. The TurboReg
software then automatically completed the registration process
through rigid transformations, though affine transformations
were also available. However, the results displayed relatively high
registration errors: they were computed on 150 landmark pairs
distributed across the H&E and IHC VSs and had median errors
of 88–114 μm (depending on the stains) and a maximal error of
398 μm. Note that the cell diameter of the tissue under analysis
(prostate cancer) is in the range 15–20 μm.
The present study aims to provide an accurate image registration technique that applies to high resolutions (typically equivalent to 20× magnification) and is motivated by the need to
perform valid biomarker colocalization analyses. This need
requires registration accuracy on the scale of a few cells. To this
end, a two-step registration strategy where a global lowresolution registration initializes the registrations of separate
FOVs seems appropriate. The independent registration of FOVs
increases the registration accuracy by locally reducing alignment
errors. However, this technique requires as many additional
registrations as the number of independent FOVs. Therefore, a
fast registration method is essential. In addition to the performance in terms of accuracy and speed, the accurate analysis of
biomarker colocalization necessitates the preservation of pixel
size in the VSs (and FOVs) after transformation. Considering all
of these reasons, we decided to extend the work presented in
Mueller et al,17 which demonstrated fast and promising results.
We use parametric transformations that preserve the shape and
dimension of the pixels, and we characterize the registration
errors on high-resolution FOVs for different types of tissue by
imaging serial IHC slides as well as slides that have been stained
multiple times with IHC (using our SIMPLE-like procedure).
MATERIALS
All the slides in the study were scanned at 20× magnification
(NanoZoomer HT 2.0, Hamamatsu, Hamamatsu City, Japan)
Moles Lopez X, et al. J Am Med Inform Assoc 2014;0:1–11. doi:10.1136/amiajnl-2014-002710
Research and applications
and annotated by a pathologist by means of NDP.view
(Hamamatsu). The first dataset, referred to as SERIAL, consisted
of eight groups of four serial slides (4 μm thick) from colorectal
cancer samples. For each group, the first slide was stained with
E-cadherin, the second with FHL-2 (four-and-a-half LIM
domains protein 2), the third with B-catenin, and the fourth
with vimentine (Vim) (see figure 1B). For each group, we
selected five ROIs on the FHL-2 VS, which was chosen as the
reference for the annotations because of its central location
within the stack of slides. For each ROI of the FHL-2 VS, a
pathologist placed six control points (CPs) at remarkable loci
and segmented six histological structures, totaling 30 CPs and
30 segmented contours (SCs) per slide. The pathologist manually matched the CPs and SCs on the other VSs of the stack to
constitute the complete supervised dataset made of 24 pairs of
annotated slides.
We also constructed a second dataset made of six pairs of VSs
consisting of six tissue slides from tonsil and brain tissue
samples, each sequentially stained to identify two different IHC
markers using our SIMPLE-like technique (see Introduction).
The sequential pairs of IHC markers targeted membranous
CD3 and CD20 expression, cytoplasmic glial fibrillary acidic
protein (GFAP) and Vim expression, and nuclear p53 and Ki67
expression. The VS marked with the first biomarker of each pair
(ie, CD3, CD20, P53, Ki67, GFAP, or Vim) was used as reference to distribute five ROIs. Similar to the SERIAL dataset, a
pathologist manually annotated six CPs and six SCs for each
ROI and matched them on the VS with the second biomarker
(CD20, CD3, Ki67, P53, Vim, or GFAP, respectively).
Finally, we constituted an independent dataset, referred to as
TEST, for validation purposes. The TEST dataset was made of
seven groups of two serial slides (targeting Ki67 and epidermal
growth factor receptor, respectively) of high-grade glioma tissue
samples. For each VS a pathologist manually annotated CPs in
five ROIs (six CPs per ROI).
METHODS
Because the method presented in Mueller et al17 had a nearly
real-time registration performance and was efficiently applied to
various IHC-stained VSs, we were motivated to further investigate the possibility of using the elastix framework to identify
biomarker colocalization. The different components of the registration available for customization in this framework are presented in figure 2 and detailed below.
