Automatic segmentation and 3D feature extraction of protein

aggregates in Caenorhabditis Elegans

Pedro L. Rodrigues1,2, Antnio H.J. Moreira1,2, Andreia Teixeira-Castro1,2, Joo Oliveira1,2,
Nuno Dias1,2,3, Nuno F. Rodrigues3,4, Joo L. Vilaa1,2,3
1

Life and Health Sciences Research Institute, SHS, University of Minho, Portugal
ICVS/3Bs - PT Government Associate Laboratory, Braga/Guimares, Portugal
3
DIGARC Polytechnic Institute of Cvado and Ave, Barcelos, Portugal
4
HASLab / INESC TEC University of Minho, Braga, Portugal

ABSTRACT
In the last years, it has become increasingly clear that neurodegenerative diseases involve protein aggregation, a
process often used as disease progression readout and to develop therapeutic strategies. This work presents an image
processing tool to automatic segment, classify and quantify these aggregates and the whole 3D body of the nematode
Caenorhabditis Elegans.
A total of 150 data set images, containing different slices, were captured with a confocal microscope from animals
of distinct genetic conditions. Because of the animals transparency, most of the slices pixels appeared dark, hampering
their body volume direct reconstruction. Therefore, for each data set, all slices were stacked in one single 2D image in
order to determine a volume approximation. The gradient of this image was input to an anisotropic diffusion algorithm
that uses the Tukeys biweight as edge-stopping function. The image histogram median of this outcome was used to
dynamically determine a thresholding level, which allows the determination of a smoothed exterior contour of the worm
and the medial axis of the worm body from thinning its skeleton. Based on this exterior contour diameter and the medial
animal axis, random 3D points were then calculated to produce a volume mesh approximation. The protein aggregations
were subsequently segmented based on an iso-value and blended with the resulting volume mesh.
The results obtained were consistent with qualitative observations in literature, allowing non-biased, reliable and
high throughput protein aggregates quantification. This may lead to a significant improvement on neurodegenerative
diseases treatment planning and interventions prevention.
Keywords: Caenorhabditis Elegans, image processing, segmentation, quantification

1. INTRODUCTION
Neurodegenerative and infectious disorders, including Alzheimer's and Parkinson's diseases, amyotrophic lateral
sclerosis, and stroke are rapidly increasing as population's age. Alzheimer's disease alone currently affects 4.5 million
Americans, and more than $100 billion is spent per year on medical and institutional care for affected people. Such
numbers will double in the ensuing decades, if no treatment or preventing interventions are discovered [1].
Aging is by far the major risk factor for numerous neurodegenerative disorders. Although several cure pathways
have been discussed in biomedical research, their contribution to disease susceptibility remains unclear. In recent years,
it has become increasingly clear that many of these disorders involve protein aggregation, a process that has become a
widely accepted disease marker and is often used as readout for the development of therapeutic strategies. Furthermore,
protein aggregates are found in the majority of animal and cell models such as the nematode Caenorhabditis Elegans
(C. Elegans) and are quite useful for the recreation of these diseases.
C. Elegans nematode is among the most widely studied genetically-tractable experimental organisms, due to its both
anatomical and genetically simplicity. One major advantage of C. Elegans is its body transparency which, by means of
fluorescence techniques, allows easy visualization and monitoring of cellular processes throughout its life cycle.

