ZEBRAFISH

Volume 11, Number 4, 2014

ª Mary Ann Liebert, Inc.
DOI: 10.1089/zeb.2014.1002

Automatic Zebrafish Heartbeat Detection

and Analysis for Zebrafish Embryos

Christian Pylatiuk,1 Daniela Sanchez,1 Ralf Mikut,1 Rüdiger Alshut,1 Markus Reischl,1
Sofia Hirth,2 Wolfgang Rottbauer,2 and Steffen Just 2

Abstract

A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized
zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and
imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat
intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual
detection of the heart rates of wild-type zebrafish embryos 36–120 h postfertilization and of embryonic hearts
with bradycardia and pauses in the cardiac contraction.

Introduction to avoid movement of zebrafish embryos during videoing.

Adding compounds that prevent embryo movement such as

D uring the last decade, the zebrafish (Danio rerio) has

emerged as an indispensable vertebrate model organism
in drug discovery1 and drugs known to cause QT prolonga-
tion; hence, bradycardia in humans shows similar effects in
zebrafish embryonic hearts.2 In addition, the zebrafish has
also emerged as a powerful and reliable model organism in
large-scale forward and reverse functional genetics ap-
proaches, which has led to the identification of novel disease
genes and pathomechanisms of cardiac disease. Several of
these zebrafish mutant lines or morphants have become es-
tablished vertebrate models for defined human cardiac dis-
orders such as cardiomyopathies or arrhythmias.3–5 In this
context, the knockdown of the short-stature homeobox gene 2
(shox2) results in severe cardiac dysfunction and bradycardia
in the zebrafish model6 that is mediated by a loss of Islet1 in
cardiac pacemaker cells,5 establishing Shox2- and Islet1-
deficient zebrafish as models for studies of human sick sinus
syndrome. The screening for bioactivity of small molecules
and their therapeutic potential to modulate heart rate requires
methods for high-throughput screening (HTS). Up to date, an
assessment of the heart rate is often practiced manually by
counting heartbeats from slow motion replay of videotape
recordings, what is neither practical for large numbers of
hearts to be analyzed nor is it accurate when determining
beat-to-beat intervals as needed for detection of arrhyth-
mias.7 Existing software for semiautomated analysis of
heartbeats7–13 has several limitations. One is that the embryos
have to be anesthetized by adding Tricaine11,14 or MS-22212
anesthetics, sedatives, or neuromuscular junction blockers
can alter the heart rate.8 A second limitation is that the
embryos have to be fixed and oriented manually in an aga-
rosegel before the video recording,11,14,15 which is a time-
consuming and tedious task. Additionally, the oxygen supply
in agarose can differ from the one in water.15 Another method
for automatic detection of heartbeat can only be performed by
using a transgenic zebrafish line Tg(cmlc2:GFP) that ex-
presses the green fluorescent protein (GFP) in the myocar-
dium.8 Recording of the electrocardiogram of zebrafish
hearts has also been demonstrated.16 However, the method is
technically complex and not suited for HTS.
To improve the throughput of cardiotropic small-molecule
screens, methods to automatically evaluate the heart rate and
arrhythmias from video recordings of nonanesthetized and
nonfixed zebrafish embryos are needed.
Materials and Methods
Zebrafish strains
Wild-type and transgenic zebrafish were maintained ac-
cording to Westerfield.17 Eggs were collected from pairwise
and batch crossings. Morpholino antisense oligonucleotide-
mediated gene knockdown of Shox and Islet was performed,
as described previously.5 For the morpholino injection pro-
cedure, the TE4/6 wild-type strain was used. At 24 h post-
fertilization (hpf ), the embryos were treated with 0.003%
PTU (1-phenyl-2-thiourea) to prevent pigment formation.

1
Institute for Applied Computer Science (IAI), Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany.
2
Department of Internal Medicine II, University of Ulm, Ulm, Germany.

379
380 PYLATIUK ET AL.