The two steps in our registration strategy use the elastix
framework, in which the input images are selected with increasing resolution levels (ie, a pyramidal registration approach). To
register VSs labeled with different antibodies, we chose the
luminance image as the input for the low-resolution step. We
tested various color transformations as the input for the highresolution step. The executable and the C++ code of elastix are
available on the elastix website (http://elastix.isi.uu.nl).
The resolution levels are controlled by the ‘pyramid’ component that reduces the images by several factors using
Figure 1 (A) Sequence of steps involved in our SIMPLE-like method compared to the standard serial slide staining procedure. (B) Illustration of the
annotation process on serial slides. For each group of four slides, a reference slide in the center of the stack was chosen (ie, FHL-2). On the
reference slide, five ROIs were distributed and the pathologist annotated six CPs and six SCs in each ROI. Then, she selected the corresponding CPs
and SCs on the three other slides of the stack by matching them with the reference slide. CP, control point; ROI, regions of interest; SIMPLE,
sequential immunoperoxidase labeling and erasing.
Moles Lopez X, et al. J Am Med Inform Assoc 2014;0:1–11. doi:10.1136/amiajnl-2014-002710
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Research and applications
Figure 2 Basic registration components of the elastix framework (adapted from Klein et al34). At each registration step (low- or high-resolution), a
pyramidal registration procedure optimizes the parameters (μk) of the transformation (Tμ) to minimize the value of the cost function (C). Our
benchmark application tests different combinations of the elements in red (see details in the main text).
Gaussian-like downsampling.36 The result is a (Gaussian)
pyramid from which the full-resolution image constitutes the
base level (level 0), and the lowest-resolution image corresponds
to the highest level of the pyramid. All the experiments related
to the global low-resolution registration step were conducted
using a Gaussian pyramid with four levels corresponding to the
following magnifications: 0.125×, 0.25×, 0.5×, and 1×. For
the experiments related to the local, high-resolution registration
steps, we also chose a Gaussian pyramid with four levels, but
the corresponding magnifications were 2.5×, 5×, 10×, and
20× (needed for the biomarker colocalization analysis).
The interpolator component of elastix computes the pixel
values of the moving image at the locations that correspond to
the pixels of the fixed image. This correspondence is established
by means of a parametrized transform (Tμ), which must be optimized. In Mueller et al,17 the authors used a B-spline transform
as the first, deformable transformation. Although it seemed to
provide a satisfactory result for the simultaneous viewing of two
slides, optimizing the parameters of this first transformation is
computationally demanding. Furthermore, we observed strong
distortions, such as wraps, on our VSs that are not desirable for
biomarker colocalization analysis. Therefore, we modified the
method presented in Mueller et al17 and tested the possibility of
using only linear transformations (affine and Euler transforms)
for both registration steps.
The values of the corresponding pixels in the fixed and
moving images are used to determine the value of a similarity
function, S. For the low-resolution registration step based on
the luminance images (which are impacted by the different
stains), we chose Mattes’ mutual information (MI) as the similarity function S because it is one of the most popular image
similarity measures for the registration of multimodality
images.37 In contrast, the registration of images in the same
modality can be considered for higher-resolution images after
color deconvolution. Indeed, by using the method detailed in
Ruifrok et al,38 we are able to separate the brown staining
(DAB, i.e. 3, 30 -diaminobenzidine) from the hematoxylin (blue)
counter-staining and to use the hematoxylin channel (hem) for
the registration of the high-resolution FOVs. These findings led
us to also test the normalized cross-correlation (NCC) as function S, which assumes an affine relation between the pixel intensity values of the two images.34
The optimizer component is a stochastic gradient descent
(SGD) method that searches the linear transformation parameters (μk) to minimize the cost function C=−S. We also investigated the convergence speed for the different combinations of
input image types and cost functions by testing different
4
numbers of iterations (1000 or 2000) as well as the maximum
step lengths (25, 50, 75, 100, 125, 150, or 300) for each optimization stage. In the end, the transformation parameters μk are
modified according to the optimization result. The newly parametrized transformation serves as the initialization of the next
optimization stage, using the images at the following level of the
pyramid, which have a higher resolution.
