2236 IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 37, NO.
10, OCTOBER 2018
3-D + t Human Sperm Flagellum Tracing
in Low SNR Fluorescence Images
Paul Hernandez-Herrera, Fernando Montoya, Juan Manuel Rendón-Mancha,
Alberto Darszon, and Gabriel Corkidi
Abstract — Tracing tubular structures from biomedical
images is important for a wide range of applications. Partic-
ularly, the spermatozoon is an essential cell whose flagella
have a tubular form. Its main function is to fertilize the
egg, and the flagellum is fundamental to achieve this task
which depends importantly on the dynamics of intracellular
calcium ([Ca2+ ]i ). Measuring [Ca2+ ]i along the flagellum in
3-D is not a simple matter since it requires: 1) sophisti-
cated fluorescence imaging techniques dealing with low
intensity and signal to noise ratio (SNR) and 2) tracing
the flagellum’s centerline. Most of the algorithms proposed
to trace tubular structures have been developed for multi-
branch structures not being adequate for single tubular
structures with low SNR. Taking into account the prior
knowledge that the flagellum is constituted by a single
tubular structure, we propose an automatic method to trace
and track multiple single tubular structures from 3-D + t
images. First, an algorithm based on one-class classifica-
tion allows enhancement of the flagellum. This enhanced
3-D image permits guiding an iterative centerline algorithm
toward the flagellum’s centerline. Each sperm is assigned
an ID to keep track of it in 3-D + t. Our algorithm was
quantitatively evaluated using a ground truth 564 semi-
manual traces (six 3-D + t image stacks) comparing them
to those obtained from state-of-the-art tubular structure
centerline extraction algorithms. The qualitative and quan-
titative results show that our algorithm is extracting similar
Manuscript received February 2, 2018; revised May 8, 2018; accepted
May 18, 2018. Date of publication May 23, 2018; date of current version
October 1, 2018. The work of P. Hernandez-Herrera was supported
by the Consejo Nacional de Ciencia y Tecnología under Postdoctoral
Scholarship Grant 291142. The work of F. Montoya was supported by the
Dirección General de Asuntos del Personal Académico through
the Universidad Nacional Autónoma de México under Grant
CJIC/CTIC/4898/2016. The work of A. Darszon was supported in part
by the Consejo Nacional de Ciencia y Tecnología under Grant Fronteras
71 39908-Q, by the Dirección General de Asuntos del Personal
Académico through the Universidad Nacional Autónoma de México
under Grant IN205516 and in part by the National Institutes of Health
under Grant RO1 HD38082. The work of G. Corkidi was supported by
the Consejo Nacional de Ciencia y Tecnología under Grant 253952.
(Corresponding author: Gabriel Corkidi.)
P. Hernandez-Herrera and J. M. Rendón-Mancha are with the
Centro de Investigación en Ciencias, Instituto de Investigación en
Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de
Morelos, Cuernavaca 62209, México (e-mail : phernandez7@uh.edu;
rendon@uaem.mx).
F. Montoya and G. Corkidi are with the Laboratorio de Imágenes y
Visión por Computadora, Departamento de Ingeniería Celular y
Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma
de México, Cuernavaca 62250, México (e-mail: jjfmn@uaem.mx;
corkidi@ibt.unam.mx).
A. Darszon is with the Departamento de Genética del Desar-
rollo y Fisiología Molecular, Instituto de Biotecnología, Universidad
Nacional Autónoma de México, Cuernavaca 62250, México (e-mail:
darszon@ibt.unam.mx).
Color versions of one or more of the figures in this paper are available
online at http://ieeexplore.ieee.org.
Digital Object Identifier 10.1109/TMI.2018.2840047
0278-0062 © 2018 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission.
See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
traces as compared with ground truth, and it is more robust
and accurate to trace the flagellum’s centerline than multi-
branch algorithms.
Index Terms — Sperm, flagellum, centerline, segmenta-
tion.
I. I NTRODUCTION
C OMMUNICATION between male and female gametes
is a requirement for sexual reproduction. This dialog
uses the language of changes in membrane ion permeability.
The main sperm functions, including motility, involve the
operation of ion channels and transporters. Sperm must swim
to fertilize the egg and the flagellum is key to this function,
which depends crucially on the concentration of intracellular
calcium ([Ca2+ ]i ) [1]. Understanding sperm swimming neces-
sitates knowledge about flagellar beating patterns (typical,
helical, hyper-helical, hyper-activated or chiral ribbons).
Therefore, determining how [Ca2+ ]i modulates the flagellum
movement is a fundamental endeavor requiring high spatial
and temporal resolution. This is not a simple matter since
complex fluorescence microscopy techniques are necessary
and they yield images that have low intensity and SNR (as
low as 1.8 and as high as 5.0) [2]. This problem is augmented
in 3D+t (three-dimensional space over time) sequences,
as human sperm flagella beat at an average frequency of 15 Hz.
Measuring its [Ca2+ ]i from an image requires tracing the
flagellum’s centerline, a time consuming task if done manually.
In addition to this task, there are several applications related to
sperm dynamics analysis requiring extracting the flagellum’s
centerline such as [3]–[7]. No algorithm has been developed
to automatically trace the flagellum’s centerline in this type
of 3D+t images.
As the flagellum is similar to a tubular structure, algorithms
developed to segment tubular structures should be suitable to
trace its centerline. Such algorithms for 3D tubular structures
include vessels [8], airways [9], neurons [10]; furthermore,
machine learning algorithms have also been proposed to
perform this task [11]–[16]. These algorithms learn a model
from a training set with extracted features, they usually
differ in the features and the algorithm used to train. Then,
the centerline is extracted from the segmented volume using a
skeletonization algorithm (usually minimal-cost path). These
algorithms have been proved to perform well in several
fields, however they usually have difficulties in segmenting
tubular structures with low SNR. Other approaches use the
fast marching algorithm [17]–[22] where a cost function is
used to propagate a wave and the centerline is extracted by
HERNANDEZ-HERRERA et al.: 3-D + t HUMAN SPERM FLAGELLUM TRACING 2237

Fig. 1. Algorithm overview. (a) A volume rendering of the 3D image stack (all sub-images have an identical scale bar); (b) contrast enhancement:
the original 3D image stack is pre-processed to improve the contrast; (c) a one-class classification algorithm is employed to enhance the flagellum
where the scale represents the probability that a voxel belongs to a tubular structure; (d) an iterative algorithm is employed to extract the flagellum’s
centerline; blue and green segments depict different iterations and red path depicts the last iteration; (e) extracted centerline for the 3D image stack
where the scale depicts the z-coordinate from the centerline; (f) flagellum’s centerline for different time points; each color identifies a different time
point. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

back-tracing from terminal points. The main drawback of
these approaches is the need to set the initial and terminal
point, which usually produces shortcuts when tracking a large
centerline if the two points are close.