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Until now, authors have been studying aggregation phenotypes by counting aggregates manually on a limited cell
sample or neuronal subtypes [2]. Manual quantification is time-consuming, user-dependent, error-prone, subjective, and
requires expert knowledge to yield accurate and robust results. Frequently, due to the large amounts of aggregates, it is
not possible to perform quantitative analyses, leaving researchers limited to qualitative observations and to recreate
diseases in non-neuronal tissues to simplify quantification [3-5].
This problem could be resolved by the introduction of software tools to improve productivity from precise, fast,
repeatable and objective quantification of protein aggregates in C. Elegans models. In particular, image processing
techniques could play an important role in this domain, allowing reliable analyses that are not possible by eye screening.
Until now, several strategies for automatic tracking of the head, tail and entire body movement of the nematode
have been described [6-9]. These existing tracking algorithms share some similarities. Commonly, the centroid and the
skeleton of the worm are constantly identified using intensity algorithms, morphological operations and on head and tail
recognition using pattern recognition algorithms. Locomotive features of C. Elegans, such as speed, motion and body
posture, have also been extracted using different classifiers, such as the random forests classifier in [9]. Other authors
[10] developed an automated micro-positioning system, based on a Gaussian Pyramid Level that allows online tracking
at 30 Hz with a high resolution for revealing subtle differences in locomotive behaviour of C. Elegans. None of these
works tried to quantify protein aggregates widely used by biological researchers as disease marker. Regarding this issue,
Andreia et al. [11] developed an image processing application tested in a Caenorhabditis elegans (C. elegans) model of
Machado-Joseph disease. This work only analyses 2D images to quantify different ratios: (1) the area of aggregates per
total body area; (2) the number of aggregates per total body area.
Therefore, the work described in [11] does not take into account the 3D dimensionality concerning the worm body
or its protein aggregates volume. This paper attempts at filling this gap, by proposing a novel design and implementation
of an image processing application to quantify several worm features, such as: total area, perimeter or volume; distance
from head to tail; thickness of the worm; volume, area and perimeter of its protein aggregates. It might be a step forward
to quantitatively evaluate the behavioural phenotypes of novel mutants, gene knockout lines and pharmacological
treatments, by analysing the 3D distributions of all protein aggregates.
The reminder of this paper is organised as follows. Section 2 describes all algorithms to reconstruct the worm body
and its proteins aggregates, as well as its 3D quantification. Section 3 presents and discusses some simulation and
statistical results and Section 4 presents some conclusions and future developments.

2. METHODS
To effectively reconstruct three-dimensionally the C. Elegans body and its protein aggregates, it was necessary to
acquire a sequence of images from which the animals position and posture was static. Culturing of C. Elegans was
performed as described in [1]. Then, several image processing methods were used and implemented under
C++/Qt/OpenGL allowing noise reduction, 2D aggregates and worm body segmentation, 3D reconstruction, and an
automatic quantification of several worm parameters as described above.
In the end, a user interface was also developed, enabling fast modifications of computing parameters, image
processing outputs and statistical ratios such as the area or volume of protein aggregates per total area or volume per
worm body respectively, which may facilitate high-throughput studies in the search for modifiers of protein aggregation.
2.1. Image acquisition
All images were captured on an Olympus FV1000 (Japan) confocal microscope, under a 63x water or 60x oil
objective, respectively. Z-series (Fig. 1 (a)) imaging were taken of all the C. Elegans lines generated, using 514/515 nm
laser excitation for YFP (yellow fluorescent protein), 458 nm for CFP (green fluorescent protein) and 593 nm for
mCherry fusion proteins. The pinhole was adjusted to 1.0 Airy unit of optical slice, and a scan was taken every ~0.5 m
along the Z-axis.
Immobilised 4- or 10-day-old animals were subjected to fluorescence recovery after photo-bleaching (FRAP), as
previously described [12], with the following modifications: relative fluorescence intensity (RFI) was determined using
the following equation: RFI=(Tt/Ct)/(T0/C0), in which T0 is the total intensity of the region of interest (ROI) prior to
photo-bleaching, and Tt is the intensity of the same area at a given time after bleaching. The intensities were normalised
against a no bleached ROI within the same cell (C0, intensity of the control area prior to bleaching, and Ct at any time
after) as a control for general photo-bleaching and background fluorescence [12].
After being captured, all images were added and normalized, yielding to a 2D stack image (Fig 1 (b)).

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Figure 1. (a) Representation

R
of image acquiisition step; (b) the stacked 2D
D image of all 2D
D slices in (a); (c) image enhan
ncement with a
isotropic diffusiion of the imagee gradient of (bb).

