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Automated comet analysis

Article in Cytometry March 1999
DOI: 10.1002/(SICI)1097-0320(19990201)35:23.0.CO;2-9 Source: PubMed

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r 1999 Wiley-Liss, Inc.

Cytometry 35:134144 (1999)

Automated Comet Assay Analysis

W. Bo
cker,* W. Rolf, T. Bauch, W.-U. Mu
ller, and C. Streffer
Institut fu
r Medizinische Strahlenbiologie, Universitatsklinikum Essen, Essen, Germany
Received 20 June 1997; Revision Received 26 August 1998; Accepted 29 September 1998

Background: Recently the comet assay or single-cell

gel electrophoresis assay has been established as a
sensitive method for the detection of DNA damage and
repair. Most of the software now available to quantify
various parameters for DNA damage requires the interaction of a human observer. In this report, we describe an
automated analysis system that is based on self-developed
software and hardware and needs minimal human interaction.
Methods: The image analysis is divided into two parts: 1)
automatic cell recognition and comet classification and 2)
quantification of desired comet parameters. Image preprocessing, segmentation, and feature classification were
developed with algorithms based on mathematical morphology. To enhance evaluation speed, we have introduced parallel processing of data under the Windows NT
operating system (Microsoft Corporation, Redmond, WA).
Use of an analogue real-time autofocus unit (Bo
cker et al.:

Phys Med Biol 1997;42:19811992) allows for faster analysis.

Results: Our recognition software shows a sensitivity of
95.2% and a specificity of 92.7% when tested on test
samples from routine work with DNA damage by low-dose
radiation (02 Gy). The parallel hardware and software
concept enables us to analyze 100 comets on one slide in
less than 15 min.
Conclusions: A comparison of measurements made on
the same samples by manual and automated analysis
systems revealed that there are no significant differences.
The slope of the dose-response curves and the repair
kinetics are very similar and demonstrate that automatic
comet assay analysis is possible. Cytometry 35:134144,
1999. r 1999 Wiley-Liss, Inc.

The comet assay permits the quantification of DNA

damage (e.g., after x-ray exposure) in individual cells on
the basis of the migration of DNA structures in an electrical
field. As a result of this migration, comets are formed, and
the amount of DNA in the comet tail can be a measure of
damage (1) (Fig. 1).
stling and Johanson (2), who first described this assay
O
nearly 13 years ago, measured the fluorescence intensity of
stained DNA with a microscope photometer by using a
small diaphragm in the center of the comet head and at a
certain distance in the direction of DNA migration. The
ratio of both intensities was considered to be a measure of
the amount of DNA damage. Subsequently, other investigators (1,316) reported an improvement in the method by
the introduction of image analysis and additional parameters such as comet length and comet tail distances.
Usually, manual analysis of the comet assay consists of
three parts (17): 1) selection of the cell nucleus to be
measured, 2) image segmentation with interactive thresholds, and 3) comet quantification. Despite its ease, manual
comet analysis has some crucial disadvantages. There is a
need for experienced personnel (the first two points
involve several sources of errors). The variable skill of
different investigators makes a direct comparison of their

data difficult; as investigators become tired, their recognition and concentration abilities may decrease, resulting in
increased errors.
We have therefore developed an automated comet-assay
technique that allows rapid and autonomous evaluation of
the relevant comet assay parameters. The image analysis is
divided into two parts: 1) automatic cell recognition and
comet classification and 2) quantification of desired comet
parameters. Image preprocessing, segmentation, and feature classification were developed with algorithms based
on mathematical morphology. Quantification of fluorescence intensity was carried out after application of shading correction on each comet image.
To enhance the processing speed, the comet scoring
procedure was divided into three parallel software tasks
distributed to three different hardware components: 1)
searching and focusing, 2) grabbing and administration,
and 3) recognition and analysis (Fig. 2). The patternrecognition classifier was tested with routine biological

Key terms: DNA damage; comet assay; automated analysis; image processing; mathematical morphology; quantitative fluorescence microscopy

*Correspondence to: Wilfried Bo

cker, Institut fu
r Medizinische Strahlenbiologie, Universitatsklinikum Essen, Hufelandstr. 55, 45122 Essen, Gemany.
E-mail: wilfried.boecker@uni-essen.de

AUTOMATED COMET ANALYSIS

135

FIG. 1. Camera gray-level image of human lymphocyte comets captured with an intensified target camera. Left: Control sample. Right: Irradiated with
2 Gy.

