High content quantification of foci

spatial organization in the nucleus

Sylvain V. Costes
Lawrence Berkeley National Laboratory

Funding:
NASA – LBNL NSCOR
National Aeronautics and Space Administration Grant no. T6275W, NASA
Specialized Center for Research in Radiation Health Effects
A
Why Quantify Biological ImagesB?

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 Fig. A, C Fig B, D
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 Quantitative Biology and imaging:
A growing trend
Number of publications
on “Fluorescence Microscopy”
• In the U.S. there are between 0.5-1
million people in the academy who
1400 have to deal with storing and accessing
scientific microscope images.
1200 – There are over 2600 accredited
universities in the US.
1000 – Most universities have at least one
imaging core facility. Many have
800 multiple from individual investigators
who own their own microscopy system.
600
– Core facilities usually serve about 50 to
400 100 research groups. Average research
group size is usually 4 people.
200

0
• There is also the industrial sector
(biotech, medical devices, etc.)
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Earth’s magnetic field protects us
from cosmic radiation
Space radiation – HZEs are predominant

From Zeitlin and Cuccinotta

Estimated 1 out of 50 cells will be traversed by Fe ion during a 3-year

Mars Mission. Curtis and Letaw, 1989
HZE = High Energy and Charged particles
 Physical properties of HZE particles
50% of energy of an 1 GeV/amu Fe is deposited via
excitation, and the other half as ionization.
Excitation energy is dissipated locally leading to high
dose in the core, whereas ionization leads to emission
of high energy electrons, called delta-rays which will
rc = 9 nm dissipate the energy radially with a 270 µm range.
rp = 270 µm
Radial profile of energy deposition, perpendicularly Example: Dose deposited in the
to HZE traversal (1 GeV/amu Fe) core of 1 GeV/amu Fe
Rc~9 nm, LET~150 keV/µm
D = Ecore/Mcore
Ecore = 0.5xLET/L (half of LET is going
into excitation, L is the considered length of
Dose profile in traversal)
penumbra range, Mcore = ρ.L.πRc2 (Mass of core, assume
~1/r2 water)
D = 0.5xLET/ (ρ.πRc2)
D = 0.5x1.5e5x1.6e-19/(π[9e-3]2.1e-15)
D ~ 47,000 Gy (close to reported 48 kGy)
 Nucleus in silico to predict radiation damage *

• One monomer = 2 kbp

• Each chromosome =
about 100,000
monomers
• Sizes of
chromosomes are
precise
• 1.6 million monomers
in the whole genome
• 3.2 Gbp in the whole
genome

* Track model: Cucinotta, F. A., Katz, R., Wilson, J. W., Dubey, R. R., 1995, Radial dose distributions in
the delta-ray theory of track structures. Proceedings of two-center effects in ion-atom collisions, AIP
Conference Proceedings (T.J. Gray and A.F. Starace Eds.), Woodbury, NY, 1995, pp. 245-265.
Cucinotta, F. A., Nikjoo, H., Goodhead, D. T., 1999, Applications of Amorphous Track Models in Radiation
Biology. Radiation and Environmental Biophysics, 38, 81-92.
Chromosome model:
A.L. Ponomarev, H. Nikjoo, F.A. Cucinotta, NASA Radiation Track Image GUI for Assessing Space
Radiation Biological Effects , American Institute of Aeronautics and Astronautics (AIAA), 2005
Comparison between simulated and experimental images for
different types of radiation

Real RIF 2 µm pseudoRIF

[0.69,0.82] RIF/µm 0.7 +/- 0.2 pRIF/µm

Costes et al, PLoS Computational Biology, Aug 2007

Predictions need to be based on real
nuclear patterns
• DSB generation is stochastic with DNA density as a
weighting function
– DNA pattern in a real nuclear image is unique
– Therefore, DSB pattern is unique
• We use DNA imaging pattern to predict DSB pattern
for any given nuclear image
– Propose an image-based modeling of DNA damage pattern
– Validated it using theoretical images generated with
NASA DSB code (Costes et al, PLoS Comp. Bio. Aug
2007)
 Image-based modeling of DNA damage
pattern in real images
Initially detected RIF for Predicted RIF pattern (red)
corresponding green profile