To increase the registration speed and robustness (by avoiding
local minima37), C is not evaluated on the complete images but
on randomly sampled pixels; we also tested the number of randomly sampled pixels in our benchmark (2500, 4000, 5000,
7500, 9000, or 10 000). The randomization and retrieval of the
pixel values is made by the sampler in a fixed image or by the
interpolator in a moving image. For the experiments conducted
in this work, we chose a B-spline interpolator.34
For each parameter set (PS)—which consisted of the values
provided to the elastix framework in terms of the number of
iterations, the maximum step length and the number of sampled
pixels used to evaluate C—we evaluated the low-resolution
registration accuracies using the root mean square error (RMSE)
computed on the CPs. For the high-resolution step, the PS also
included the color transformation (luminance or hem channel)
and similarity function S (MI or NCC). For high resolutions, we
also computed the Hausdorff distance (HD) on the SCs (see the
Materials section).
To identify the best PSs, we compared the RMSE (and HD)
values that were computed before (RMSE-O, HD-O) and after
the registration (RMSE-R, HD-R) using the T statistic from the
Wilcoxon matched-pair test provided in the Statistica package
(StatSoft, Tulsa, Oklahoma, USA). If the registration procedure
is efficient, a large majority of the differences of the RMSE (and
HD) values before and after registration should be negative,
which would lead to a small T value (T=0 indicates that all differences have the same sign).39 For the low-resolution registration step we also computed RMSE values provided by
TurboReg, a method previously used to register images of
IHC-stained slides.12 It should be noted that TurboReg uses the
Levenberg–Marquardt optimization method to minimize the
squared difference of pixel intensities.35
To determine the quality of the best results provided by
elastix, we compared them with those obtained using the supervised (rigid or affine) registrations based on the CP pairs. To this
end, we used the CorrespondingPointsEuclideanDistanceMetric
included in elastix. This supervised registration approach can be
viewed as providing an optimal RMSE reference, but is not
practical because it requires an expert to locate (as exactly as
possible) several CP pairs for each FOV to be registered.
Moles Lopez X, et al. J Am Med Inform Assoc 2014;0:1–11. doi:10.1136/amiajnl-2014-002710
Research and applications
The elastix PSs selected on the basis of the SERIAL dataset
were applied to the TEST set. The fully commented parameter
files required by elastix are provided in the online supplementary material.
RESULTS
We benchmarked the combinations of the elastix components
(shown in red in figure 2) on the two datasets (SERIAL and
SIMPLE) and for two different resolution steps (low resolution:
1×, high resolution: 20×). We first present the results on the
SERIAL dataset, which is an example of the most general situation for biomarker analysis on VSs acquired from serial IHC
slides. The low-resolution registration results are presented first,
with the goal of finding a combination of the elastix components that enables the robust optimization of the linear transformation parameters (μk). We then present the high-resolution
registration results that are obtained after initialization by the
low-resolution step. Finally, the results of the benchmarks conducted on the SIMPLE dataset, which illustrates an improved
situation for biomarker analysis in which multiple VSs are
acquired from the same tissue section, are presented.
To illustrate the differences between these two IHC
approaches (SERIAL vs SIMPLE), we computed the RMSE of
the original conditions before any registration (RMSE-O) for
the 30 CPs for each image pair. To register the SIMPLE-like
VSs, we register the same tissue slide that is stained twice
(including an elution step to remove the first staining). Thus, it
is not surprising that we observed strongly lower RMSE-O
values for the SIMPLE dataset compared to the SERIAL dataset
(see figures 3A and 4A).