material, e.g., human lymphocytes, analyzing several thousands of comets with different DNA damage status. Both
the sensitivity (95.2%) and specificity (92.7%) are quite
excellent and allow investigations of cell DNA damage and
its repair (DNA repair kinetics) at low doses (02 Gy).
Automated comet analysis following our strategy is comparable in speed with manual analysis. The same microscope
slides of comets of human lymphocytes were measured
automatically as well as manually to compare both methods for the determination of dose-response curves (02
Gy) as well as for DNA-repair kinetics (DNA damage after 2
Gy; repair time 0180 min). All results obtained from
automated comet analysis show good agreement with
those obtained from manual scoring.
MATERIALS AND METHODS
Cell Preparation and Gel Electrophoresis
The experiments were carried out with unstimulated
human peripheral blood lymphocytes taken from healthy
donors. Cells were isolated by using the Ficoll-Hypaque
technique (18).
The cells were kept in tissue culture flasks (25 cm2;
Becton Dickinson) in incubators (Heraeus, Hanau, Germany) at 37C, 5% CO2 and 90% relative humidity. For
each sample, 100 l of a cell suspension were prepared
containing 10,00060,000 cells (optimal cell number per
sample: 50,000 cells/100 l). The cells were irradiated
with a Stabilipan x-ray machine (Siemens, Erlangen, Germany) at 15 mA and 240 kV at a dose rate of 1 Gy/min. In
the repair experiments the samples were incubated for
various times after irradiation.
In the first preparation step of the comet assay technique, regular microscope slides were precovered with
500 l of 0.1% agarose (in phosphate-buffered saline [PBS];
Serva, Heidelberg, Germany) and dried at 45C on a hot
plate for 1 h. The cell samples (100 l) were mixed with

500 l of 0.75% agarose (in PBS, 45C) and spread on the

agarose-coated slides. After gelling at 4C on a cooling
plate for 5 min, the cells were lysed in a solution of 2.5%
sodium dodecyl sulfate (SDS; pH 9.5) (87 mM SDS, 34 mM
N-lauroyl-sarcosine, 25 mM EDTA; Sigma, Deisenhofen,
Germany) for 15 min at 18C. After a 5-min wash in
distilled water at 18C, the electrophoresis was carried out
in TBE-buffer pH 8.4 (117 mM Tris, 91 mM Borate, 2.5 mM
EDTA) at 2.5 V/cm and 16 mA for 4 min at 10C by using a
modified flat-bed electrophoresis apparatus with electrode
spacing of 6 cm (FBE-3000; Pharmacia, Freiburg, Germany). To investigate the lower dose range (02 Gy), we
optimized the conditions of the electrophoresis in such a
way that some of the DNA of the control nuclei showed up
in the tail (see Fig. 7a and b). Thus, we were sure that the
conditions were sufficient to move damaged DNA into the
tail, so that even all comets of unirradiated cells had
noticeable tails (Fig. 1a). The gels were washed in distilled
water for 510 min at 4C and dried at 45C on a hot plate
for 1530 min. These slides could be stored in a humidified
container at 4C up to 2 weeks with only minor qualitative
changes. Before measurement, the microgels were rehydrated in distilled water for 8 min at room temperature.
The slides were then covered with 120 l of the staining
solution (2.5 105 M propidium iodide, pH 7.5; 1.2 g
per slide).
Instrumentation
Fluorescent comet images were taken with a Leitz
microscope (MPV II; Leitz, Wetzlar, Germany) with a
100-W mercury lamp and a 530560-nm band-pass filter
for excitation. The dichroic beam splitter reflected light
below 580 nm to the sample and passed light of longer
wavelength. A 590-nm long-pass emission filter transmitted only light with wavelengths longer than 590 nm to the
camera (filter block E2). For highly sensitive and fast

BOCKER ET AL.