1D projection of DNA Reshuffle RIF position

density profile (blue) using DAPI profile as
probability function (done
iteratively)

• Typical field of view with cells that have been traversed with 1 Gy of 1
GeV/amu Fe ions.
• After selecting a track in a semi-manual way, foci detection is done
automatically via in-house image algorithm
• Expected pattern is predicted by simply reshuffling randomly position of
detected foci
 Comparison of image prediction and Monte
Carlo simulation for DNA damage distance
distribution
25

20 pRIF
Reshuffled pRIF
% frequency

DSB 2.1 µm 1.6 µm 1.3 µm

15

10 Correlation = 0.95 1.9 µm 2.2 µm 1.1 µm

DSB/pRIF/blurred DNA
0
0 1 2 3 4 5 6
Maximum Intensity Profile
Distance between consecutive foci along track (um)
along white box

DSB
Blurred DNA
Blurred DSB (pRIF)

0.16 µm 0.32 µm
How to detect tracks on large data set?
 Track Analysis

• User draws “rough” estimates through nuclei containing tracks.

• Automated program fine tunes the angle of the track and detects all
occurrences within the nuclei. Only considers nuclei with an estimated track
drawn through.
• 1.5 min/image. 200 images/data set. 600 min (5 hours)
 Track detection
Sum projection Detect peaks in profile:
- Position should match
track location

Keep angle with following criteria:

-Minimum number of peaks
-Highest peaks
 Comparing theoretical and experimental
DNA damage pattern along 1 GeV/amu
Fe tracks – γH2AX
25 25
4 independent experiments RIF 4 independent experiments RIF
20 501 tracks, 0.69+/-0.03 RIF/um Predicted damage 20 232 tracks, 0.61+/-0.02 RIF/um Predicted damage
50 iterations for reshuffling 50 iterations for reshuffling
% frequency

% frequency
Correlation = 0.60+/-0.04 Correlation = 0.45+/-0.07
15 15

10 10
4.5 min post IR 35 min post IR
5 5
A B
0 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Distance between consecutive foci along track (um) Distance between consecutive foci along track (um)

1. Increase deviation from randomness of measured distances between

foci
2.Exclusion of close-by foci
Non-random position of foci
ATM

53BP1
γH2AX

10 mn post 1 GeV/amu Fe
 Unfolding DNA
DNA double helix 2 nm

“Beads-on-a-string” 11 nm
form of chromatin

Chromatin fiber of 30 nm
packed nucleosomes
Extended section of
chromosome 300 nm

Condensed section of
chromosome with 700 nm
rhombic loops
Metaphase 1400 nm
chromosome
 Chromatin structure and damage
The high-order structure of DNA is shown on the level of
chromatin fiber. The source is from
http://sgi.bls.umkc.edu/waterborg/chromat/chroma09.html
A piece of DNA depicted is about the size of one monomer that will
be a building block for the whole genome in this work.
 Illustration of Rdna and Rgrad
measurements
A C E
∑ I (i)
i = focus
DAPI

N focus
Rdna =
∑ I (i)
i = nucleus
N nucleus
Rdna = 1.20 Rdna = 0.94 Rdna = 0.81

B D F
Gradient(DAPI)

∑ ∇I (i)
i = focus
N focus
Rgrad =
∑ ∇I (i)
i = nucleus
N nucleus

Rgrad = 0.30 Rgrad = 1.88 Rgrad = 0.29

Foci at high DAPI Foci at interface Foci at low DAPI

 Monitoring localization of RIF after
exposure to 1 Gy of 1 GeV/u Fe
1.15
*(5) Rgrad
*(4)
*(4)
Ratio

1.00

* Rdna
*
* γH2AX
A 0.85
0 20 40 60
Time post IR (min)
1.15
*(11)
*(5) Rgrad
*(5)
Ratio