2.2. Outter contour seegmentation

The graddient of this im
mage, calculaated with a Soobel operator was
w input to an
a anisotropicc diffusion alg
gorithm (Fig 1
(c)), which prreserves sharpper boundariess. The behavioour of this alg
gorithm was dependent on tthree parameteers: number of
iterations, edgge parameter ()
( and a diffuusivity functioon. These valu
ues were expeerimentally caalculated in ord
der to obtain a
conveniently filtered imagee. In this workk, the image was
w diffused over
o
80 iteratiions, where thhe edge param
meter (
t Tukeys biiweight as edgge-stopping diiffusivity funcction
(
(Equation
1):
was used by the
(Equation 1))
Further information
i
reegarding this process can be
b found in [13]. Using thiss algorithm alllowed to redu
uce the animaal
transparency,, since most of the regions in
i Fig. 1 (b) appear
a
as blacck, allowing ann automatic ddetermination of the exterior
contour of thee nematode C.
C Elegans.
For thatt, the median value, determ
mined through the histogram of the diffused image, was used to
o dynamicallyy
determine a global
g
threshoold level, whiich segments the C. Elegan
ns as white annd the backgrround image as black. Thiss
binary imagee was also inpput to morphoological openinng and closin
ng operations (using 3x3 sqquare kernels)) that removess
background noise
n
from thee image and clleaned up som
me spots insidee the worm boody.
Followinng the morphhological operrations, the biinary image allowed
a
the deetermination of a contour for the wholee
white objectss in the image - C. Elegans object - basedd on an iso-co
ontour and splline approxim
mation function
n according too
the Catmull-Rom spline method[14]. This counterr was later sm
moothed withh a Gaussiann distribution,, producing a
shrinking effeect. If necessaary, the user caan interactively modify the contour, by selecting
s
the ppoints along it, and updatingg
the slice apprroximation. Fig. 2 shows some examplees of the worm
m exterior conntour determinned with this methodologyy,
and Fig. 3 shoows the externnal contour foor the animal represented
r
in Fig. 1 (c).
b
reconstruction
2.3. 3D body
The outeer contour of the worm waas used to deffine a binary image, wheree the points w
within the conttour appear ass
white and thhe other points as black. Then, this im
mage was useed to determiine a medial axis of the body using a
skeletonizingg algorithm deescribed in [115]. Briefly, this algorithm
m is divided into two sub-iterations thaat remove thee
boundary andd corner pointts of the whitee object, untill only the skeeleton remainss as white pixxels. If some short
s
branchess
arise from thee medial axis, due to some animal irreguularities, they are
a eliminatedd through the sselection of th
he longest axiss
by thinning thhe skeleton (F
Fig. 3 (b)). Thee skeleton length allows thee quantificatioon of the worm
m distance from
m head to taill.

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Figure 2. Illuustration of diffferent outer deliineation of diffferent animals.

Figure 3. Overrview of the diffferent results achieved

a
on diffferent image pro
ocessing stagess: (a) outer conttour segmentation; (b) skeletonn
deetermination; (cc) 3D random points; (d) volum
me reconstructio
on of the wholee body and its pprotein aggregattes.

Using thhe medial axiis informationn, a randomizzed spherical point cloud (Fig 3 (c)) w
was generated
d around eachh
skeleton poinnt, where the radius
r
of thesee spheres was calculated according to thee diameter of the 2D outer contour
c
(Fig 3
(a)). Based onn these 3D ranndom points, a 3D triangle mesh was callculated, usingg an algorithm
m variant to th
he well-knownn

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Marching Cuubes algorithm

m [16]. The linnks or surface parts between
n the random points
p
were coonstituted betw
ween all cubicc
grids during the processinng, using a trri-linear interppolation (Fig 3 (d)). Finallly, the proteinn aggregation
ns were easilyy
segmented baased on a threeshold level annd blended with
w the resultiing volume mesh,
m
since theey appear in th
he image withh
higher intensiity levels thann soluble proteeins and the worm
w
body. Su
uch volumes allow
a
the quanntification of the
t total worm
m
body volume and its proteiin aggregates.
In orderr to facilitatee the usage of
o the previoously describeed analysis, a user interfaace was deveeloped, whichh
encapsulates all the algoritthms and allow
ws the researccher to (1) control all proceessing operatiions; (2) navig
gate through a
list of image files with diffferent formats from the contents of a specified
s
direectory; (3) ediit contours off the object of
interest by moving,
m
copyinng, pasting annd performingg undo/redo operations;
o
(4)) visualize andd save to a daata file all thee
segmentationn results.