136

FIG. 2. Overview of the three different software tasks (enclosed in dotted rectangles) for automated comet assay. 1: Searching and autofocus (upper left).
2: Grabbing and administration (middle left). 3: Recognition and analysis (lower left). The processing time of the respective tasks within the cycle is given
on the right. The searching and grabbing (s&g) cycle starts with xy movement (movement to a new image field of the slide) followed by autofocusing. On
finding bright objects within the image, the autofocus triggers the frame grabber to acquire the image, otherwise it triggers the xy stage to move to the next
field. After acquisition and shading correction, the image is placed on the so-called image stack. The recognition and quantification (r&q) cycle fetches an
image from the stack for further segmentation, feature extraction, classification and quantification. After completion, the cycle restarts with the next image.
It stops when either sufficient comets have been analyzed or the stack is empty (e.g., when the s&g unit cannot provide further images). This may occur
when the s&g cycle scans areas having no or only few comets. On the other hand, the s&g cycle stops if the image stack is occupied, so that the s&g cycle has
to wait until the r&q cycle has gathered a new image from the stack. Note: If the stack is neither occupied nor empty, both process cycles run independently
of each other and parallel in time.

(video cycle) image acquisition, we used an imageintensified target camera (Proxitronic HL1; Proxitronic,
Bensheim, Germany). The analogue video output was
interfaced to an IBM-compatible PentiumPro 200 computer with a built-in frame-grabber board (Matrox Pulsar,
Matrox Ltd., Dorval, Quebec, Canada). The captured
microscope images (250 microscope magnification, 25
Fluotar objective, numerical aperture 0.7) were sampled
with 768 576 pixels with an intensity quantization of
1,024 gray levels (10 bits).
Image Processing
Image processing mainly consists of three parallel software tasks (dotted rectangles in Fig. 2) distributed to three
different hardware components. To synchronize the process timing, the software tasks communicate with each
other via interrupt channels. After initialization, the searching and grabbing (s&g) part (running on the autofocus
hardware unit) starts by scanning the microscope slide. If
the autofocus task detects bright objects within the entire
field, an image is acquired on the frame grabber and stored
in a so-called image stack (in the memory of the PC host).
The time of this cycle (xy movement autofocusing
grabbing shading correction; Fig. 2, upper left) is 2.3 s.
Apart from this, images in the image stack are processed

and analyzed with the recognition and analysis (r&a) task

(running on the host central processing unit; Fig. 2, lower
right) independently of the s&g procedure. The process
starts by fetching an image from the stack. After segmentation, feature classification, and quantification (total process time 4.9 s) the results of calculated tail/head fluorescence are stored, thereafter starting the next cycle. The
process stops when either the stack is empty or enough
comets have been analyzed. The s&g process (Fig. 2, upper
right) is repeated unless the image stack is full or sufficient
comets have been analyzed. The parallel processing of
both cycles in combination with the common image stack
saves evaluation time with respect to the overall analysis.
The function of the image stack is that of a buffer, which
is advantageous in our implementation because of the
different process times of both cycles. When comet
density on the microscope slide is high, more images can
be acquired than analyzed (the s&g task is roughly two
times faster than the r&a task), so that putting all the
images on the stack avoids retardation of the s&g tasks. On
the other hand, when comet density is low, the s&g task
sometimes needs more than one cycle to locate an
adequate image field for acquisition. In these cases,
obtaining a new image may require more time than a
complete r&a cycle. As a consequence, gathering images

AUTOMATED COMET ANALYSIS

from the stack avoids retardation of the r&a task in such

cases.
Searching and Focusing
The positioning part of the automated comet analysis
consists of automated stage movement and automated
focusing, developed as an autonomous unit, permitting
automated x, y, z positioning without any requirement for
processing time of the host computer. All instructions
necessary for automated positioning are generated on a PC
plug-in multipurpose board (Multi-Lab 2h; Sorcus, Heidelberg, Germany) with an onboard X86-compatible processor, 512 kB of memory, and an on-board real-time multitasking operating system (OsX; Multi-Lab 2h; Sorcus).
Stage movement. The microscope slide was moved
with an automatic scanning stage for x and y positioning
(Marzhauser, Wetzlar, Germany) with a step size of 2.5 m
and a device for z positioning (Leitz) with a step size of
0.25 m. Four different scan modes (single-field scan, line
scan, meander scan, and spiral way scan) can be selected
by the operator at the beginning of automatic analysis. By
default, the start position of a scan is the center of the
microscope slide, but the operator is also able to define an
arbitrary start position. The step size of the scan is
dependent on the microscope magnification and was so
calculated that successive images belonged to adjacent
areas without any overlapping. The autofocus is triggered
for every new image.
Autofocusing. To develop a fast autonomous autofocus for fluorescence microscopy, we constructed an
electronic board (19) for the determination of edge
information and contrast measurements by using analogue
video processing (20). During the routine work, focusing
was completed within 1 s.
Grabbing and Administration
Startup procedure. Several startup procedures such
as automatic camera gain and black level adjustment,
shading calculation, and stepping motor reset have to be
carried out before a slide is scanned meanderlike over a
fixed part of the slide area. The automatic gain adjustment
is necessary to avoid saturating the camera as well as to use
the largest possible dynamic range of the image digitizer.
Furthermore, it must be ensured that the camera transmission characteristic is linear.
Stepping motor reset is essential for every slide, whereas
shading calculation and gain adjustment should only be
carried out after changes are made in the microscope
illumination conditions, e.g., after any readjustment of the
excitation lamp. For clarity, we distinguish between shading correction and shading calculation. The latter involves
the acquisition of the cameras black-and-white image,
subsequent low-pass filtering, and storage of the calculated
shading mask, whereas the former is carried out for every
acquired comet image to compensate for the photometrics nonlinearities by use of the shading mask.
In routine analysis, no significant change in illumination
is detectable during the first 500 h of excitation lamp
lifetime, thus necessitating no further gain adjustments or
shading calculations to provide almost identical experimen-