Additionally, the transgenic strain Tg(cmlc2:GFP) that ex-
pressed GFP exclusively in the myocardium8 was used.
The present study was performed under institutional ap-
provals that conform to the Guide for the Care and Use of
Laboratory Animals published by the US National Institute of
Health (NIH Publication No. 85-23, revised 1996).
Imaging setup
Two different imaging setups were used: First, for evalu-
ation of the heartbeat detection rate, series of overview im-
ages of the front quarter of zebrafish embryos were
automatically recorded with a frame rate of 30 frames per
second by using an automated microscope.18 Videos were
captured for 15 s. The image size was 640 · 480 pixels and
parts of the head and yolk sac were displayed next to the heart
of the zebrafish embryos on the images. Each image was
labeled with a timestamp and the corresponding coordinate of
the 96-well plate. Recordings were performed at 28C. Em-
bryos that moved during video recording were discarded.
Second, an inverted microscope (Leica DMIL LED) was
used in combination with an objective of 20 · magnification
to image the transgenic fluorescent strain and half of the
Shox2-MO and Isl1-MO zebrafish embryos. Videos of the
heart region were recorded with a digital camera (Leica DFC
400) and the imaging software Leica Application Suite V3.7.
The original image resolution of 1392 · 1040 pixels was re-
duced to 640 · 480 pixels to decrease the time for image
analysis. The frame rate was 30 frames per second and videos
were captured for 30 s. These zebrafish embryos were ori-
ented manually in 2.5% methylcellulose on a slide and re-
cordings were performed at room temperature (21C).
Heart detection by digital motion analysis
Two different movement detection algorithms written in
Matlab (The MathWorks, Inc., Natick, MA) have been
combined to accurately detect the zebrafish heart as the re-
gion of interest (ROI), as shown in Figure 1. First, all images
of a series (Fig. 1A) are transformed in the CIE 1976 (L*, a*,
b*) color space (CIELAB standard of the International
Commission on Illumination). The CIELAB includes all
perceivable colors and its coordinates are based on a cube
root transformation of the color data to avoid light intensity
influence. In this color space, an image is represented by three
matrices: brightness, a green-red, and a blue-yellow axis.
Then, the change of brightness of two successive images is
compared and the absolute difference between two digital
images represents pixels, whose intensity has changed, thus
indicating movement (Fig. 1B). All images are compared. As
each result is different from one frame to the other, to acquire
a unique region of the heart, all single segmentations are
summed up (Fig. 1C). By using a threshold value method and
morphological operations for Figure 1C, irrelevant move-
ments are removed and a unique area is found (Fig. 1D). The
boundaries of the detected ROI are depicted in the original
image (Fig. 1E).
The second algorithm aims at identifying red areas within
the zebrafish, which correspond to accumulations of eryth-
rocytes in the zebrafish heart. During a heart contraction
cycle, the red area within the heart’s ventricle alternately
increases in the diastole and decreases in the systole. This
area is isolated from the digital image by a combination of
different segmentation operations. As red is the feature of
interest, the green-red axis is selected (Fig. 1F). By a
threshold value method and a median filter, pixels with the
most relevant information are left (Fig. 1G). Several mor-
phological operations, such as dilatations and erosions, are
implemented to remove small particles and obtain a non-
interrupted area (Fig. 1H). This procedure is implemented in
each frame and the results are summed up as in the previous
method and can be used as a mask across the original image to
show the heartbeat region of a frame (Fig. 1I).
Both methods for detection of the zebrafish heart are
combined using an AND logical operation (Fig. 1J). How-
ever, in case an image sequence does not present red colors
(like most of the videos of the Islet1 and Shox2 morphants
used in the experiment), the color segmentation, evidently,
does not yield any results. Hence, the heartbeat area is de-
termined only by the motion detection algorithm. In both
cases, if more than one object results as the heartbeat area, the
largest object is chosen as the expected heartbeat area.
Heartbeat detection
The standard deviation of the ROI in each frame is cal-
culated. The calculation of this feature yields a time series