Hernandez-Herrera et al. [14] recently proposed a one-class
classification and a machine learning algorithm (MESON) to
trace the centerline from tubular structures. Tubular structures
were segmented using the classifier and the fast marching
algorithm allowed extraction of their centerline. An alternate
iterative method (RIVULET2) to extract the centerline from
tubular structures was used by Liu et al. [20], also based
on the fast marching algorithm. Because these approaches
extract multi-branch structures by design, they can also be
used to extract single tubular structures. Indeed, multi-branch
algorithms can extract correctly single tubular structures if the
images have high contrast and high SNR. However, multi-
branch algorithms usually produce spurious branches from low
contrast images (as those in this work) when a single structure
should be detected.
Here we propose a tracing algorithm designed to extract
single tubular structures from a 3D+t low contrast and noisy
image stack. We obtain the centerline from the tubular struc-
tures using a machine learning approach combined with a
minimal path algorithm (described in Sec. II C-D). The initial
and terminal point to propagate the fast marching are auto-
matically detected by the algorithm. The algorithm has been
designed to trace several single tubular structures avoiding
the shortcuts produced by classical fast marching approaches.
First, the MESON [14] algorithm is applied to each 3D stack
to enhance tubular structures. Then, the center of the sperm’s
head is automatically detected as described in other works for
2D+t images [23]–[27], extending here the method for 3D+t,
and it is used as the starting point of the centerline algorithm.
The algorithm searches for the best minimal path over a prede-
fined distance of approximately the maximum flagellum’s
diameter (using the fast marching algorithm), just to move
a small distance from the starting point and then it stops. The
cost to travel across points near the detected minimal path
is forced to increase (in order to avoid detecting the same
minimal path), consecutively indexing the terminal point as the
starting one; this procedure is iterated to complete the whole
flagellum’s centerline, avoiding detection of erroneous paths,
specifically at the more curved parts. Our team has already
presented a preliminary work on sperm flagellum tracing [28].
Compared to MESON and RIVULET2, our algorithm does
not rely on the segmentation of the tubular structures to
trace the centerline, instead it relies on the enhanced stack
to guide the tracing. Furthermore, those algorithms propagate
the fast marching until all the points in the segmentation
had been visited, whereas our algorithm propagates the fast
marching until a pre-defined length has been reached, avoiding
shortcuts, specifically at the most curved part of the tubular
structure. Finally, those algorithms back-trace from the local
maximums (there can be many of them) of the geodesic
distance generated by the fast marching, instead our algorithm
only back-traces from the global maximum (a single point).
Therefore, our algorithm is better suited to trace the flagellum
since it traces a single branch, whereas the others may trace
many branches.
The main contributions of this paper are: (i) a novel method-
ology to trace the centerline from multiple single tubular
structures with high curvature and low SNR, allowing to trace
2238
Algorithm 1 Pre-Processing
Input: A 3D image stack V
Output: A 3D image stack Vlog with the contrast enhanced
Step 1: Logarithmic transformation of the 3D image stack
the sperm flagellum’s centerline, (ii) a criterion to decide
if a detected path belongs to a tubular structure, allowing
to stop the iterative algorithm, (iii) a detailed justification
of each algorithmic step, making possible the replication of
the methodology and the results, and (iv) a wide variety
of experiments to demonstrate the accuracy of the proposed
approach.
II. M ETHODS
Fig. 1 depicts an overview of the algorithm’s main steps.
In this section, each step is described in detail.
A. 3D+t Image Acquisition Setup
An Olympus IX71 inverted microscope with an Optronis
5000 camera acquiring 3000 images/sec and a resolution
of 512x512 pixels was used to obtain the datasets. A 3D+t
stack was recorded using a piezoelectric device (Physics
Instruments), allowing focal plane changes at 90 Hz and an
amplitude of 20 micrometers (see Corkidi et al. [29] and
Silva-Villalobos et al. [30] for details). Sperm were obtained
and prepared as described in Corkidi et al. [2].
B. Pre-Processing
The images acquired with the microscope have low SNR
because of the small number of photons collected by the
camera focal plane sensor and high frame rate (frames
per second). Hence, it is necessary to pre-process the image
stacks to improve their quality. There are several approaches
in image processing to improve image contrast [31], [32].
Here, the logarithm transform is used as a pre-processing.
The log transformation (Vlog (x ) = log(V (
x ) + 1)) is applied
to the image stack V to shrink the range of intensity values.
This transformation allows more compression of high intensity
values than those of lower intensity. Thus, it increases the
contrast of low intensity values such as those occurring at the
flagellum’s tail. Fig. 1(a) depicts a volume rendering of the
3D image stack, whereas Fig. 1(b) shows the pre-processed
stack.
C. Flagellum Enhancement
Hernandez-Herrera et al. [14] recently proposed
the MESON algorithm to segment bright tubular
structures (neurons) from a 3D image stack. This algorithm
could also be suitable to segment the sperm’s flagellum since
it locally resembles a tubular structure.
We employed this algorithm to enhance tubular struc-
tures (the flagellum). The enhanced image is used to guide
a minimal path algorithm towards the center of the flagellum.
In addition, the algorithm was employed to stop an iterative
IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 37, NO. 10, OCTOBER 2018
Algorithm 2 Enhancement Algorithm
Input: A 3D image stack I and scales σr , r = 1, 2, . . . , l
Output: Enhanced image stack with values in the range [0,1].
Values near to 1 for points (voxels) with high confidence to
belong to the tubular structure while near to zero for a low
confidence.