137

tal conditions. On the other hand, measuring comets

without readjustment of the camera gain could result in
saturation of the image digitizer, which must be avoided at
all times. In principle, saturation would be avoided completely if gain adjustment at the time of start-up is carried
out with the brightest comets from the whole measurement series. Obviously, this is not feasible in routine work
because 1) it cannot be guaranteed at start-up that a
particular slide possesses the brightest comet intensity and
2) we have no knowledge of comet brightness in advance.
We noticed, however, that comet heads of control
samples usually show the brightest fluorescence intensities compared with irradiated comets. Moreover, we
primarily focus our investigations on unstimulated human
lymphocytes, which are mostly in the G0/G1 phase, so that
the maximum value of fluorescence intensity (Imax) of the
respective comets varies less significantly than Imax values
obtained from asynchronous cells. In addition, we have
estimated variations of Imax occurring as a result of preparations and staining.
From various experiments investigating DNA damage in
human lymphocytes, we observed that the total variation
of Imax is less than 30%. Hence, for gain adjustment, this
variation is taken into consideration in such a way that the
maximum gray level of a control comet does not exceed
values above 70% of the maximum value of the digitizer
(dmax 1,024). This restriction of the dynamic range of the
digitizer is acceptable because the remaining gray-level
range is still large enough to provide sufficient sensitivity
and, moreover, it ensures that no saturation occurs.
It is also of some importance to avoid gray-level clipping
of faint comet areas (e.g., the right part of the tail) as the
result of an inappropriate camera black level. For precise
segmentation, the gray-level range of the transition between comet regions and background must lie within the
digitized gray-level interval. Optimal segmentation results
can be obtained if the black level is adjusted in such a way
that the mean gray-level of the image background gb
(calculated from the corresponding gray-level histogram)
is approximately 5% of gmax.
Before the regular start of the automated analysis, some
very bright comets of the control samples (these usually
exhibit the brightest fluorescence) need to be positioned
into a software window on the monitor. An automatic
procedure evaluates a certain gain, for which the highest
detectable gray-level value gmax does not exceed the
experimentally determined upper limit (70% of dmax,
gmax 700). This procedure is repeated for five different
comets at very different positions on the slide. Finally, the
gain of the brightest comet is used for the subsequent
analysis. The whole procedure lasts about 23 min. The
selection of very bright comets at different positions on
the slides as well as the restriction of gmax to 70% of dmax
ensures that the vast majority of comets analyzed automatically will not saturate the camera. In those very rare cases
in which saturation arises, such comets will be rejected
from further analysis.
Recognition and Analysis
For reliable results to be obtained, the measurement of
DNA damage must be restricted to intact comets; any

138

BOCKER ET AL.
B

FIG. 3. A: Comet structures obtained from human lymphocytes (irradiated with 2 Gy) after gel electrophoresis in a weak electrical field. The DNA of the
cell nuclei was stained with a fluorescent dye (propidium iodide). The image was taken with a fluorescence microscope by using an image intensified target
camera. The extent of the comet tail is dependent on the amount of cellular DNA damage. After staining the slides should be analyzed at the same day to
avoid degradation in image quality. Comets shown here were stained two days before analysis and stored in between in a refrigerator at 4C to demonstrate
the loss in image contrast and a degradation in comet shapes. Note: In contrast to this see Figure 1, where analysis was carried out 2 h after staining.
B: Three-dimensional-perspective fluorescence intensity distribution (in false-color presentation) of comets in A. Note: The comet tail runs into the plain of
the background. The image preprocessing must reliably detect the comet boundary to calculate the overall fluorescence intensity.

