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
Content Introduction Material and Methods Results Summary and Discussion
Introduction Biofilm
Biofilm
X
Y
Z
Introduction Biofilm
Histogram of a volume block
Introduction Biofilm
Biofilm (projection in Y)
Surface
IntensityDecay in depth
X
Z
Y
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
Introduction First observations Tissue section (projection in Y)
Surface
Intensitydecay
X
Z
Y
Introduction Observations
Intensity decay
depth
frequency
Intensity
Introduction Observations
Intensity decayundermorph.openingandexpon.fitting
after opening 1
after opening 2
after opening 3
Introduction Idea
Correction by the decay function for reliable segmentationProblem: Estimation Limitations
Introduction Goals
Reliable segmentation for - Volume estimationand - estimation of local relationship of objects and of objects in clusters
Introduction Partial Solutions
Iterative methodsusing approximated correction after Visser et al. and Roerdink et al.
Depth table correction (Rigaut & Vassy).
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)
Material Flowchannel
Position ofprobe
Direction of flow
01 07 13
19
X
Y
Z
...
Methods Model
Decay caused by - the surrounding medium - occlusion (shadowing)
Methods Model
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
Methods Depth
- histograms - quantiles (p=.999) - offset by saturation - intensity decay fit
Frequency
Depth
Intensity
Results Measurements
Volume: RED Volume: GREEN Volume: GREEN in RED Volume: GREEN in dilated(RED) Volumes in Clusters gen. from RED
Results Measurements
Volumes in voxel
RED
GREEN
Results Measurements
RED
GREEN
GREEN in RED
GREEN in dilated(RED)
Gain by correction
Summary and Discussion Disadvantages Advantages
Discussion Disadvantages
Scaling problems Verification of correction Possible errors by deviations from
the assumptions
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
Discussion Advantages
Simplicity multiplicative correction (table operation)
Little computational effort a histogram above all data 255 quantiles