Step 1: Detect a training set of background points
Step 2: Extract local shape information (features) from the
3D image stack I
Step 3: Estimate a one-class classifier (decision function)
for each detected training set and apply the classifiers to the
3D image stack
centerline extraction algorithm. Algorithm 2 describes the
steps used to enhance tubular structures such as the flagellum.
The enhancement algorithm is based on the one-class clas-
sification approach where a training set of points belonging
to the background is automatically detected using a Laplacian
filter. A density function (one-class classifier) is learned from
the feature vectors (eigenvalues of Hessian matrix) of the
points in the training set.
1) Multi-Scale Laplacian to Detect a Training Set: The Lapla-
cian of the 3D image stack is used to detect the training set
consisting of background points. The Laplacian of the 3D
image stack (I ) at scale σ is given by:
n,σ (ξ )
F
L n,σ (I ) = F −1 {−ξ 2 ·
I (ξ )}, (1)
σ2
where F −1 is the inverse Fourier transform, −ξ 2 is the
Laplacian operator, Fn,σ is a low-pass filter [14] in the
frequency domain, σ is the scale (radius) used to compute
the low-pass filter, n is a parameter used to control the
decay of the low-pass filter, 
I is the Fourier transform of
the 3D image stack I and “·” denotes the product between
two numbers. The multi-scale isotropic Laplacian (L) of the
3D image stack is given by: L(I )( x ) = L n,σmax (x ) (I )(
x ),
where
σmax (
x) = argmax {|L n,σ (I )(
x )|}
σ ∈{σ1 ,σ2 ,...,σl }
is the scale at which the Laplacian of the 3D image
stack (Eq. (1)) reaches the maximum absolute value, x is
a point (voxel) in I and {σ1 , σ2 , . . . , σl } are the expected
radii of the tubular structures to enhance. The points in the
3D image stack with positive multi-scale Laplacian belong
to the background of the 3D image stack (see [14]). Hence,
a training set consisting of background points is detected as:
T = {
x |L(I )(
x ) > 0}.
For each scale σr , a subset of points from T is selected such
that the maximum absolute value of the Laplacian corresponds
to scale σr :
Tσr = {
x |
x ∈ T ∧ (σmax (
x ) = σr )}, r = 1, 2, . . . , l.
The training set (T ) only corresponds to a fraction of
the background points (approximately 40%), we are unable
to detect 100% of the background points using only the
HERNANDEZ-HERRERA et al.: 3-D + t HUMAN SPERM FLAGELLUM TRACING
Laplacian. Hence, from the remaining voxels that were not
detected in the training set, some of them belong to the
background and some others to the foreground. The one-class
classifier is used to assign low values for those remaining
voxels in the background while high values for the voxels
in the foreground.
2) Feature Extraction: Frangi et al. [33] demonstrated that
the eigenvalues (|λ1 | ≤ |λ2 | ≤ |λ3 |) of the Hessian matrix
provide local shape information of the 3D image stack. Specif-
ically, it can be shown that for a bright tubular structure λ2 , λ3
are negative and have large absolute values and |λ1 | is low,
for a bright blob structure λ1 , λ2 and λ3 are negative and have
large absolute values, for a bright plate-like structure |λ1 |, |λ2 |
have a low value and λ3 is negative and has a large absolute
value, and for a noisy structure (background) |λ1 |, |λ2 | and
|λ3 | usually have low values. Hence, the eigenvalues are
good features to differentiate a tubular structure from a noisy
structure.
3) One-Class Classifier Construction: For each scale σr
provided by the user, a one-class classifier is constructed (λ1
is not taken into account here). The one-class classifier is built
from the distribution of the eigenvalues from the training set
Tσr by using a 2D histogram. The 2D histogram bins contain
the votes of the training set eigenvalues. If the feature vector
from a point in the 3D image stack is assigned to a bin with
high value, then the point should correspond to a background
point. On the other hand, the feature vector from a point
inside a tubular structure should correspond to a bin with low
value in the 2D histogram since |λ2 | ≈ |λ3 |, |λ2 | >> 0 and
none or few vector in the training set must have high values.1
Therefore, the 2D histogram can be used to enhance tubular
structures. The 2D histogram is transformed to have values in
the range [0,1] and smoothed to have low transitions between
neighboring bins. Let’s call Dσr the final 2D histogram. Dσr
can be used for classification purposes by: computing the
eigenvalues (λ2 and λ3 ) for a given position x in the 3D image
stack, calculating the bin position of the eigenvalues in Dσr
and selecting the value of Dσr in the assigned bin position.
Finally, the assigned value can be thresholded and everything
above the threshold would be detected as belonging to the
background, otherwise it would be detected as belonging to
foreground. Instead of using Dσr for classification purposes,
here Dσr is used to enhance the foreground.
4) Enhanced 3D Stack Construction: The scale σmax ( x ) used
to compute the Laplacian is detected for each x in the 3D
image stack. Thus, the feature vector (eigenvalues λ2 and
λ3 ) is extracted and the assigned value on the 2D histogram
Dσmax is calculated for the feature vector. Let’s call this value
R(x ), then according to our previous analyses R( x ) must have
high value (close to one) for points in the background but
low (close to zero) for the flagellum. Hence, the function
R f (
x ) = 1 − R(x ), is used to enhance the flagellum. Fig. 1(d)
depicts an example of flagellum enhancement step. Note that
the flagellum’s full structure can be visualized since low values
are given in the background while high in the flagellum.
1 The points in the training set correspond to the background of the 3D
image stack and its eigenvalues should be low (|λ2 | ≈ |λ3 | ≈ 0)
2239
Algorithm 3 Iterative Centerline Tracing
Input: 3D original image stack V , pre-processed 3D image
stack I , 3D enhanced image stack R f , iteration length l0
and threshold T
Output: centerline tracing as a graph representation
Step 1: Detect starting point sk for tracing sperm k
Step 2: Compute fast marching algorithm starting at sk until
it reaches a length l0
Step 3: Extract the centerline by back-propagating from the
point pk that reached the length l0
Step 4: Increase the values of cost-function F( x ) for points
inside a tube. This tube is constructed from the centerline
detected in previous step and the radius of the flagellum
Step 5: Compute the orientation and probability of the
centerline to be inside the flagellum. If the probability
is smaller than 0.5 or orientation is larger than 110◦ stop
algorithm; otherwise return to Step 2 where starting point
sk is set to pk .