Low-resolution step on serial VSs
During the low-resolution registration step, we tested both affine
and rigid transformations that were optimized with MI as the similarity function S. The value of S was estimated using Ns pixels
sampled from the luminance of the equivalent 1× magnification
images. The number of iterations, Ni, of the SGD optimization
procedure is fixed, so there is no early stop, and the maximum displacement (in pixels) allowed at each iteration is given by the
Figure 3 Box plots of the RMSE and
HD values (in μm) for the original
conditions (RMSE-O and HD-O) and for
those obtained after registration with
the parameter sets (PSs) used in the
low- (A) and high-resolution (B and C)
steps for the SERIAL set. The boxes
show the IQRs, and the medians are
shown as thin lines in each box. The
whiskers show the non-outlier ranges,
and the outliers are drawn with the ‘+’
symbol. (A) RMSE-O and RMSE-R
values obtained with the 30
low-resolution PSs identified by
applying condition (1). The continuous
horizontal line shows the 60-μm limit
to compare with percentiles 75. (B)
RMSE-O and RMSE-R values obtained
with the seven high-resolution PSs
identified by applying condition (2).
The original condition for the
high-resolution registration step is the
result of the low-resolution step (ie,
PS19 in A). The horizontal lines show
the 20- and 31-μm limits that
approximately correspond to percentile
25 of the RMSE-O and to the
maximum RMSE-R value encountered,
respectively. (C) HD-O and HD-R values
obtained with the seven PSs in B. The
horizontal line shows the median HD-O
value (249 μm). HD, Hausdorff
distance; RMSE, root mean square
error; IQR, interquartile range.
Moles Lopez X, et al. J Am Med Inform Assoc 2014;0:1–11. doi:10.1136/amiajnl-2014-002710
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Research and applications
Figure 4 Box plot of the RMSE-O
and RMSE-R values (in μm) obtained
with the parameter sets (PSs) used in
the low- and high-resolution
registration steps for the SIMPLE set.
The boxes show the IQRs, and the
medians are shown as thin lines in
each box. The whiskers show the
non-outlier ranges and the outliers are
drawn with the ‘+’ symbol. (A)
RMSE-O and RMSE-R values obtained
with the 24 low-resolution PSs,
completed using the 12 additional PSs
(PS25 to PS36) tested with smaller Ns
values (see main text). The continuous
horizontal line shows the 20-μm limit.
(B) RMSE-O and RMSE-R values
obtained with the seven
high-resolution PSs exhibiting, at most,
two FOVs with RMSE-R values higher
than the RMSE-O values. The
continuous horizontal line shows the
5-μm limit (see main text for details).
FOV, field of view; RMSE, root mean
square error; SIMPLE, sequential
immunoperoxidase labeling and
erasing; IQR, interquartile range.
maximum step length (MSL). The tested values of the parameters
of the optimization procedure are listed in table 1. Additionally to
these tests with elastix, we also applied TurboReg with parameters
similar to those used in Metzger et al,12 that is, a rigid transformation with the luminance image used as input.
For each of the resulting 168 PSs, we compared the RMSE
values computed from the 30 CPs after the equivalent 1× magnification images of the entire slides were registered. All PSs provided a large majority of RMSE-R values that were lower than
the RMSE-O values. We then used the T statistic of the
Wilcoxon matched-pair test to filter out the best performing
combinations. We isolated 30 PSs for which all but one of the
24 differences (RMSE-R − RMSE-O) are negative and for
6
which the rank of the sole positive difference is either 1 or 2
(ie, among the smallest values; see online supplementary table
S1) by applying the following selection condition:
T 2
ð1Þ
The RMSE-R value distributions obtained with these PSs are
shown in figure 3A, and the corresponding parameter values are
detailed in online supplementary table S1. It should be noted
that TurboReg did not satisfy condition (1) because it provided
a T value of 25 (with a p75RMSE of 124 mm), that is, the fifth
highest T value among all the tested PSs of elastix.
Moles Lopez X, et al. J Am Med Inform Assoc 2014;0:1–11. doi:10.1136/amiajnl-2014-002710
Research and applications
Table 1 Tested values for the parameters of the stochastic
gradient descent optimization procedure for the low- and
high-resolution steps applied on the SERIAL dataset
Parameter
Low-resolution
High-resolution
Tμ
S
Ns
Ni
MSL
Input
Affine, rigid
MI
2500, 4000, 5000, 7500, 9000, 10 000
1000, 2000
25, 50, 75, 100, 125, 150, 300
lum
Affine, rigid
MI, NCC
8000
2000
5, 25, 50, 100
lum, hem
Tμ is the parametric transformation model. S is the similarity function. Ns is the
number of samples (ie, pixels) used to estimate the value of S. Ni is the number of
iterations of the procedure. MSL is the maximum displacement (in pixels) allowed at
each iteration. Input indicates the input image (lum=luminance image,
hem=hematoxylin channel) resulting from the color deconvolution.