1.00

* 53BP1 Rdna
*
γH2AX, 3 min after 1 GeV/amu Fe
B 0.85
0 20 40 60

1.15 Time post IR (min)

*(4)
Rgrad
Ratio

1.00

* Rdna
ATMp

C 0.85
0 20 40 60
Time post IR (min) Costes et al, PLoS Computational Biology, Aug 2007
 Conclusion and future directions
• RIF are detected in locations different from where damages
occur.
– However foci frequency along tracks match prediction (not true for low
LET)
• If RIF contain DSB, does this reflect
– DSB movement
– Chromatin remodeling
• Impact?
– Including DSB active movement as a function of chromatin
organization in current risk assessment models is an important step
towards mechanistic models for radiation-induced cancer risk
calculations.
• Can we resolve some uncertainties using live cell imaging?
Raw image
Step 1: registering reference, i.e.
nucleus
 Registration process
• Z-stack are collapsed into a sum projection
• Consecutive 2D frames are then registered using a third
party algorithm:
http://www.cs.dartmouth.edu/~farid/research/registration.html
• Third party registration algorithm (Matlab):
– the transformation between images is modeled as locally affine but
globally smooth
– local and global variations in image intensities are taken into
account
– Translation, rotation and scaling matrices are obtained between
consecutive time frames
• Transformation applied to all channels with Z left invariant
Applying registration to signal of
interest: foci
 1.8

1.6
ratio value

1.4
Slide31_pos4 rdna ratio
1.2 Slide31_pos4 rgrad ratio
Log. (Slide31_pos4 rgrad ratio)
1

0.8
5.00 10.00 15.00 20.00
time (min)
 Live cell with 53BP1-GFP - early
V = 2 µm/min

1 µm

2 min + 6 sec + 12 sec + 36 sec

1 µm

2 min + 6 sec + 42 sec + 84 sec

1 µm
Final time: 3.5 min
+ 6 sec + 24 sec + 42 sec +84 sec

2 min post IR, 10 frames/min

 Live cell with 53BP1-GFP - late
1 µm

26 min + 6 min + 9 min + 50 min

1 µm

26 min + 9 min + 50 min + 2h40min

26 min post-IR
2,10 and 30 min time interval
100% Low-LET High-LET High-LET
(Foci/DSB) 100% (Relative foci/ µm)
80% 100%
80% (Foci/Track)
60%
60%
40% 40% 75%
20% Foci
DSB 20%
0% 0% 50%
0 20 40 60 0 20 40 60 0 20 40 60
Time (min) Time (min) Time (min)
Z Z Z

Y Y Y

X
A B C
DAPI-γH2AX overlay

5 µm
γH2AX

2 min, RT 10 min, 37oC 60 min, 37oC 48 hrs, 37oC

 CONCLUSION
• These data support the hypothesis that RIF relocalize to
specific nuclear sub-domain within the first minutes
following exposure to radiation
• Integration of biophysical model with high content
quantitative data has led to a better understanding of the
biological consideration affecting foci formation
• Live cell imaging is necessary to resolve the complex
dynamic involved in sensing DNA damage. More
elaborate live cell experiments will be conducted soon.
• Foci seem to mark a change in the chromatin state.
Discuss model and how this shifts from usual paradigm…
 Chromatin status at foci location

Small chromatin Large chromatin

expansion (15 Mbp) Permanently modified chromatin
expansion (2-4 Mbp)

•Senescence
4-96 hrs Days •Growth arrest
OR

• Complex DSBs in •Mis-repaired DSB

heterochromatin •Altered chromatin
•All DSBs in euchromatin

Aberrant
phenotype
Acknowledgements
Chair of NSCOR:
Mary Helen Barcellos-Hoff

NASA collaborators:
Artem Ponomarev
Francis Cucinotta

LBNL - imaging:
James L. Chen
Damir Sudar

LBNL – constructs:
Philippe Gascard
Berbie Chu
David Nguyen