D. Iterative Centerline Tracing
Algorithm 3 depicts the main steps of our iterative approach
for centerline tracing.
1) Starting Point Detection: Our approach to track sperm is
similar to previous algorithms [23], [34], [35] with the main
difference that the other algorithms were designed to track
the sperm head in 2D+t images, whereas our approach is
developed for 3D+t images. The starting point sk used to
initialize Algorithm 3 at sperm k is selected at the center of
the sperm’s head (the algorithm can trace more than one sperm
per stack). Sperm heads are usually the brightest structures in
the 3D image stack, this information is used to detect each
seed point sk . The heads are segmented by applying a binary
threshold T . The threshold value should be low enough to
allow segmentation of the voxels in the head but high enough
to avoid segmentation of voxels in the flagellum. A connected
components algorithm is employed in the segmented stack to
identify each connected component Ck which corresponds to
each sperm’s head. The starting point sk is computed for each
connected component as the centroid from the 3D coordinates
of the voxels in each connected component. To keep track of
sperm, an ID is assigned to each sk at the current time point (t)
which depends on the IDs from the previous time (t − 1).
To this end, the nearest neighbor algorithm is used to do
the assignment. Let rk be the seed points detected in the
previous time. Then, the starting point sk is assigned with
the same track ID as sperm i from the previous time if the
minimum Euclidean distance between sk and all the seed
points in the previous time rk corresponds to sperm i (seed
point ri ). Our algorithm fails when two sperm are crossing
since the segmentation produces a single connected component
instead of two and one head disappears. After the sperm cross,
the algorithm would detect two heads and the one that was not
detected will be assigned a new ID.
Fig. 2(a) depicts a 3D image stack with two nearby sperm.
Fig. 2(b) depicts the sperm’s head segmentation in gray
while green points depict the centroid sk for each connected
2240
Fig. 2. Seed points detection. (a) volume rendering of the 3D image
stack (all sub-images have an identical scale bar); (b) gray depicts
the segmented head, green depicts the detected seed point and red
the detected point in the previous time point. (For interpretation of the
references to color in this figure legend, the reader is referred to the web
version of this article.)
component and red points depict the seed points rk from the
previous Time Point (TP). The red points are used to assign
the sperm’s ID to the green points using the nearest neighbor
algorithm.
2) Minimal-Cost Path: Our algorithm to trace the flagellum’s
centerline is an iterative approach based on the minimal-cost
path where the first iteration initializes at the starting points sk
and it stops when the paths reach a length l0 . The process is
repeated and the terminal points from the previous paths are
used as the new positions for the starting points. This process
is repeated until the probability of the detected path to be in
the flagellum is lower than 0.5 or there is a sharp change of
orientation in the path.
Given an initial point sk , a terminal point pk and a function
F(x ) that determines the cost of traveling across point x,
the minimum-cost path to travel from sk to pk is given by
a minimization problem
 pk
T ( pk ) = min F(C(s))ds
C sk
where C(s) is a curve in R3 . Among all the possible paths
to travel from sk to pk the path C(sk , pk ) with minimum
value is called the minimum-cost path. Given a point pk ∈ R3 ,
the minimum-cost path C(sk , pk ) can be obtained by solving
the Eikonal equation  which  is a non-linear partial differential
 
equation given by  ∂T∂t(x )  = F(
x ), with T (sk ) = 0. The fast
marching algorithm [36] is used to get an approximation to
the solution T ( x ) of the Eikonal equation.
3) Extract the Centerline by Back-Propagating: Given a
starting point sk and a terminal point pk , the minimum cost
path can be detected by backtracing from pk along the gradient
of T (x ) until sk is reached2 (see [37]). The fast marching
algorithm is initialized at sk and the point pk is automatically
detected as
 p
pk = argmin{ F(C(s))ds such that C = l0 },
p sk
2 Get all the neighboors of p
k and select the neighboor c0 with minimum
value T (x ). Get all the neighboors of c0 and select the neighboor c1 with
minimum value T ( x ). Repeat until sk is reached
IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 37, NO. 10, OCTOBER 2018
thus, among all the paths starting at sk and length l0 (there are
many paths satisfying this condition), we select the path with
minimum-cost (the one ending at pk ).
The cost function F( x ) plays an important role in the
detection of the minimum cost path since it guides the path.
The path will travel through low values of F( x ) instead of
higher. Hence, the function F( x ) should be constructed with
low values in the flagellum’s centerline and high values for
points distant from the centerline.
In our experiments the cost function F( x ) is defined as:
x ) = exp−60·G σa ∗[V (x )·R f (x )] ,
F( (2)
where V is the raw image stack with values in the range [0, 1],
R f is the enhanced flagellum, G σa is a Gaussian kernel and
“∗” represent the convolution, respectively. The function V
forces the minimum-cost path to travel across high intensity
values of V (flagellum’s center) while the function R f discour-
ages the path travelling outside the flagellum (R f assigns
low values to the background). The Gaussian smoothing G σa
is employed to reduce noise and have smooth cost values
between neighboring points, this creates smooth minimum cost
paths, the σa was set to [1,1,1] equal to the minimum radius
to be detected; a higher value may result in over-smoothing
structures with radius smaller than σa . The value 60 was found
by parameter optimization using grid search and selecting the
value that stabilizes the highest average F-score using few
stacks for training.
The previous approach is designed to trace multiple sperm.
The algorithm is initialized at each seed point sk corresponding
to the center of each sperm’s head and the algorithm stops
when all the paths C(sk , pk ) reached a length l0 .