MI, mutual information; MSL, maximum step length; NCC, normalized
cross-correlation.
A rigid transformation was included only for PS1, PS2, and
PS3, all of which exhibited relatively high RMSE-R values (see
online supplementary table S1). In contrast, 75% of the registrations made with PS19, PS25, and PS30 had RMSE-R values
smaller than 60 μm, whereas the RMSE-O values had a median
of 1299 μm (see online supplementary table S1). The latter
three PSs used an affine transformation with at least 7500 pixels
(Ns) to estimate S and an MSL of either 100 or 150 pixels (ie,
920 μm or 1380 μm). From these three equivalent PSs, we
chose PS19 to initialize the upcoming high-resolution registration step because it is the least computationally expensive
(Ns=7500 and Ni=1000).
High-resolution step on serial VSs
During the high-resolution registration step, we once again
tested affine and rigid transforms that were optimized using
either MI or NCC as the similarity function S. In light of the
good optimization results obtained from the low-resolution
step, we chose Ns=8000 pixels to evaluate S and the number of
iterations Ni=2000 for the SGD optimization procedure. The
value of S was thus estimated from 8000 pixels sampled from
either the luminance (lum) or the hematoxylin channel (hem) of
the equivalent 20× magnification of each FOV. The maximum
displacement (in pixels) allowed at each iteration is given by the
MSL (see table 1).
For each of the 32 PSs, we compared the RMSE and HD
values obtained for each FOV after registration. The RMSE
values were computed on the six CPs, while the HD values
were computed on the six SCs, but both sets were computed
after the registration of the equivalent 20× magnification
images of the FOVs. Again, we used the T statistic to determine
the best-performing combinations with respect to the RMSE
(TRMSE) and HD (THD). We used the following selection condition to isolate seven PSs (see online supplementary table S2) for
which at most 11 out of 95 FOVs exhibited RMSE-R (HD-R)
values larger than the RMSE-O (HD-O) values:
ðTRMSE , 350Þ ^ ðTHD , 350Þ
ð2Þ
Compared to the low-resolution step, a higher RMSE threshold
value was used both because the T statistic was evaluated for 95
pairs (in place of 24 at low resolution) and because the lowresolution step used as initialization already provided low
RMSE-O (and HD-O) values (cf. figure 3B, C). Interestingly,
online supplementary table S2 shows that all of these PSs used
NCC as the similarity function S, regardless of the input. In
addition, five out of seven PSs used a rigid transform, supporting the findings of Mueller et al17 that a simple transformation
produces high accuracy at high resolutions. Overall, the best performances were provided by PS5 (see online supplementary
table S2, figure 3B, C), although it failed to correctly register an
FOV showing a tissue fold (see figure 5A, B) that was the largest
outlier in figure 3B, C. This result illustrates the impact that a
tissue defect may have on the registration results. In the discussion section, we suggest methods to identify registration errors
on specific FOVs to omit them from subsequent analyses (or to
manually correct it, if it is absolutely required).
To provide an optimal but realistic reference, we also applied
supervised registrations that use the CP pairs to optimize the
rigid and affine transformations. The results obtained from
these two optimal registrations were very similar and, comparatively, our completely unsupervised method using PS5 provides
very satisfactory results (see online supplementary figure S1 and
figure 5C, D).
Low-resolution step on the SIMPLE-like VSs
The results obtained from the SERIAL dataset show that affine
transforms are preferable for low-resolution registrations, that
Ns should be sufficiently large (greater than 5000), and that
serial VSs are registered appropriately with Ni=1000. However,
as mentioned above, SIMPLE-like VSs are easier to register than
serial VSs, so we used smaller values of the MSL parameter in
these tests (see table 2).