other kind of objects (subsequently termed as artifacts)

such as attaching and abnormal comets, staining artifacts
in the gel, etc., have to be rejected. This requires a
pattern-recognition software that is able to distinguish
between intact measurable comets and artifacts. Before
object classification, image segmentation must be carried
out to extract image objects from the image background.
Comet segmentation and feature classification.
Before segmentation, the camera image is filtered with
morphological alternating sequential filters to remove
acquisition noise (21) followed by a shading-correction
procedure (17,22). The first step in comet segmentation is
the transformation of the gray-level image into a binary
image. This step, which is one of the major sources of
errors, must be carried out as precisely as possible. Good
segmentation results can usually be achieved [watersheds
(23), histogram analysis, neural networks (24)] if the
object contrast is sufficiently high. Precise segmentation
becomes more difficult when the tail of the comet structure shows no contrast or only low contrast with respect
to the background. Common statistical techniques such as
histogram analysis, entropy maximization (24), and kmeans clustering, yield unsatisfactory results because of
the large background noise of the comet images. Contourbased procedures also fail because of the low contrast of
the comets.
Thus, we have developed a simple two-step procedure
for threshold calculation based on image-content analysis.
In the first step, a global threshold, t1, is determined by
counting threshold-dependent noise pixels in the binary
image. The assumption made here (and this holds for the
described image-acquisition system) is that the background of the camera image is contaminated with randomly distributed Gaussian pixel noise (Fig. 3). Depending
on the threshold selected, noise pixels from the image
background may appear in the thresholded image. Upon
threshold reduction, the binary image background becomes noisy and results in a drastic increase in the number

of objects (NO) (Fig. 4). Figure 5 shows a typical example

of the dependence of the NO on the threshold parameter.
The explosion-like increase in the NO with decreasing
threshold indicates the occurrence of noise pixels in the
corresponding binary image. A reproducible global threshold is found if the NO exceeds a predefined threshold
(NOt 300; Fig. 5).
In the second step, we have introduced an adaptive
threshold to improve the segmentation results. The procedure is known as threshold with hysteresis (25) or
threshold with reconstruction. Let I(x,y) be a gray-level
input image, O(x,y) a binary output image, and t1 and t2
two gray levels where
t1 denotes the threshold determined in the first step and
t2 is empirically set to t2 t1 5;
if I(x,y) t1 O(x,y) 1 (retained area)
if I(x,y) t2 O(x,y) 0 (rejected area)

if I(x,y) t2

O(x,y) 1 if points are

connected to the retained area (fuzzy area).

(O(x,y) 0 0 else

The effect of threshold with reconstruction is illustrated in Figure 6B. The darker regions of the comet image
are the contributions of global thresholding (step 1),
whereas the lighter regions around the comet structures
are due to the reconstruction procedure (step 2). The
combination of both steps enables us to extract the comet
areas more precisely without a significant increase in
background area (noisy area). Our experiments show that
it is not necessary to determine a global threshold (step 1)
for each input image. Routinely, the same global threshold
is used for the next nine incoming comet images, followed
by a renewed step-1 threshold procedure.
In addition to the binary image, a marker image is also
generated from the gray-level input image, including only
the white spots of true comets. This image (Fig. 6C) is later

AUTOMATED COMET ANALYSIS

139

FIG. 4. The gray-level input image (A) has been binarized with different thresholds (t) after an object-counting procedure (B). t 80, number of objects
(NO) 101. C: t 65, NO 307; D: t 55, NO 1,485. The occurrence of noise pixels is utilized to calculate an optimal threshold, which is essential for
the following quantification of fluorescence.