4) Increase Cost-Values for Already Visited Point: In order
to discourage future iterations to travel across the already
detected paths C(sk , pk ), the cost function is increased for
points close to C(sk , pk ). Recall that the flagellum resembles
a tubular structure and the path C(sk , pk ) should travel across
the center of this tubular structure. Hence, a tube is constructed
to reduce the cost-function inside the tube. The tube is
constructed such that the starting point of the tube is the point
sk , the terminal point of the tube is pk and the center of the
tube is given by the path C(sk , pk ), the radius of the tube is
r0 and it is given by r0 = 2 · max{DT (C(sk , pk ))}, where
DT ( x ) is the distance transform [38] from the segmented
flagellum (obtained by applying a threshold of 0.5 to R f ( x ))
and the radius r0 corresponds approximately to twice the
maximum radius of the flagellum in the detected centerline
C(sk , pk ). The cost function F( x ) is updated as:

∞ if x belongs to any tube
F(x) =
F(x ) otherwise,
the new cost function F( x ) discourages the minimal-cost path
to travel across points in the tube since the new cost value for
traveling across those values is infinitely expensive (∞).
Fig. 1(d) depicts the different iterations of the algorithm for
a 3D image stack with one sperm, the first iteration starts at the
sperm’s head and is depicted by a green path, the second by
a blue path, the third by a green path and so on. The red path
HERNANDEZ-HERRERA et al.: 3-D + t HUMAN SPERM FLAGELLUM TRACING 2241

depicts the last iteration of the algorithm and it was rejected

since it does not belong to the centerline of the flagellum.
The distance transform DT from a binary stack B( x ) is
defined as:

min{ x − xb |B(
x b ) = false} if B(
x ) = true
DT (x) =
0 otherwise.
Given a point x in the binary stack B, the distance transform
computes the closest distance from x to the points in the
background. The binary stack B corresponds to a rough
segmentation of the flagellum and it is calculated from the
enhanced image R f by applying a threshold:

true if R f (
x ) > 0.5
B(x) =
false otherwise,
by construction R f ( x ) is close to 1 inside the flagellum and
it is close to 0 in the background. Hence, values close to
Fig. 3. Stopping criteria. (a,c) Maximum intensity projection (MIP) from a
0.5 should be near the boundary of the flagellum. Note that 3D image stack (all sub-images have an identical scale bar) at time point
the flagellum is roughly a tubular structure, thus the distance 28 and 22 for a 3D+t image stack, respectively. (b,d) Example of stopping
transform gives approximately the radius of the flagellum for criteria due to low “probability” and wrong orientation, respectively. Red
path depicts the last iteration. (For interpretation of the references to
points near the center of the flagellum. color in this figure legend, the reader is referred to the web version of
5) Stopping Criteria: The algorithm has two criteria to stop this article.)
the iteration of the minimal-cost path detection. It stops if the
detected minimum-cost path has a low possibility of being
inside the flagellum or there is a sharp change in orientation. points (usually 5) in the neighborhood of xi and belonging to
a) Low possibility of being inside the flagellum: recall that C(sk , pk ) using Principal Component Analysis (PCA). The
the function R f ( x ) (see Sec. II-C 4)) assigns values close to direction d( x i , C(sk , pk )) is given by the principal component
1 to points inside the flagellum while low values and close to from the points in the neighborhood of xi . The change of
0 in the background. Hence, for each xi ∈ C(sk , pk ) the value orientation at seed point sk between the two detected minimal
R f (
x i ) can be interpreted as the “probability” of the point paths is given by:
xi to be inside the flagellum. The 50 percentile of the values  sk , C(rk , sk )), d(
 sk , C(sk , pk )) >,
θ (sk ) = cos−1 < d(
{R f (x i )|
x i ∈ C(sk , pk )} is used as a measure of the path
to be inside the flagellum. This measure is less sensitive to where < a , b > denotes the dot product between the vector
the gaps and low values such as those at the flagellum’s  The path C(sk , pk ) is detected as a continuation of
a and b.
end. Hence, if the 50 percentile of the values of the path is path C(rk , sk ) if θ (sk ) (the maximum change in orientation
higher than 0.5, then the path is detected as belonging to the was set to 110◦ since changes in orientation larger than
flagellum, otherwise, the path C(sk , pk ) is detected as being this values were not observed among the sperm analyzed),
outside the flagellum. Fig. 3(a) depicts the Maximum Intensity otherwise the path is detected as an incorrect continuation
Projection (MIP) of a 3D image stack at time point number 28, and it stops. If any of the two criteria to stop the algorithm is
the flagellum’s end is usually the most difficult part to segment satisfied, then the algorithm stops. Otherwise, the algorithm
due to the low contrast. Fig. 3(b) depicts the detected minimum returns to Step 2: Minimal-cost path where the new starting
cost path at each iteration as segments green, blue and red. point sk for the next iteration is set as the terminal point pk
The segments assigned with green and blue are above the of the current detected minimal path.
50 percentile, whereas the red ones are below. Thus, red path is Fig. 3(c) depicts the maximum intensity projection (MIP) of
rejected as being inside the flagellum and the algorithm stops. a 3D image stack at time point number 22. Fig. 3(d) depicts the
Note that the red segment is indeed outside the flagellum. detected minimum-cost paths as segments with green, blue and
b) Orientation: The sperm’s flagellum is a smooth red. The segments assigned green and blue have an orientation
structure which does not have sharp changes in orientation. smaller than 110◦ with respect to the previous path. However,
This information is included in our algorithm to stop the the red path has an orientation larger than 110◦ with respect to
iteration if there is a sharp change in orientation. The its previous path (blue path). Thus, the red path is rejected as
orientation of the trace is measured at each starting point sk being a continuation of the blue path and the algorithm stops.
from the detected minimum-cost path. In order to measure
the orientation, we require the current detected minimal-cost
path and the previous minimal-cost path. Let C(sk , pk ) be the E. Sub-Voxel Centerline
current detected minimal-cost path and C(rk , sk ) the previous The flagellum’s centerline has voxel coordinates due to 3D
detected minimal-cost path. The direction d(  x i , C(sk , pk )) Cartesian representation of the stack. Hence, a post-process
for a point xi in path C(sk , pk ) is calculated from a few of the centerline is required to have sub-voxel accuracy.
