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