FIG. 5. Dependence of the number of objects

(NO) within the binary image on the selected
threshold. The exponential growth of NO at the
right tail of the distribution results from binarized
noise pixel (each isolated pixel is regarded as an
object). Noise pixel in the binary image indicates
that the threshold level reaches the image background value. Experiments with different comet
images reveal that a suitable threshold will be
found when the NO just exceeds 300. The
procedure begins with a sufficiently high threshold value estimated from the corresponding graylevel histogram (typical t 100) to guarantee
that NO calculation will begin at the right tail of
the maximum. Thereafter, the threshold is repetitively lowered while the NO value is being
calculated. Note: The decrease in NO at lower
threshold values is due to object merging.

used to reconstruct only comet areas with an intact comet

head (artifacts such as ragged comet tails sometimes show
similar morphological features but contain no highlighted
spots and will be therefore rejected during object recon-

struction). Thereafter, the binary comet image is filtered

with a morphological Opening and Closing to reject small
artifacts (Fig. 6D). Objects intersecting with the image
boundary are rejected with the so-called border-kill algo-

140

BOCKER ET AL.

FIG. 6. Main steps of image processing. The comet input image (A) is transformed into a binary image by using a threshold, which is previously calculated
by using the NO algorithm (B). Additionally, a second, improved segmentation step is necessary to extract the low contrast transition region between comet structure
and image background. This is carried out with an adaptive threshold called threshold with reconstruction. The dark gray-comet structure area in the figure is
segmented with the global threshold, whereas the additional comet areas, which are segmented due to the threshold with reconstruction, are labeled light gray. The
union of the two areas is used for further analysis (D). A marker image (C) obtained from a high threshold value (10% below the maximum gray-level) is used to
reconstruct only structures with an intact comet head, so that isolated tails, possibly occurring as a result to apoptosis are rejected. The boundary of the remaining
objects is smoothed with morphological thickening (E) to enhance the reproducibility of classification. The result of feature classification is shown in image
(F). Finally, two masks (head and tail mask) must be calculated (G) and the total gray-level intensities within the two areas are evaluated (H).

AUTOMATED COMET ANALYSIS

rithm (26) (Fig. 6F). The remaining objects are reconstructed (21) from the marker image shown in Figure 6C.
It is advantageous to smooth object boundaries (Fig. 6E) to
enhance the precision of feature classification as well as
the calculation of the comet head region. This is achieved
by use of the morphological thickening (26) algorithm.
Finally, the image is labeled and analyzed for several object
features.
Comet classification. The purpose of comet classification is the decision of whether a certain object within the
labeled image is a comet or an artifact. The necessary
information for classification is provided for by different
object features (feature extraction). Altogether, 20 different features (size parameters such as perimeter and area,
morphological parameters such as inertia and intercepts,
and intensity parameters such as gray-level mean and
gray-level variance) are calculated to create the feature
space, in which comet classification takes place. Each
object can be represented as a 20-dimensional vector in
which every component represents a specific feature. The
feature space is a multidimensional hyperspace with
dimension 20, where similar objects (vectors) build a
cluster. Before automatic analysis was carried out, we
created a comet and an artifact cluster within the feature
space obtained from feature vectors measured from a
sufficient large pool of training samples (500 intact comets
and 500 artifacts). During analysis, the software calculates
whether the certain feature vector belongs to the comet or
to the artifact cluster.
There are several procedures in pattern recognition (27)
and multivariate (28) analysis for cluster analysis. The
approach used in our development is splitting the feature
space into two 10-dimensional subspaces and carrying out
different classification procedures in both spaces one after
the other to optimize recognition performance. One
subspace, 1 (used for preclassification), includes sizedependent object features (area, perimeter, convex perimeter, moment in vertical direction, ferets in the vertical and
horizontal directions, and intercepts in four directions);
the other subspace, 2 (used for main classification),
includes shape and gray leveldependent features (compactness, roughness, number of holes, orientation, feret_max_angle, feret_min_angle, minimum, maximum, mean,
and stddev.- graylevel). Before the automatic analysis,
minimum and maximum values of each feature were
calculated from the training set for the 1 subspace and
stored on the computers hard disk. During cell inspection, an object is preclassified if each value of the 10
size-dependent features lies within its corresponding minimummaximum range; otherwise, it is rejected from
further analysis. Thereafter, main classification is carried
out for all preclassified objects by using the Mahalanobis
classificator (16) for shape and gray leveldependent
features in 2.
The Mahalanobis distance (d) of a certain object class
(; comet or artefact), is defined as
d (x i) (x i )T Cov1
i 2
(x

(1)