2242
Fig. 4. 3D centerline of a time stack. (a) Volume rendering of a
3D image stack (all sub-images have an identical scale bar); (b) red
segment depicts the first iteration of the algorithm, yellow segment
corresponds to second iteration and the final centerline (flagellum’s
length truncated) corresponds to green path. The centerlines are shifted
for visualization purposes. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
A sub-voxel representation can be useful to have smooth
traces, specifically when the image has low resolution such
as the flagellum’s tail. Cubic smoothing spline is applied to
the detected centerline to have a smooth centerline where
the control points correspond to the 3D coordinates of the
extracted centerline (start point corresponds to the flagellum’s
head and end point to the last point of the extracted centerline)
and the coefficients of the cubic spline were calculated using
the Matlab function ‘csaps’. Fig. 1(e) depicts the sub-voxel
centerline of the original centerline (Fig. 1(d)). Note that the
sub-voxel centerline is smoother.
F. Centerline of the 3D+t Image Stack
Algorithm 3 for 3D centerline extraction is applied to each
time point t of the 3D+t image stack to extract the flagellum’s
centerline of the 3D stack and track each sperm. Algorithm 3
could have stopped prematurely in the tracing of the sperm
due to gaps (low intensity similar to the background) in the
flagellum. Hence, Algorithm 3 is re-run for stacks with a
detected sperm’s centerline length of less than lt units, lt is
computed using a histogram from the detected flagellum’s
length across all the time points of the 3D+t image stack.
For those stacks, the algorithm is re-run with the parameter
l0 increased to twice its initial value and the sensitivity of
the one-class classifier is increased to allow detection of
finer structures. Finally, the total length of each centerline is
truncated to a maximum length automatically detected as the
bin with maximum value in the centerlines length’s histogram.
Fig. 4(a) depicts a volume rendering of a 3D image stack at
time point number 53. Fig. 4(b) depicts the centerline detection
for the first run, second run and truncated length as red,
yellow and green segments, respectively. Note that the red path
corresponds to an under-estimation of the flagellum’s center-
line, the yellow path corresponds to an over-estimation of the
flagellum’s centerline while the final centerline corresponds to
an accurate representation of the flagellum’s centerline.
III. R ESULTS
In this section, qualitative and quantitative results are
presented for eight 3D+t image stacks. Results were obtained
using a workstation with a 64-bit processor, 16 cores and
IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 37, NO. 10, OCTOBER 2018
240 GB of RAM. The 3D+t image stacks, our software and
documentation are available upon request to the authors.
Parameter Setting: Our approach requires three main para-
meters σr , l0 and T . The parameter σr represents the scales to
be used to compute the multi-scale isotropic Laplacian. This
parameter was set to σr = {1.5, 2, 3, 4} voxels for all the
experiments since the radii of the flagellum is expected to fall
in these values. The parameter l0 was set to 10 voxels for all
the experiments since this value is larger than the maximum
diameter that we are expecting to detect, except for the 3D+t
image stack Fluo-4 DS3 , having a large gap, which was
set to 20 voxels. Finally, the parameter T depends on the
sperm’s head brightness which can be as low as 35 and as
high as 250. The raw 3D image stack, containing an average
of up to 5 sperm was cropped to a region of interest (big
enough to cover the sperm to be traced and usually containing
a single sperm), the cropped image was used as input to each
algorithms to speed up the processing.
A. Qualitative Results
In this subsection, qualitative results are presented for two
large 3D+t image stacks (Calcein DS3 and Calcein DS4,
568 time points) and three other ones (two stacks loaded
with Fluo-4 and one stack with Calcein -the first one being a
Calcium sensitive dye and the second one being insensitive-).
The 3D+t image stacks treated with Calcein have a higher
SNR than the treated with Fluo-4, thus they are easier to trace.
Supplementary video MOVIE 1 (available in the supple-
mentary files /multimedia tab) depicts a full centerline tracing
for Calcein DS3 a 3D+t image stack (150 time points) with
a single sperm. It shows a volume rendering of the 3D image
stack with an overlay of the tracing (gray line). Our algorithm
is able to correctly trace the flagellum. Fig. 5 depicts an
example of multiple sperm tracings. Fig. 5(a) depicts a volume
rendering from Calcein DS4 TP 263 a 3D stack loaded with
Calcein, the heads are the brightest structures from the stack
and the sperm’s flagellum is difficult to visualize because
it has low fluorescence as compared with that of the head.
Fig. 5(b) depicts the trace for each sperm in the 3D stack
where the colormap indicates the z-coordinate of the flagellum.
For example, the head for the two sperm at the upper-right
corner is at the bottom of the stack since it has assigned a low
value (blue) while the tail of the flagellum is at the top of the
stack since it has assigned a high value (red). Supplementary
video MOVIE 2 (available in the supplementary files /multi-
media tab) depicts the full centerline tracing (418 time points)
for Calcein DS4 a 3D+t image stack with multiple sperm.
Note that the algorithm fails to trace the sperm appearing at
the middle-left of the stack, this is because the flagellum is
not in the field of view of the camera. The algorithm stabilizes
when the sperm is in the field of view. Tracing multiple sperm
simultaneously in the same frame is a challenging task because
algorithms are usually developed to trace bright structures and
the sperm’s head is the brightest structure. Then, our previous
approach [28] failed to trace multiple sperm since the trace
jumped from one head to another if two sperm were near.
This problem is corrected by detecting an initial point for each
HERNANDEZ-HERRERA et al.: 3-D + t HUMAN SPERM FLAGELLUM TRACING
Fig. 5. Qualitative results. (a) Volume rendering of a 3D stack with multiple sperm (all sub-images have an identical scale bar). (b) trace for each
sperm, colormap depicts the value of the extracted flagellum’s z-coordinate. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
sperm’s head and propagating the fast marching algorithm for
all these points. This allows the algorithm to avoid jumping
to another sperm. An even more difficult task is to trace
the flagellum when two sperm are in close proximity and
the flagellum are touching or crossing, our current approach
cannot handle this task.
Fig. 6 depicts qualitatively results againts state-of-the-
art tubular structures tracing algorithms (MESON [14] and
RIVULET2 [20]) where green, yellow, blue and red lines
represent the semi-manual trace, our trace, MESON trace
and RIVULET trace, respectively. An exhaustive grid search
for the best parameter configuration at each time point was
performed for the MESON and RIVULET2 algorithms.