FIG. 1. Heart region detection by combining two methods: upper row: method to detect change of pixel intensity: (A)
native digital image, (B) differential image of two successive images, (C) sum of all differential images, (D) image after
threshold adjustment, erosion, and dilatation, (E) original image with marked detected region of interest (ROI). Lower row:
method to detect the heart region by color segmentation: (F) a matrix of the CIELAB color space, (G) image after threshold
adjustment, (H) image after median filtering, erosion, and dilatation, (I) original image with marked detected ROI after
masking and summing all detected ROIs, (J) original image with combined detected ROI.
AUTOMATIC ZEBRAFISH HEARTBEAT DETECTION AND ANALYSIS
with a periodic behavior, which corresponds to the periodic
heartbeat. From this periodicity, a frequency can be deter-
mined. For this, a function of the Matlab toolbox Gait-CAD
[http://sourceforge.net/projects/gait-cad/files/] was applied.
This function determines the relevant local maxima of a
curve and their respective times of occurrence. The period of
time between two maxima corresponds to the beat-to-beat
interval of the zebrafish heart. The median value for all de-
tected beat-to-beat intervals is calculated and its reciprocal
value (heart frequency) is also computed and multiplied with
60 representing the heartbeats per minute (bpm). All detected
beat-to-beat intervals and the heartbeat rate are saved to-
gether with a graphic display of the data (Fig. 2) and an image
of the identified ROI (Fig. 3 and Supplementary Video;
Supplementary Data are available online at www.liebertpub
.com/zeb). The data can be imported into spreadsheet pro-
grams for further analysis. To improve the ease of use, we
have implemented the Matlab software into LabVIEW (Na-
tional Instruments, Austin, TX) that provides a Graphical
User Interface.
381
FIG. 2. Example of detected
heartbeats of an Isl1-deficient em-
bryo with frequent pauses in the
cardiac contraction. The graph il-
lustrates the dynamic change of
pixel intensity in the ROI. Peaks
indicate individual heart contrac-
tions (highlighted by asterisks). In
this example, the first pause (P)
occurs after nine heartbeats and
lasts for 2.2 s.
Measurement of heartbeat regularity
According to the guidelines of the European Society of
Cardiology and the North American Society of Pacing and
Electrophysiology,19 the root mean square of successive
differences (RMSSD) is a time-domain method that can be
applied for the short-term assessment of heart rate variability
(HRV). The RMSSD is calculated by the square root of the
mean of the sum of the squares of the successive differences
between adjacent beat-to-beat (RR) intervals:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
n1
+i ¼ 1 (RRi  RRi þ 1 )2
RMSSD ¼
n1
Where n indicates the total number of RR interval terms
and i the duration of the i-th interval.
There is a lack of normative data for short-term measures
of HRV, but elevated RMSSD values indicate arrhythmic
events.19 To compare RMSSD values of two different mor-
phants with sinus bradycardia and frequent pauses in cardiac
FIG. 3. Different orienta-
tions of zebrafish and detected
ROI: (left): dorsoventral posi-
tion, (right): lateral position.
382 PYLATIUK ET AL.

Table 1. Results of Automatic Heartbeat Detection

Object Age of development Detection rate Correlation with visual counting
WT embryos 36–120 hpf 96% (204/213) R = 0.99
WT embryos in lidocaine 50 and 100 lg/mL 60–72 hpf 95% (88/93) R > 0.97
Transgenic fluorescent strain 72 hpf 100% (14/14) R = 0.99
Morphants with bradycardia and frequent 60–72 hpf 95% (69/73) R = 0.98
pauses in the cardiac contraction
Rates of automatic heartbeat detection and correlations with visual counting are given for wild-type (WT) zebrafish embryos, WT
embryos in two different concentrations of the cardiotoxic drug lidocaine, a transgenic fluorescent strain, and two different morphants.

contraction (Islet1-Mo and Shox2-Mo) with a wild-type
control group, the RMSSD values were averaged for each
individual and standard errors were calculated. These mean
values were tested for significance in comparison to the
control group.
Validation of the heartbeat detection rate
Validity of the method for automatic detection of the heart
rate was performed by a comparison with visual counting of
slow-motion videos. Wild-type zebrafish embryos 60–72 hpf
were dispensed in a 96-well plate, and volume was leveled to
50 lL with an E3 buffer solution containing lidocaine at
two different concentrations (50 and 100 lg/mL). Embryos
were exposed for 15 min before a heart rate assessment.
Additionally, heartbeats of two different morphants were
analyzed and compared with controls. Shox2 deficiency in-
terferes with the pacemaking function in zebrafish em-
bryos.10 Injection of Shox2-MO leads to sinus bradycardia,
and Isl1-deficient embryos also exhibit frequent pauses in the
cardiac contraction.
Statistical analysis
Statistical analysis was performed with MatLab and Mi-
crosoft Excel. Comparisons between means of two groups
were performed using the unpaired t test. Differences were
considered significant at p < 0.05. Correlations between two
datasets were calculated with Pearson’s correlation coefficient.
In this case, R = 1 is a total positive correlation, R = 0 is no
correlation, and R = - 1 is a negative correlation.
heart rate of embryos treated with lidocaine 50 lg/mL was
152.2 bpm (SD: 12.2 bpm) and lidocaine 100 lg/mL was
133 bpm (SD: 8.3 bpm), respectively. Moreover, the beat-
to-beat interval variability increased as shown by an increase
in RMSSD values in one third of the treated embryos. Fur-
thermore, videos from supplementary material given in other
publications11,16 were analyzed. In all cases, the heart rate
was automatically detected and significant decreases were
found in treated embryos.
On first pass, the method scored 100% heart rates of the
fluorescence videos from heartbeats of a transgenic strain
[Tg(cmlc2:GFP)] that expressed the GFP exclusively in the
myocardium.8 Additionally, videos of fluorescent embryonic
zebrafish hearts from supplementary material given in20 were
also detected automatically.
Heartbeats were also detected automatically in videos of
two different zebrafish morphants with sinus bradycardia and
frequent pauses in cardiac contraction. The mean heart rate
was the slowest in the Islet1-MO group with 73.1 bpm
( – 16 bpm), followed by the Shox2-MO group with 95.5 bpm
( – 11.1 bpm), whereas it was 140.7 bpm ( – 11.8) in the control
group. A comparison of the mean RMSSD values from con-
trols with those of Shox2 morphants exhibited significant dif-
ferences ( p = 0.0001). Correspondingly, this also applies for a
comparison of the mean RMSSD values from controls with
those of Islet1 morphants ( p = 0.0002) (see Fig. 4).

