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ISBIq: A Framework for Simulation of Cell Cycle
in Fluorecence Microscopy
Vladimı́r Ulman & David Svoboda
Centre for Biomedical Image Analysis, Masaryk University
Brno, Czech Republic
PV182 – CBIA seminar
March 14, 2013
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
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Motivation
Historically, gtgen and Static simulator were,
quite independently, first published/born in 2007.
Recently, joint proper investigation of this field has begun:
Generation of Synthetic Image Datasets for Time-Lapse Fluorescence
Microscopy. David Svoboda, and Vladimı́r Ulman. ICIAR 2, volume 7325 of
Lecture Notes in Computer Science, page 473-482. Springer (2012)
We faced several requests for time-lapse datasets during conference
meetings and discussions when presenting CytoPacq (static
simulator).
ISBI Cell Tracking Challenge 2013 ,
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
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M
ito
sis
near th
a related phase called G0) doing their job in the organism—a
propha
Task nerve cell carries impulses, for example.
tubule
Mitosis is conventionally broken down into five stages:
two ce
The prophase,
aim was to prometaphase,
simulate how themetaphase,
cell looks during
the cell
anaphase,
andcycle:
site en
tubule
the cen
INTERPHASE
Each
some h
with s
S
G1
tromer
(DNA synthesis)
site di
microt
is s
kineto
e
G2
kin
tached
o
t
Cy
MIT
crotub
(M) OTIC
PHA
When
SE
microt
pole fr
movem
posite
Figure 12.6 The cell cycle. In a dividing cell, the mitotic (M)
Ulman & Svoboda
(CBIA)
March 14, 2013
3 / 18 is
next
phase alternates
with interphase, aISBIq
growth period. The first part of
Problem Analysis
Cell cycle can be split into Interphase and Mitosis:
Interphase (95%) . . . simple ,
G1 phase (50%)
S phase (30%)
G2 phase (15%)
Mitosis (5%) . . . tricky /
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis
Note: 100% ≈ 24 hour cell cycle length
Ulman & Svoboda (CBIA)
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March 14, 2013
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Problem Analysis
Cell cycle phases step by step
G2 phase
G2 of Interphase
Centrosomes
(with centriole pairs)
Chromosomes
(duplicated,
uncondensed)
Prophase
Early mitotic
spindle
Aster
Prom
Centromere
Fragments
of nuclear
envelope
cell gather nutrients/energy for
mitosis
nuclear envelope still encloses
nucleus
chromosomes have not yet been
condensed
creation of centrosomes
Nucleolus
Nuclear
envelope
Plasma
membrane
G2 of Interphase
• Ulman
A nuclear
envelope
encloses the nucleus.
& Svoboda
(CBIA)
start of mitosis
Chromosome, consisting
of two sister chromatids
Prophase
• The
chromatin fibers become more
ISBIq
Kinetochore
Prom
• 2013
The nuclear
enve
March 14,
5 / 18
Problem Analysis
Cell cycle phases step by step
Prophase Prophase
Prometaphase
s
us.
Early mitotic
spindle
Aster
Centromere
Fragments
of nuclear
envelope
Nonkinetochore
microtubules
nucleoli disappear
mitotic spindle begins to form
(itself)
centrosomes move away to
opposite poles of cell
chromatin condensates
Chromosome, consisting
of two sister chromatids
Prophase
• Ulman
The chromatin
become more
& Svoboda fibers
(CBIA)
each chromosome is composed
of two sister
chromatids
Kinetochore
Kinetochore
microtubule
Prometaphase
• The
nuclear envelope fragments.
ISBIq
March 14, 2013
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e
Problem Analysis
Cell cycle phases step by step
Metaphase
Prometaphase
Nonkinetochore
microtubules
Fragments
of nuclear
envelope
nuclear envelope fragments
each chromatid is attached to
one of two centrosomes
chromosomes become more
condensed
Kinetochore
Kinetochore
microtubule
Prometaphase
•Ulman
The nuclear
envelope
& Svoboda
(CBIA)fragments.
