depth intensity correction of biofilm volume data from clsm karsten rodenacker 1, martina hausner 2,...
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Depth Intensity Correction of Biofilm Volume Data From CLSM
Karsten Rodenacker1, Martina Hausner2, Martin Kühn2, Stefan
Wuertz2, Sumitra Purkayastha3
1GSF-IBB, 2TU München, Germany3ISI, India
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Content Introduction Material and Methods Results Summary and Discussion
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Introduction Biofilm
Biofilm
X
Y
Z
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Introduction Biofilm
Histogram of a volume block
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Introduction Biofilm
Biofilm (projection in Y)
Surface
IntensityDecay in depth
X
Z
Y
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Introduction Assumptions
Grown from surface (substratum) layer
Similar attachment of fluorescent stain, especially independent on depth
Semi-3-D arrangement (umbra type),some continuity in depth
Material distributed in all depths
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Introduction First observations Tissue section (projection in Y)
Surface
Intensitydecay
X
Z
Y
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Introduction Observations
Intensity decay
depth
frequency
Intensity
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Introduction Observations
Intensity decayundermorph.openingandexpon.fitting
after opening 1
after opening 2
after opening 3
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Introduction Idea
Correction by the decay function for reliable segmentationProblem: Estimation Limitations
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Introduction Goals
Reliable segmentation for - Volume estimationand - estimation of local relationship of objects and of objects in clusters
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Introduction Partial Solutions
Iterative methodsusing approximated correction after Visser et al. and Roerdink et al.
Depth table correction (Rigaut & Vassy).
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Material Hybridized biofilm
- FISH (Fluor. In Situ Hybridization) - EYFP (Enh. Yellow Fluorescent Protein)
Image (volume) acquisition with Zeiss LSM410 (voxelsize .0625 µm3=.25x.25x1. (µm)3)
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Material Flowchannel
Position ofprobe
Direction of flow
01 07 13
19
X
Y
Z
...
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Methods Model
Decay caused by - the surrounding medium - occlusion (shadowing)
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Methods Model
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Methods Model
1'1
'1
'2
0
1
)(log
)(log
QTT
ttT
T
tT
tT
teQTT 20
'2
t3
T0
T1 (t3)
T2 (t3)
Depth
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Methods Depth
- histograms - quantiles (p=.999) - offset by saturation - intensity decay fit
Frequency
Depth
Intensity
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Results Measurements
Volume: RED Volume: GREEN Volume: GREEN in RED Volume: GREEN in dilated(RED) Volumes in Clusters gen. from RED
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Results Measurements
Volumes in voxel
RED
GREEN
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Results Measurements
RED
GREEN
GREEN in RED
GREEN in dilated(RED)
Gain by correction
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Summary and Discussion Disadvantages Advantages
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Discussion Disadvantages
Scaling problems Verification of correction Possible errors by deviations from
the assumptions
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Discussion Disadvantages
+ Grown from surface (substratum) layer+ Similar attachment of fluorescent stain,
especially independent on depth
? Semi-3-D arrangement (umbra type),some continuity in depth
? Material distributed in all depths
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Discussion Advantages
Simplicity multiplicative correction (table operation)
Little computational effort a histogram above all data 255 quantiles