Results and Discussion

The heartbeat detection rate of the software was evaluated
with different wild-type (WT), induced morphants, and
transgenic zebrafish embryos at 36–120 h postfertilization
(hpf ) (Table 1).
Although the orientation of the zebrafish was random be-
tween dorsoventral to lateral, in 95% or more of all videos,
the heartbeat was detected automatically by our software and
the heart rate determined by the algorithm correlated highly
with the result found by visual counting. Repeated mea-
surements of the heart rate with our method in the same
animal were in the range of 2%. In addition, the detection
method was validated by adding a known cardiotoxic drug,
the sodium channel blocker lidocaine, which induces bra- FIG. 4. Mean root mean square of successive differences
dycardia in zebrafish.2,20 A significant decrease in heart rate (RMSSD) values and standard errors of Islet1-Mo, Shox2-
with increasing concentrations of lidocaine was found Mo and the control group. ***Indicates highly significant
( p < 0.01). The mean heart rate in untreated zebrafish em- differences between the RMSSD values of the control group
bryos 72 hpf was 170.6 bpm (SD: 13.9), whereas the mean and those of the morphants ( p < = 0.001).
AUTOMATIC ZEBRAFISH HEARTBEAT DETECTION AND ANALYSIS
The method described here demonstrates the feasibility of
fully automatic analysis of zebrafish embryo heartbeats for
application in small-molecule HTS or in toxicological assays.
However, a number of factors that can influence the auto-
matic detection have been identified. These are as follows:
 Movements of the zebrafish.
 Movement of erythrocytes in large blood vessels such
as the aorta or the vena cava.
 Strokes of the pectoral fins in the first second of the
video, where the ROI is identified.
 High-image resolution ( > 640 · 480 pixels) signifi-
cantly slows down the detection procedure and reduces
the number of sequential images that can be analyzed.
 Low-image resolution ( < 160 · 120 pixels) reduces the
number of image information and consequently the
number of correctly identified ROIs.
 Pigmentation of the zebrafish can superimpose the heart
region and consequently no heart movement will be
detected.
At the moment, only heart rate and beat-to-beat variability
can be assessed automatically. More detailed assessments of
cardiac function, such as automatic analysis of the ejection
fraction, remain challenging. However, the development of
automated assays will accelerate both the search for active
compounds that may milder cardiac diseases and the identi-
fication of toxicological levels of compounds. We are cur-
rently applying the technology to zebrafish embryos in both
applications.
Acknowledgments
Funding was provided by the Helmholtz Association pro-
gramme ‘‘BioInterfaces: Molecular and Cellular Interactions
at Functional Interfaces’’ and by the European Union (EU
COST Action BM0804: ‘‘EuFishBioMed’’).
Disclosure Statement
The authors declare that they have no competing financial
interests.
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Address correspondence to:
Prof. Christian Pylatiuk
Institute for Applied Computer Science (IAI)
Karlsruhe Institute of Technology (KIT)
Hermann-von-Helmholtz-Platz 1
76344 Eggenstein-Leopoldshafen
Germany
E-mail: pylatiuk@kit.edu