Fig. 6(a-c) depicts the volume rendering for three different
time points (Fluo-4 DS3 TP 6, Fluo-4 DS1 TP 28 and
Calcein DS1 TP 50) from three 3D stacks with low, high and
medium curvature. Fig. 6(a,b) corresponds to sperm loaded
with Fluo-4 while Fig. 6(c) corresponds to a sperm loaded
with Calcein. Note that the images corresponding to sperm
loaded with Fluo-4 have more noise and have less contrast than
the sperm with Calcein. The head is the brightest structure of
the 3D stacks and the flagellum is very difficult to visualize
since it has very low contrast. Fig. 6(d-f) depicts the semi-
manual trace for each time point as a green line. The semi-
manual trace for each time point was extracted using the
plugin Simple Neurite Tracer [39] from the open source image
processing application FIJI [40]. To this end, the user selected
an initial point at the sperm head and another terminal point
in the flagellum. Then, the algorithm extracts the minimum-
cost path between these two points. The user selected another
point in the flagellum and the minimum path is detected. The
user repeated this process several times (to avoid shortcuts)
until the tail of the flagellum is reached. Then, the user has
to proceed with the next time point. Fig. 6(g-i) depicts the
centerline traced by our approach as a yellow line. Note that
the trace highly resembles the semi-manual trace in the three
time points. Fig. 6(j-l) depicts the centerline traced by the
2243
MESON algorithm as a blue line. Note that the algorithm
stops prematurely for Calcein DS1 TP 50 (Fig. 6(l)) while
it produces extra branches for Fluo-4 DS1 TP 28 (Fig. 6(k)).
This algorithm ignores the a-priori knowledge that there is
only one tubular structure to trace. Fig. 6(m-o) depicts the
centerline extracted by the RIVULET2 algorithm as a red
line. Note that RIVULET2 creates extra branches for the three
stacks and it stops prematurely for Fluo-4 DS3 TP 6 and Fluo-
4 DS1 TP 28 (Fig. 6(m,n)). Multi-branch algorithms such
as MESON and RIVULET2 can be modified to extract a
single structure by selecting the largest branch. However, this
approach may fail since there are time points where the largest
branch does not correspond to the flagellum’s centerline.
Furthermore, the required time to perform this modification
would be even more expensive than manual trace since it
requires to do a grid search for parameter configuration (this
step can produce a lot of traces depending on the number of
parameters, the interval limits and step size), then manually
select the best centerline among all the traces and finally
selecting the best branch. Indeed, the best traces depicted
in Fig. 6 for the MESON and RIVULET2 algorithms have
different parameter setting for each time point.
B. Quantitative Results
In this section, quantitative results are presented for
six out of eight 3D+t image stacks. Calcein DS3 and
Calcein DS4 were disregarded from this analysis
because the semi-manual trace was unfeasible for these
large 3D+t image stacks (used only for qualitative
analysis). The metrics Precision (P), Recall (R), and
the F-score (F) are employed to measure the quality
of the reconstruction, P = SC /(SC + Sextra ), R =
SC /(SC + Smiss ), F = 2(P · R)/(P + R), where SC is
the total length of the automatic trace that was correctly
traced (true positive), Smiss and Sextra are the total length of
the missing (false negative) and extra segments (false positive)
2244 IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 37, NO. 10, OCTOBER 2018

Fig. 6. (a-c) Volume rendering from 3D stacks, the first two were treated with Fluo-4 while the third was treated with calcein (each column has an
identical scale bar); (d-f) semi-manual trace for each 3D stack; (g-i) centerline traced by our iterative algorithm; (j-l) centerline traced by MESON
algorithm; (m-o) centerline traced by RIVULET2 algorithm. (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)

of the automatic trace, respectively. The values for each metric
are in the range [0,1] with a low value corresponding to
a wrong tracing while high values correspond to good
tracings. A point pc in the trace is classified correctly if
there is a point pm in the semi-manual trace such that
 pc − pm  < C, where C is a constant defined by the user.
In our experiments, C was set to 7.5 voxels since this value
is the maximum flagellum’s diameter in our dataset. The last
metric is Aver age − Di splacement − Err or (ADE) which
measures the average distance error between the manual
trace and the automatic trace. Note that Precision penalizes
the automatic trace if it deviates (it has extra segments)
HERNANDEZ-HERRERA et al.: 3-D + t HUMAN SPERM FLAGELLUM TRACING 2245

from the semi-manual trace, Recall penalizes the automatic TABLE I

trace if it missed some parts of the semi-manual trace while P ERFORMANCE E VALUATION ON SIX 3D+ T I MAGE S TACKS .
A: O UR A LGORITHM , B: FLE, C: FLR, D: MESON [14],
F-score penalizes the automatic trace if it missed or has
E: RIVULET2 [20], F: FRANGI+RIVULET2
extra branches with respect to the semi-manual trace. Thus,
the F-score metric is the best indicator of the similarity
between the automatic trace and the semi-manual trace since
it takes into account the two possible errors produced by
automatic traces.
Our algorithm was compared against two variations of our
iterative algorithm called minimal-cost path Fixed Length
with Enhancement (FLE) and minimal-cost path Fixed
Length Raw (FLR), two state-of-the-art multi-branch algo-
rithms MESON and RIVULET2, and one variation of
RIVULET2 called FRANGI+RIVULET2. FLE corresponds
to one iteration of our algorithm where the parameter l0 sets
the length of the trace. FLR corresponds to one iteration of
our algorithm where the cost function (Eq. 2) depends only on
the raw image F( x ) = exp−60 I (x ) , and the parameter l0 sets
the length of the trace. FRANGI+RIVULET2 corresponds to
pre-processing the 3D image stack using Frangi vesselness
measure [33] and then applying the RIVULET2 algorithm to
trace the flagellum’s centerline.
Table I depicts the average values (μ) and the standard
deviation (σ ) for the metrics Precision, Recall and F-score
for the six methods in six 3D+t image stacks. Each metric
was calculated for every time point in a given 3D+t image
stack, then the μ and σ were computed. The first two 3D+t
image stacks were from sperm loaded with Calcein and have
98 and 83 time points, respectively. The third to sixth 3D+t
image stacks were loaded with Fluo-4 and have 73, 94,
108 and 108 time points, respectively. Hence, we compared
the six methods using a total of 564 semi-manual traces.