For all the tested PSs (listed in online supplementary table S3)
and all the registered image pairs, the RMSE-R values were significantly smaller than the RMSE-O values (see figure 4A).
Consequently, the T statistic was equal to zero for each PS.
Interestingly, online supplementary table S3 shows a dramatic
increase in RMSE-R for MSL values above 10 (except for PS4),
and this effect increases with the value of Ns. Thus, we also
tested smaller values of Ns, Ns=2500 (PS25 to PS30), and
Ns=4000 (PS32 to PS36) in addition to the six MSL values
detailed in table 2. We observed the same negative impact of
large MSL values on the results, but the effect was reduced due
to the smaller Ns (see figure 4A, where PS25 to PS36 are shown
first for illustration purposes). All PSs with MSL≤10 have a
maximal RMSE-R value of 19 μm, which is approximately two
pixels on the equivalent 1× magnification image used in the
low-resolution registration step.
In conclusion, we chose PS27 with Ns=2500 (faster computation time) and MSL=10 to initialize the following highresolution registration step.
High-resolution step on the SIMPLE-like VSs
The results on serial VSs show that rigid transformations offer
good accuracy at high resolutions. Therefore, we only tested
rigid transformation models for the high-resolution registration
step on the SIMPLE dataset. The transformation models were
optimized using either MI or NCC as the similarity function S,
which was evaluated using either the lum or the hem channel of
the equivalent 20× magnification of each FOV. We also used the
same values for Ns (8000 pixels) and Ni (2000) as on serial VSs.
The tested values for MSL are listed in table 2.
Figure 4B shows seven of the 16 PSs tested with the 30 different FOVs (listed in online supplementary table S4); these seven
were selected (in bold in online supplementary table S4)
because they exhibited at most two FOVs for which the
RMSE-R (HD-R) value was larger than the RMSE-O (HD-O)
value. As shown in online supplementary table S4, six of these
Moles Lopez X, et al. J Am Med Inform Assoc 2014;0:1–11. doi:10.1136/amiajnl-2014-002710
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Figure 5 Illustration of two pairs of FOVs (seen at 8× equivalent magnification) registered using PS5. The first column (A and C) shows the FOVs
used as the fixed images as blue rectangles. Six control points (CPs) and six manually segmented contours (SCs) per image are also shown in blue.
The second column (B and D) shows the corresponding FOVs used as the moving images (identified by the low-resolution registration step) as red
quadrilaterals and where the green CPs and SCs (corresponding to those in A or C) were manually defined by a pathologist (‘ground truth’). The
registration is initialized with the low-resolution step, transforming the blue CPs and SCs of the image from the first column (A or C) to those shown
in black in the second column (B or D). A subsequent high-resolution registration step is then applied by using PS5, resulting in the red CPs and
SCs. The RMSE (HD) values were calculated between the green and red CPs (SCs). The first row (A and B) shows an incorrect FOV registration for
which the final RMSE (HD) value was 226 μm (887 μm). The second row (C and D) shows a successful FOV registration for which the final RMSE
(HD) value was 17 μm (119 μm). FOV, field of view; HD, Hausdorff distance; RMSE, root mean square error.
PSs used NCC as the similarity function S, even when registering the luminance images. In fact, we observed that the input
image had less impact on the registration accuracy than the MSL
parameter (see online supplementary table S4 and figure 4B).
With the SIMPLE VSs, PS7 performed better than all other PSs
and all registered FOV pairs exhibited RMSE-R values smaller
than 5 μm.
Table 2 Tested values for the parameters of the stochastic
gradient descent optimization procedure for the low- and
high-resolution steps for the SIMPLE dataset
8
Application of the PSs selected for serial slides
on the TEST VSs
Parameter
Low-resolution
High-resolution
Tμ
S
Ns
Ni
MSL
Input
Affine
MI
5000, 7500, 9000, 10 000
1000
1, 5, 10, 20, 30, 40
lum
Rigid
MI, NCC
8000
2000
1, 5, 25, 50
lum, hem
See table 1 for the meaning of the parameters.