3. RESUL
LTS
ges from worrms with diffe
ferent genetic modificationss
This imaage processinng methodologgy was studieed in 84 imag
acquired as described
d
in seection 2.1 at the Life and Health
H
Sciencces Research Institute (ICV
VS Universiity of Minho)).
For all the annimals, a 3D reeconstruction was made alloowing the quaantification off several param
meters regardiing the proteinn
aggregates (vvolume, area and
a number) innformation orr the worm bo
ody (volume, area,
a
animal llength). Tablee I summarizess
some quantifiication resultss for the 4 worrms illustratedd in Fig. 4.
Taable 1. Results summary
s

Agggregates

Woorms
Num
mber
(Figg. 4)

Volume
V
( 3)
(m

Area m2)
(A

Number

Volume (m
m3)

Area (
m2)

a
a)
bb)
c
c)
d
d)

302
137
71
57

107
71
33
76

126
121
88
76

34757
43772
34247
27227

32566
35333
3118
22733

Aniimal
Len
ngth
(m)
35
50
36
65
32
24
35
56

Figure 4. Voolume renderingg of different 3D volume apprroximations resuults.

t main probblems of this work was thhe animal tran

nsparency. It was
w firstly reduced using an
a anisotropicc
One of the
diffusion algoorithm which input was ann image gradieent magnitudee determined with
w a Sobel operator (Fig. 1). This stepp
allowed the definition
d
of thhe worm outer contour by enhancing
e
inteensity changees and reducinng the image darkness
d
alongg
its body. Thiss result was thhen input to oppen and closee morphologiccal operations that were essential to reduce small noisee
points within the animal annd in the imagge backgroundd.

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This outcome allowed an automatic delineation with a smoothed Catmull-Rom spline method. It produced a
smoothed effect with fewer oscillations and preserved the data monotonicity (Fig. 2). Only a few number of times, it was
necessary to interactively modify the contour on the head region.
The levels of the animal transparency were also a challenge to fully reconstruct the 3D worm body. Therefore, 3D
random points were generated according the animal medial axis and the 2D outer animal contour. Although this
reconstruction was determined using 3D random points, the worm body volume was similar when compared to ones
characterized under microscopy [1].
Concerning biological issues, this application has been tested and validated with C. Elegans models in order to
evaluate genes with potential to modify protein aggregation. Recent data from our lab has shown that with this
application it will be easily determined the influence of different genes to control and decrease the protein aggregates
responsible for neuron cells death (unpublished results).

4. CONCLUSIONS
Whether protein aggregates are a cause or a result of cellular degeneration is still a controversial issue. In either
case, quantification of these protein aggregates is used to evaluate the effect of gene and drug therapies for many
conformational disorders. As so, this novel imaging processing application, which allows non-biased, reliable and high
throughput quantification of protein aggregates, may contribute to a significant improvement on the prognosis of
treatment effectiveness for these group of disorders. It already proves to be a valuable tool by decreasing the total
number of decisions, time-consumption and user dependence, while increasing the quantification efficiency and
robustness [1].
Hereupon, new research paths based on this work have to be further investigated, such as the optimization of the
diffusion algorithm. Although this algorithm works well for almost all images, new algorithms can be implemented to
automatically determine and optimize the diffusion algorithm parameters to reduce computation time as well as to test
and validate new diffusivity functions. Future research paths comprise the development of a new algorithm to
automatically segment protein aggregates and reduce all thresholding inherent shortcomings and dependences.

ACKNOWLEDGEMENTS
The authors acknowledge to Foundation for Science and Technology (FCT) - Portugal for the fellowships with the
references: SFRH/BD/74276/2010; SFRH/BD/68270/2010; and, SFRH/BPD/46851/2008. This work was also supported
by FCT R&D project PTDC/SAU-BEB/103368/2008.

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