The values shown in the Table I for the algorithms FLE and
FLR correspond to the length l0 that reached the highest
average Precision, Recall and F-score value. These values
for the Calcein dataset correspond to l0 = 95, 95 for FLE
and l0 = 100, 90 for FLR. The values of l0 for the Fluo-
4 dataset correspond to l0 = 70, 55, 90 and 90 for FLE while
l0 = 90, 60, 105 and 95 for FLR. Note that our algorithm
and FLE have similar and the highest performance for the
Calcein DS1 because this 3D+t image stack has a high SNR
and the flagellum’s beating has low curvature. RIVULET
has very low Precision and F-score value compared to our
approach because those metrics are penalized if there are
extra segments in the tracing. FLR has the highest Precision
value for the 3D+t image stack Fluo-4 DS1, however it has
low Recall and F-score values, hence indicating that it was
not able to accurately trace the flagellum. Our algorithm has and they usually produce spurious branches. Observe that our
higher Precision and F-score values compared to FLE for algorithm, FLE and FLR have an ideal metric value for Fluo-
Fluo-4 DS1, thus indicating that it produces better traces for 4 DS2, whereas FRANGI+RIVULET2 has an almost ideal
the flagellum. Our iterative algorithm has better performance metric value, this is because the extracted flagellum from this
because the flagellum’s beating for this 3D+t image stack has 3D+t image stack has low curvature and high SNR.
a high curvature and the classical fast marching algorithms The average values (μ) for the metrics Precision, Recall
such as FLE, FLR, MESON and RIVULET2 are not able to and F-score for the six methods show that our algorithm has
capture the curvature as they usually take shortcuts due to the the highest Precision, Recall and F-score values indicating
propagation of the fast marching in all directions. Note that that it produces more accurate tracings than FLE, FLR,
MESON and RIVULET2 have low performance, this is due MESON, RIVULET2 and FRANGI+RIVULET2. Note that
to the fact that they are not designed to track a single branch RIVULET2 has the lowest F-score value indicating that it
2246
performed the worst for the 3D+t image stacks and therefore
it is discouraged to use it to trace a single branch struc-
ture. Pre-processing the 3D image stack using the Frangi
vesselness measure increases the average metric values for the
RIVULET2, however it still has low metric values indicating
incorrect tracings. Comparing the average value between our
approach and the two variations of our algorithm (FLE and
FLR), we note that FLR has the lowest F-score (0.92) value
indicating that it performed the worst for the 3D+t image
stacks and therefore it is discouraged to use the raw data to
guide the minimal-cost path. FLE highly increases the metrics
values, it has an average F-score of 0.94, hence adding the
enhanced image to guide the minimal-cost path is highly
recommended since it results in better tracing than using the
raw image. Finally, adding the iterative approach (our algo-
rithm) also increases the accuracy, it has an average F-score
of 0.97, thus for 3D+t image stacks with flagella beating
with high curvature is recommended to included an iterative
approach. Finally, our algorithm has an average ADE value
of 2.28, thus, indicating that the average distance between
the automatic trace and the semi-manual trace is very near
to 2 voxels which suggest that the traces are very close to
each other.
The running times to trace the 564 stacks are: 1 hour (h)
8 minutes (min) for our algorithm, 1 h 1 min for FLE,
12 min for FLR, 131 h 32 min for MESON, 12 h 26 min
for RIVULET2 and 16 h for FRANGI+RIVULET. MESON
required the highest reconstruction time because it needs to
tune three parameters, hence a grid search for the best trace
requires an excessive amount of parameter combination (847).
The lowest time was achieved by FLR with 12 min since
it only requires one run of the fast marching algorithm to
obtain the best trace for each 3D stack. Note that FLE is
approximately 5 time slower than FLR. This is mainly because
the construction of the enhanced image stack R f ( x i ) is a
time consuming task due to the calculation of the feature
vector (eigenvalues of the Hessian matrix). The most time
consuming task of our algorithm is the construction of the
enhanced image stack representing 67% of the total running
time (Sec. II-C, since it required the calculation of the Lapla-
cian, feature vector and one-class classifier). Algorithm 3 has
to be re-run for traces that stopped prematurely, on average this
value was 15% of the total traces. The maximum percentage
was for Fluo-4 DS1 that required to re-run the algorithm 3
in 25 traces from a total of 73 traces (34%), whereas the
minimum percentage was for Calcein DS1 which required
to re-run the Algorithm 3 only 6 traces from a total of 98
traces (6%). The total of traces to be re-run depends on the
quality of the stacks; if there are large gaps within them,
then many will stop prematurely. This can be usually fixed
by increasing the initial value l0 . However, increasing l0 to
a large value would induce the algorithm to miss the most
curved parts of the flagellum.
IV. C ONCLUSIONS
An iterative algorithm for the tracing of the sperm’s
flagellum from a 3D+t image stack of very low SNR fluo-
rescence based images has been developed. There are no
IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 37, NO. 10, OCTOBER 2018
previous algorithms able to extract the centerline from this
type of fluorescent images. Other algorithms able to extract
the centerline from tubular structures have been designed to
extract more than one branch, however there is a prior knowl-
edge that the flagellum is a single structure. The quantitative
and qualitative results show that the multi-branch algorithms
MESON and RIVULET2 produce poor results when tracing
a single structure, whereas our algorithm is producing similar
results as a manual tracing. The qualitative results using a
total of 564 semi-manual traces highly discourage using the
raw data to guide the minimal-cost path (Table I, method
C) and they encourage the use of the enhanced image to
keep the trace inside the flagellum (Table I, method B).
Furthermore, the iterative approach increases the accuracy of
the tracing (Table I, method A), specifically at the more curved
parts of the flagellum. The proposed method is capable of
solving a major problem related with the analysis of the 3D
motility of sperm cells in fluorescence images with very low
intensity and low SNR.
ACKNOWLEDGMENT
The author would like to thank P. Torres and E. Mata for
helpful assistance with biological procedures and A. Bribiesca
for computational support.
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