MI, mutual information; MSL, maximum step length; NCC, normalized
cross-correlation; SIMPLE, sequential immunoperoxidase labeling and erasing.
Even after an accurate low-resolution registration step, the
high-resolution registration step remained useful and provided
significantly better registration, as illustrated by the T statistic
values reported in online supplementary table S4 (associated
with p values <10−4). Figure 4B confirms that all of the FOVs
registered with PS7 exhibited RMSE-R values below 5 μm,
which is approximately the diameter of a cell nucleus in the
tissue samples.
We registered the TEST VSs by successively applying the lowand high-resolution steps with the PSs giving accurate results on
the SERIAL dataset (see result sections above). We characterized
the registration accuracy by using the control points placed
manually by a pathologist and computing the RMSE values after
the low (RMSE-RL) and high-resolution (RMSE-RH) registration steps. It should be noted that the brain tumor samples constituting the TEST dataset presented very few histological
structures. The VSs were thus less textured and more difficult to
register, especially for the high-resolution step, compared with
the colonic tissue samples constituting the SERIAL dataset.
Moles Lopez X, et al. J Am Med Inform Assoc 2014;0:1–11. doi:10.1136/amiajnl-2014-002710
Research and applications
Figure 6 Example of colocalization index map computation at high resolution. (A) FHL-2 high FOV (20× magnification). (B) Corresponding (ie,
result of the low-resolution registration step) E-cadherin FOV (20× magnification), which provides a good starting point for the high-resolution
registration. (C and D) Usual staining measurements, such as the Quick-Score (ie, mean DAB intensity) as illustrated here, are computed on each tile.
FOV A is divided into squared tiles 80 μm in length, and the high-resolution transform is then used to compute the corresponding tiles in FOV B
(note the slight rotation of the tiles). The RMSE-R after the high-resolution registration for this pair of FOVs was 17 μm. (E) The local contributions
to a global colocalization index, such as the overlap coefficient as illustrated here, are computed on each tile. The value of the global overlap index
(which is the sum of the tile contributions and takes values between 0 and 1) is 0.8784 for the two FOVs. FOV, field of view; RMSE, root mean
square error.
For the 35 ROIs constituting the TEST dataset, we obtained
relatively large RMSE-RL values after the low-resolution registration step using PS19. While on the SERIAL dataset 75% of
the RMSE-RL values were below 42 mm and the maximum
value was 97 mm, on the TEST set 75% of these values were
above 40 mm and the maximum value was 246 mm. However,
this maximum corresponds to a shift of 25 pixels at low resolution, that is, about 1% only of the width of the image (2438
pixels). This low-resolution step was thus used to initialize the
high-resolution one.
As expected, the lack of histological structures more affected
the high-resolution registration step. We tested different PSs
from the seven selected in our previous experiment (see results
above and online supplementary table S2), among which were
similarly performing (PS4, PS5, and PS7) and less effective PSs
(PS1 and PS2). We also tested these five PSs with an additional
level in the downsampling pyramid, resulting in a total of 10
tested PSs. From this short comparative study, the best results
were obtained with PS4 and PS40 (ie, PS4 with the additional
level in the downsampling pyramid), giving p75RMSE-RH values
Moles Lopez X, et al. J Am Med Inform Assoc 2014;0:1–11. doi:10.1136/amiajnl-2014-002710
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Research and applications
of 86 and 79 mm, respectively, as compared to 101 mm for PS5.
These results show that smaller displacements are preferable
(MSL=50 for PS4 in place of 100 for PS5, see online supplementary table S2) when histological structures are lacking.
Nevertheless, even on tissue samples that are hard to register,
such as the brain tumor samples included in the TEST dataset, a
rapid exploration of the parameters can lead to acceptable registration results with an accuracy level corresponding to about
four cell diameters.
