Computational Modeling of

Fluorescence Loss in Photobleaching

Motivation Reaction-Di↵usion Model Simulation of FLIP images

From today’s research it is well known that diseases like To present a model allowing transport across the mem-
Alzheimer, Parkinson, Corea Huntington and Arterioscle- brane, we now distinguish between the concentrations in- To simulate the experiment, we compute
rosis are caused by a jam in intracellular membrane traffic side cytoplasm u and nucleus v, let u and v be defined on the FLIP model (5) and (4) on the mesh
displayed in Fig. 3. The mesh, consist-
[8]. Hence to improve treatment, a quantitative anal- ⌦ such that: ing of 221558 triangles (68534 in cyto.,
ysis of intracellular transport is essential. One impor- u(t, x) = 0 for x 2 ⌦N , 114 in bleaching area, 89077 in membrane,
tant method for visualization of the transport processes and 63833 in nucleus), was generated by
v(t, x) = 0 for x 2 ⌦C . (1) Gmsh [4] on the Chan–Vese contours. The
in living cells, is the so called Fluorescence loss in pho-
tobleaching (FLIP). Since FLIP is a modern and widely Consequently, c = u + v denotes the concentration of model parameters are: ✏ = 10 4, k = 104,
used microscopy method, the motivation for making an eGFP in ⌦. Here the transport across the membrane is bq/(1 + q) = 160 with di↵usion rates given
by Figure 3: Mesh
automated reliable analysis of the image data is high. modeled as a direct transport process by the reversible 8 8
3
chemical reaction equation: >
<25 if x 2 ⌦C >
<10 if x 2 ⌦C
↵ = 5 · 10 4 = 2.5 · 10 4
k+ if x 2 ⌦M , if x 2 ⌦M .
u )* v, (2) >
: 3 >
:
10 if x 2 ⌦N 25 if x 2 ⌦N
k

Fluorescence Loss In Photobleaching (FLIP) where k + and k are positive reaction rates. Initial data as seen in Fig. 4 are logistic functions across
From [7] we find that the photobleaching may be mod- the membrane. The max–values are taken as intensity
In studies of fluorescently tagged proteins one of the most eled by the di↵erential equation: averages from the initial FLIP image.
commonly used techniques is FLIP, where a cellular region @ q
is photobleached using a high-intensity laser pulse. u(t, x) = b · u(t, x) , x 2 ⌦B , t > 0 , (3)
@t q+1
In FLIP, a small cellular region is repeatedly bleached,
while images are taken in between the bleaches. This where b is the intrinsic bleaching rate constant and q is a
procedure is illustrated in Figure 1. equilibrium constant.

Figure 4: Initial Figure 5: Bleach Figure 6: t = 17 Figure 7: t = 33

In both the FLIP images and the simulation, we ob-

serve a stronger e↵ect of bleaching in the cytoplasm than
Figure 1: FLIP. Here bleaching occurs in cytoplasm, the lighter area in the nucleus — the membrane shows its protective e↵ect.
illustrates nucleus.
FLIP is often used to study the cellular structure through
the movement of proteins and is a very popular technique.
But in spite of its popularity an automated,
reliable analysis of image data is still lacking.
So far only few FLIP analyses published, include the whole
image information in the analysis [6, 7]. However it is well
known that the transport of the fluorescent protein eGFP
Figure 8: Average intensity in cy- Figure 9: Average intensity in nu-
between nucleus and cytoplasm is slower than the di↵usion toplasm. cleus.
inside cytoplasm and nucleus [3, 9].
Simulated average intensities are depicted in Figs. 8
and 9. Even though simulated intensities follow the ob-
servations, initially the intensity in the cytoplasm drops
faster than observed in FLIP images. This defect may be
attenuated by further (automated) tuning of reaction and
di↵usion rates in future work.
(a) Not bleached (b) 14.4 s (c) 30.4 s (d) 62.4 s