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March 14, 2013
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Problem Analysis
Cell cycle phases step by step
Metaphase
Metaphase
Anaphase
Telophase
Cleavage
furrow
Metaphase
plate
centrocomes are now at
opposite poles of the cell
chromosomes converge to
metaphase plate
Spindle
Centrosome at
one spindle pole
Metaphase
Ulman
& Svobodaare
(CBIA)
• The
centrosomes
now at opposite
Nuclear
envelope
forming
Daughter
chromosomes
Anaphase
Te
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March 14,
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• Anaphase
is the shortest stage of mitosis,
• 2013
Two daughter
e
10 μm
Problem Analysis
Cell cycle phases step by step
Anaphase Anaphase
Telophase and Cytokinesis
Cleavage
furrow
Nucleolus
forming
(the shortest stage in the cell
cycle)
two liberated daughter
chromosomes begin moving
toward opposite ends of cell
cell elongates
by the end of anaphase, the two
ends of the cell have equivalent
and complete collection of
Nuclear
envelope chromosomes
Daughter
chromosomes
forming
Anaphase
Ulman & Svoboda (CBIA)
Telophase
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March 14, 2013
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10 μm
Problem Analysis
Cell cycle phases step by step
Telophase
and Cytokinesis
Telophase and Cytokinesis
itosis,
Cleavage
furrow
Nucleolus
forming
two daughter nuclei form in the
cell
nuclear envelopes arise
nucleoli reappear
chromosomes become less
condensed
formation of a cleavage furrow,
which pinches the cell in two
Nuclear
envelope
forming
Telophase
• Two daughter nuclei form in the cell.
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
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Problem Analysis
Cell cycle phases step by step
Figure 12.10 Cytokinesis in animal and plant cells.
Telophase and Cytokinesis
(a) Cleavage of an animal cell (SEM)
consin
rize at
rd the
kidney
tween
te the
intact
ges in
e segose on
two daughter nuclei form in the
cell
nuclear envelopes arise
nucleoli reappear
100 μm
Cleavage furrow
chromosomes become less
condensed
formation of a cleavage furrow,
which pinches the cell in two
Contractile ring of
microfilaments
Daughter cells
(b) Cell plate formation in a plant cell (TEM)
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March 14, 2013
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Problem Analysis
Cell cycle phases step by step
G1 phase
cell grows to the regular size of
cell slowly
nucleus still contains only half of
all chromosomes
chromatin soon decondensates
cell gather nutrients/energy for
the next (S) phase
this is the longest stage in the
cell cycle
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
5 / 18
Problem Analysis
Cell cycle phases step by step
G1 phase
cell grows to the regular size of
cell slowly
nucleus still contains only half of
all chromosomes
chromatin soon decondensates
cell gather nutrients/energy for
the next (S) phase
this is the longest stage in the
cell cycle
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
5 / 18
Problem Analysis
Cell cycle phases step by step
S phase
DNA replicates
cell keeps to its volume
this is the second longest stage
in the cell cycle
Ulman & Svoboda (CBIA)
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March 14, 2013
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Design of the new simulator
Model of cell
The following data structures were used to model the cell phantom
cell . . . binary mask
nucleus . . . binary mask
nucleoli . . . binary mask
chromosomes . . . sets of dots (molecules of fluorescent dye)
Note: Aside to binary masks, lists of boundary points were used as well.
The following (basic) conditions are valid
number of chromosomes . . . 23 (46)
during interphase, the chromosomes are located inside nucleus
during mitosis, the nucleus disappears and chromosomes are spread
over the whole cell
Ulman & Svoboda (CBIA)
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March 14, 2013
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Design of the new simulator
Single cell simulation
The Cell::DoNextPhase() template
Simulates particular cell cycle phase completely and atomically.
It manages visual appearance and motion of a cell together.