141
Cov1

denote the mean vector and the

where and
inverse covariance matrix of class calculated from the
feature vectors of the training set, x i the feature vector in
the subspace 2 of the preclassified object, and ()T the
transposed vector.
From the training set, we calculated the mean vectors
and the covariance matrixes of the artifact and the comet
cluster, respectively, in the subspace 2 . These quantities
could also be calculated before regular comet analysis and
were also stored.
During analysis, the main classification step involves the
calculation of two Mahalanobis distances for each preclassified object. If the Mahalanobis distance, dcomet, of the
objects feature vector to the comet cluster is smaller than
the Mahalanobis distance dartifact to the artifact cluster, the
object is classified as comet; otherwise it is classified as
artifact. Finally, all classified comets are used for subsequent DNA-damage quantification.
DNA-damage quantification of classified comets.
Quantification of classified comets is based on the assumption that the acquired image is a superposition of the
comet fluorescence intensity distribution and a homogeneous fluorescent image background Ib (Fig. 3b). This
hypothesis was supported by three-dimensional laserscanning investigations recording chromatin distributions
inside the comet (17). It was found that the comet depth
(the extension along the optical axis) is small (5 m)
compared with gel thickness (100 m). Furthermore,
background fluorescence due to nonspecific staining is
comparatively homogeneous within the image background
and under the comet area, so that the above hypothesis
holds for the preparation technique described in this report.
As a consequence, the calculation of tail-to-head fluorescence intensity ratio must be performed after subtraction
of background fluorescence (Ib) (17). The gray level of the
background intensity is given by the corresponding threshold (T2).
Comets classified as positive are used as a binary mask
for the shading-corrected input image to calculate the
integrated gray-level intensities of the respective comet
area (Fig. 6G). Furthermore, a circular region (comet head
region) determined from the corresponding feret features
is used to evaluate the integrated gray-level intensities
within the comet head (Fig. 6H). Calculation of the
integrated gray-level intensity (Iob.) is carried out by using
the formula
gmax

Iob.

g hist

ob.

(g),

(2)

gT2

where histob denotes the gray-level histogram of the

corresponding object (ob.; comet area or comet head
area), T2 denotes the threshold value of the segmented
comet, and gmax denotes the maximum gray level within
the comet area.
Comet tail intensity (Itail) is given by subtraction of
comet head intensity (Ihead) and comet intensity (Icomet)
(17). The ratio (R) of both intensities (Itail/Ihead ) is a
potential parameter expressing DNA damage and is calcu-

BOCKER ET AL.

142

FIG. 7. A: Comet frequency distributions versus the DNA damage parameter tail/head obtained from manual analysis for controls and human lymphocytes
irradiated with 2 Gy. Note: The shift in tail/head ratio with respect to both distributions is dependent on DNA damage. B: Comet frequency distributions
versus the DNA damage parameter tail/head obtained from automatic analysis by using the same samples as in A.

lated for each comet classified:

R

Icomet Ihead
Ihead

(3)

Measurements. To estimate the quality of automated

analysis, a comparison of manual and automatic scoring
was carried out on 100 comets by using the same microscope slides. Manual analysis was carried out with a
separate image-analysis system by using comet analysis
software (1,17) established in our laboratory for routine
work for more than seven years. The order of measurement (manual-automatic, automatic-manual) of each slide
was alternated to randomize measurement errors due to

fading, and other photochemical processes. In addition,

we had previously observed that scoring a slide twice had
no significant effect on the results (unpublished data).
Figure 7A (manual) and B (automatic) show the comet
frequencies versus the DNA damage parameter tail/head
ratio for controls and 2 Gyirradiated samples, respectively. The histograms of comet frequencies from irradiated samples show a shift in this DNA damage parameter
toward higher values compared with the controls. The
shifted values in both figures represent the effect of
induced DNA damage (due to irradiation) and are very
similar for manual and automatic analysis. For the comparison of the different measurements, mean values are
calculated from the respective histograms (calculation of