Figure 2: Di↵usion-limited FLIP of eGFP in the cytoplasm of a McA

√  
cell with frame rate 1.6 sec.
Ongoing and future work
Until now we have been focusing on active transport across
the membrane. However studies indicates that eGFP
di↵uses through the Nuclear Pore Complex. By assum-
Chan-Vese Segmentation ing such a passive transport across a porous membrane,
An important step towards a reliable FLIP simulation is the higher florescence intensity inside nucleus can only
to specify the geometry of any given cell. occur if eGFP binds to other molecules. If we adapt
By Chan-Vese’s algorithm [2, 5], which automatically this idea it could mathematically be modeled by using
Assuming that bleaching only occurs in the bleaching
detect the borders, the FLIP image can be segmented into KedemKatchalsky conditions and as a consequence we can
area and eGFP has a di↵usive behavior that follows the
background and foreground that may include the cell with omit the membrane domain. In FLIP images we observe
multidimensional di↵usion equation, our time-dependent
its irregular borders, the nucleus or the bleaching area. spatially resolved structures especially within the nucleus.
PDE-model with membrane transport reads:
The result of the segmentation is a level set function By using spatially resolved reaction parameters to rep-
L, where the zero level = {x 2 R2|L(x) = 0} is the resent binding between eGFP and other molecules, the
q model will develop structures. Thus by use of a Reaction-
closed contour which describes the segmented area. Now ut = r · (↵ru) + k v k +u ✓b 1+q u ,
let I(x) be the grayscale image, c and c+ the average ⌦M ⌦B Di↵usion equation in the entire cell, Kedem–Katchalsky
inside and outside respectively. With this in mind, let conditions at the membrane and Discontinuous Galerkin
vt = r · ( rv) + k +u k v , x2⌦, t>0,
us now consider the Chan-Vese fitting energy: ⌦M for solving the system we get simulations of a cell with
(4) structures as observed on the FLIP images. The result
E(c , c+, ) =F0( ) + F1( ) + F2( ) where ✓ is a 0–1 step function simulating the high–intensity of the simulation can be seen at the center of this poster.
Z Z
=µ d + 1 (I(x) c )2dx laser pulses occurring at a frequency given by the frame Further work would be to fully describe, understand and
Z L<0 rate. Di↵usion coefficients are piecewise defined, at the analyze this model.
+ 2 (I(x) c+)2dx , cell boundary we use Neumann boundary conditions. Ex-
L>0 amples for reaction rates k ± are logistic functions
where µ 0, 1, 2 > 0 are fixed parameters. ± 1 1
By keeping c and c+ fixed and minimize the Chan- k (x) = , (5)
✏ 1 + exp (±k · dist(x xm))
Vese fitting energy with respect to L, we can obtain the References
0<✏⌧1 , k 0 .
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Dynamics and Di↵erential Equations, 9(1):93–131, (1997).
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" ✓ ◆ # keeps u = 0 in nucleus and v = 0 in cytoplasm. cessing vol. 10, no. 2, , pp. 266-277, (2001).
rL
Lt = "(L) µ div + 1(I c )2 2 (I c + ) 2 . [3] Y. Chen et al., Probing Nucleocytoplasmic Transport by Two-Photon Activation of PA-
GFP, Microsc. Res. Tech. 69:220-226, (2006).
|rL|
[4] http://geuz.org/gmsh/
[5] Ch.V. Hansen, Segmentation of Fluorescent Microscopy Images of Living Cells, Bachelor
Here we introduced an artificial Project, IMADA, SDU, (2012).
time parameter to make an ac- [6] J. Lippincott-Schwartz and G. H. Patterson Development and Use of Fluorescent Protein
Markers in Living Cells, Science 300, 87 (2003).
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of Chan-Vese segmentation, the cell Phd in Applied Mathematics, age correlation spectroscopy for quantitative analysis of endosome motility, Journal of Mi-
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segmented into cytoplasm ⌦C , nu- Mail: cvh@imada.sdu.dk Traffic, 3 (2002) 781-790.
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