DISCUSSION AND CONCLUSIONS
In this work, we showed that high registration accuracies can be
achieved with simple transformation models (affine and rigid),
even when registering VSs in the presence of non-linear deformations. The registration accuracy achieved with our approach
is suitable for biomarker colocalization analysis. Indeed, registration errors as low as 20 and 5 μm were frequently achieved for
the SERIAL and SIMPLE datasets, respectively, which approximately correspond to the diameters of a tumor cell and a tumor
cell nucleus, respectively. However, these low registration errors
on serial slides can only be achieved if histological structures are
present and conserved between adjacent slides (such as in
colonic tissue), else the registration errors may increase as
demonstrated on the TEST dataset. However, as shown with the
SIMPLE dataset including brain tissue samples, high registration
accuracy can be reached for tissue samples lacking in histological
structures by using VSs stained using our SIMPLE-like method.
This approach can still offer a good alternative to the more
limited double-staining method (see Introduction).
Compared with the results presented in Mueller et al,17 our
simpler, low-resolution registration step exhibits a comparable
error in terms of the RMSE. In contrast, the high-resolution
registration step cannot be directly compared because Mueller
et al17 estimated the high-resolution registration accuracy by
using the Jaccard and Dice coefficients on binarized images.
These coefficients must be regarded as relative measures for the
comparison of different registration methods applied on the
same FOV pair. However, these results show that, for magnifications higher than 10×, simple transformations (ie, local translations) can perform almost as well as more complex, deformable
transforms. This observation agrees with our results that showed
that rigid transforms are indeed accurate at 20× magnification.
Compared with other frameworks, elastix provide a better registration than the TurboReg software used in Metzger et al,12
which exhibited much larger registration errors. Indeed, on the
SERIAL dataset TurboReg exhibited a median error of 70 mm at
1×, as compared with 37 mm for our method. A median error
above 88 μm obtained at 20× magnification was reported for
prostate tissue in Metzger et al.12
Colocalization measurements on registered VSs can be carried
out by means of extensions of the colocalization indices usually
used to characterize the degree of overlap between two channels
in fluorescence microscopy images.40 In fluorescence microscopy, no registration is required because images are acquired on
the same sample with different wavelengths. The usual colocalization indices are overlap, Manders’ coefficients, and possible
extensions (eg, using rank-based intensity weighting41). In the
case of registered VSs, similar indices can be computed on tiles.
The tile sizes must be determined in relation to the registration
accuracy. At high resolution, colocalization on serial VSs can be
reasonably measured by using squared tiles 80 μm in length
because the corresponding cells in two registered FOVs can be
considered to be approximately 20 μm apart. On registered
SIMPLE-like VSs, this tile size can decrease to 20 μm or even be
10
directly computed after the low-resolution registration step on
tiles of 60–80 μm length in view of the good results already
obtained at this step. Figure 6 illustrates how to compute colocalization indices based on local staining measurements (via
staining area or intensity measurements, as usually employed for
characterizing global IHC staining31 42).
The simplicity of the transformation models and the
extremely parallel nature of the high-resolution registration step
enable the use of the method on large-scale studies involving
hundreds of patients. Such large-scale studies prevent the use of
CPs or SCs to evaluate the quality of a registration because it
would necessitate an enormous amount of annotation work. To
be fully efficient, an unsupervised approach, such as the one
proposed in the present study, needs a method to detect incorrect or suboptimal registrations by using only (unsupervised)
information extracted from the images, such as the value of the
similarity function S, which is indicative of image pair similarity.
Our observations on S values obtained after registration indicate
that certain threshold values could be determined (at low and
high resolution) to identify cases with insufficient registration
accuracy. These data should be confirmed on larger series of
registered image pairs.
Contributors Conceived the study objectives: IS, CD. Designed the methods and
experiments: XML, PB, CD. Contributed materials: LV, A-LT, LL, IS. Performed the
implementation and the experiments: XML, PB, Y-RVE. Analyzed the data: XML, CD.
Wrote the paper: XML, CD.
Funding This work was supported by the Télévie program of the ‘Fonds National
de la Recherche Scientifique’ (FNRS, Brussels, Belgium), the Fonds Yvonne Boël
(Brussels, Belgium), and the Fonds Erasme. The CMMI is supported by the European
Regional Development Fund and the Walloon Region. CD is a Senior Research
Associate with the FNRS.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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