AUTOMATED COMET ANALYSIS

dose-response curves, repair kinetics), (10,11). The histograms themselves supply useful information about the
spread of DNA damage values within one sample, and the
comet frequency distributions often differ for different cell
lines (1).
It is obvious that in all cases automatic analysis shows a
slightly higher DNA damage parameter compared with the
corresponding manual value due to systematic differences.
The main reasons for this bias may be the different
hardware (frame grabber) used for automatic and manual
analysis as well as the different image analysis to compute
DNA damage. On the other hand, the slopes of both
analyses (automated and manual) are very similar for
dose-response estimation as well as for repair kinetics. The
tail/head mean values were calculated for each comet
histogram sample and are presented as irradiation doseresponse curves (Fig. 8A) and DNA-repair kinetics (Fig.
8B). The spread in the histograms (Fig. 7) is mainly a result
of two different effects. First, the lymphocytes consist of
subpopulations (B and T cells) that differ considerably in
radiosensitivity (29). Because of the large mean lifetime of
the lymphocytes (several months), cell age may also
influence the DNA damage parameter. Second, the time of
lysis may affect the results. We observed (unpublished
data) that an increase in lysis time results in an increase in
the DNA-damage parameter. Therefore, it is necessary to
measure at least 100 single cells to achieve reliable mean
values.
Classification and quantification errors. The determination of classification decisions (7.3% false-positive
[specificity, 92.7%]; 4.8% false-negative, [sensitivity, 95.2%])
was processed for a test pool (slides from routine work) of
500 comet images obtained from human lymphocytes.
This included controls as well as irradiated samples. Most
of the false-positive decisions involved abnormal comets
due to apoptosis, whereas most false-negative decisions
were due to very small and faint fluorescent comet
structures.
The most relevant quantification error concerning the
DNA-damage parameter arises from the definition of the
comet area (threshold dependent) and the head area (feret
diameter dependent). The image-processing steps were
developed in such a way as to minimize both sources of
error. The total error of both procedures was estimated
from tail/head ratios obtained from 10 images of the same
comet by repetitive image acquisition (1 image per second), so that effects due to fading could be neglected. The
difference in ratio calculation was about 2.5%. Moreover,
we changed the position of the comet and repeated the
above procedure. Under these conditions, the difference
in ratio calculation was about 6.7%, although shading
correction was carried out.
DISCUSSION
We have described a self-developed image-analysis system that has been optimized for automatic comet assay
analysis. The image processing is divided into a quantification part with necessary shading correction and a recognition part for the identification of comets. The stage

143

FIG. 8. A: Comparison of dose-response curves obtained from manual

and automated analysis. The experiments were carried out with human
lymphocytes irradiated in the range of 02 Gy. The ordinate shows the
DNA damage as mean values of 100 individual comets tail/head ratios for
each data point. For better comparison, the control values (mean values
for 0 Gy) are subtracted from the corresponding tail/head ratios for both
slopes. Mean tail/head ratios of unirradiated lymphocytes were found to
be 22.5 (manual analysis) and 24 (automatic analysis) because of the
preparation conditions used for low-dose estimation (see Materials and
Methods). B: Comparison of repair kinetics obtained from manual and
automated analysis. The experiments were carried out with human
lymphocytes, that had been irradiated with 2 Gy. The radiation induced
DNA damage (tail/head ratio) measured immediately after exposure to 2
Gy was corrected for the controls (0 Gy) and set to 100%. The ordinate
shows the percentage damage in relation to this initial damage remaining
after various times of repair. Each data point represents the mean value of
tail/head ratio of the corresponding comet frequency distribution, which
was calculated from 100 individual comet measurements. For better
comparison, the tail/head ratio is normalized to the corresponding control
value.

movement and the autofocus work autonomously with

respect to the image processing and analysis; this allows
for simultaneous multitasking procedures of the scoring
process. While analyzing a grabbed image it is possible to
capture other images. Because image processing is not
finished, each grabbed image is stored on a so-called image
stack in the meantime working as FIFO (first in, first out).
Following this strategy, we could reduce the scoring time
dramatically, so that automatic analysis is as fast as (in
some cases even faster than) manual analysis. The time of
recognition and analysis of a single image (which may

BOCKER ET AL.

144

contain several comets) is carried out in less than 5 s. The

parallel hardware and software concept enables us to
analyze 100 comets on one slide in less than 15 minutes.
For quantification of DNA damage, we used the comet
tail/comet head fluorescence intensity ratio. In addition,
the quantification procedure also allows the evaluation of
other commonly used comet assay measurement parameters such as tail moment, tail length, comet length, and
comet moment to compare differences in the results.
However, calculation of these values as well as the
corresponding measurements are not presented in this
report. In a previous study we showed that the tail/head
ratio is a very sensitive parameter (17). The evaluation of
complete dose-response curves is carried out within a few
hours. A comparison of measurements made on the same
samples by manual and automated analysis systems has
revealed that there are no significant differences. The
slope of the dose-response curves and the repair kinetics
are very similar and show that automatic comet assay
analysis is possible.
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