It is controlled only by time-sampling parameter.
It is split into
“internally-induced stuff” aka local interior affairs,
“externally-induced stuff” aka global cell movement.
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
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Design of the new simulator
Single cell simulation
Local interior affairs
This stage supervises any changes that are
relevant to the specific cell cycle phase,
subject to cell current needs.
Examples: re-organization of chromatin, drifts (if desired) or other
visually apparent changes of cell nucleus or nucleoli, changes in cell
shape, etc.
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
8 / 18
Design of the new simulator
Single cell simulation
Global cell movement
This stage supervises any changes that are
(usually) commonly happening during every cell cycle phase,
(usually) influenced by cell environment.
Examples: movement and shape changes of cell within its
environment.
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
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Design of the new simulator
Cell population simulation
The Scheduler::Run()
Single cell life by means of iteratively executing
specializations of the cycle phases template function.
The Scheduler governs execution of these.
Rule: The youngest cell is the first to be processed.
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
10 / 18
Techniques we used . . .
Phases step by step
G2-Phase
Finish of cell growth, creation of
centrosomes
Brownian motion of dots
Detection of major and minor axis
inside cell shape using PCA
Location of centrosomes driven by
distance transform
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Prophase
Chromatin condensates and nucleoli
disappear
Dots belonging to one chromosome
tend to aggregate (move to mean
position) and form highly
condensed clusters
Nucleoli masks are removed form
the phantom
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Prophase
Chromatin condensates and nucleoli
disappear
Dots belonging to one chromosome
tend to aggregate (move to mean
position) and form highly
condensed clusters
Nucleoli masks are removed form
the phantom
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Metaphase
Move of chromosomes to metaphase
plate position
Ulman & Svoboda (CBIA)
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1
Nucleus membrane disappears
2
New positions for 23 chromosomes
in metaphase plate are randomly
generated
3
Old positions of 23 chromosomes,
spread over the cell, are assigned to
the new ones (min cost assignment
problem – Kuhn-Munkres
algorithm)
4
The movement of chromosomes
from old positions to the new ones
is generated
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Metaphase
Move of chromosomes to metaphase
plate position
Ulman & Svoboda (CBIA)
ISBIq
1
Nucleus membrane disappears
2
New positions for 23 chromosomes
in metaphase plate are randomly
generated
3
Old positions of 23 chromosomes,
spread over the cell, are assigned to
the new ones (min cost assignment
problem – Kuhn-Munkres
algorithm)
4
The movement of chromosomes
from old positions to the new ones
is generated
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Metaphase
Move of chromosomes to metaphase
plate position
Ulman & Svoboda (CBIA)
ISBIq
1
Nucleus membrane disappears
2
New positions for 23 chromosomes
in metaphase plate are randomly
generated
3
Old positions of 23 chromosomes,
spread over the cell, are assigned to
the new ones (min cost assignment
problem – Kuhn-Munkres
algorithm)
4
The movement of chromosomes
from old positions to the new ones
is generated
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Metaphase
Move of chromosomes to metaphase
plate position
Ulman & Svoboda (CBIA)
ISBIq
1
Nucleus membrane disappears
2
New positions for 23 chromosomes
in metaphase plate are randomly
generated
3
Old positions of 23 chromosomes,
spread over the cell, are assigned to
the new ones (min cost assignment
problem – Kuhn-Munkres
algorithm)
4
The movement of chromosomes
from old positions to the new ones
is generated
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Anaphase
Chromosomes are split and drawn to
opposite poles
Simple model of forces in which
each chromosome is pulled to one
centrosomes while the individual
chromosomes push away each other
In parallel the cell mask if slightly
expanded along major axis using
fast level set methods
Universal motion of cell without
additional deformations
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Anaphase
Chromosomes are split and drawn to
opposite poles
Simple model of forces in which
each chromosome is pulled to one
centrosomes while the individual
chromosomes push away each other
In parallel the cell mask if slightly
expanded along major axis using
fast level set methods
Universal motion of cell without
additional deformations
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Anaphase
Chromosomes are split and drawn to
opposite poles
Simple model of forces in which
each chromosome is pulled to one
centrosomes while the individual
chromosomes push away each other
In parallel the cell mask if slightly
expanded along major axis using
fast level set methods
Universal motion of cell without
additional deformations
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Telophase
Creation of nucleus membrane, nucleoli;
The chromatin is uncoiled
Masks of two new daughter nuclei
created
Masks of new nucleoli in each
nucleus created
The chromatin is uncoiled via
Brownian motion
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Telophase
Creation of nucleus membrane, nucleoli;
The chromatin is uncoiled
Masks of two new daughter nuclei
created
Masks of new nucleoli in each
nucleus created
The chromatin is uncoiled via
Brownian motion
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Telophase
Creation of nucleus membrane, nucleoli;
The chromatin is uncoiled
Masks of two new daughter nuclei
created
Masks of new nucleoli in each
nucleus created
The chromatin is uncoiled via
Brownian motion
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Telophase
Creation of nucleus membrane, nucleoli;
The chromatin is uncoiled
Masks of two new daughter nuclei
created
Masks of new nucleoli in each
nucleus created
The chromatin is uncoiled via
Brownian motion
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
Cytokinesis
Cell is pinched and new independent
daughter cells are created
PCA is used for detection for
optimal cut (minor eigenvectors
define cutting plane)
Not yet completely implemented as
this step is usually not noticeable
(too short)
Universal motion of cell without
additional deformations
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
G1-Phase
Growth of “new born daughter” cell to
full size
An expanding flow field stretches
all masks and moves dot positions
Brownian motion of dots
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
G1-Phase
Growth of “new born daughter” cell to
full size
An expanding flow field stretches
all masks and moves dot positions
Brownian motion of dots
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
G1-Phase
Growth of “new born daughter” cell to
full size
An expanding flow field stretches
all masks and moves dot positions
Brownian motion of dots
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
Phases step by step
S-Phase
Replication of DNA, i.e. growth of
nucleus
Brownian motion of dots
Dots are duplicated per partes until
all dots are duplicated
Universal motion of cell
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
11 / 18
Techniques we used . . .
. . . commonly
Universal motion of cell
It consists of
rigid: translation and rotation
non-rigid: decent local deformations of cell shape
translation is preferred towards widest escape-tunnel
in unconstrained environment: it simulates Brownian motion
rotation is Gaussian with mean 0deg and sigma 13deg
(+-15deg angles are with probability half of the probability for 0deg)
inflating-deflating flow field that preserves volume
in any case: coherency of motion is preserved
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
12 / 18
Techniques we used . . .
. . . commonly
Rendering of final images
It consists of
PSF simulation (simulates optics)
“Poissoned” gained signal (uncertainty in the number of incoming
photons)
added “little” Poisson (dark current, params from docs)
added Gaussian (read-out noise, params from docs)
two sets of params: low (cca 0.5dB) and high (cca 0.8dB) SNR
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
13 / 18
Results
demonstration of high and low SNR frames
demonstration of phanthom video
demonstration of final images video
Ulman & Svoboda (CBIA)
ISBIq
March 14, 2013
14 / 18
References
Simmons M. J., Snustad D. P. Genetika, Masarykova Univerzita, 2009
Reece J. B., Urry L. A., Cain M. L., Wasserman S. A., Minorsky P. V.,
Jackson R. B. Campbell Biology, 9th ed., Pearson Education, 2011
Krontorád-Koutná I., slides for course PV185 Biology Panorama I
(autumn 2012)
Liu L., Shell D. Assessing Optimal Assignment under Uncertainty: An
Interval-based Algorithm. International Journal of Robotics Research
(IJRR). vol. 30, no. 7, pp 936-953. Jun 2011
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March 14, 2013
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Any questions?
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March 14, 2013
16 / 18