1. Depth Intensity Correction of Biofilm Volume Data From CLSM Karsten Rodenacker 1, Martina Hausner 2, Martin Kühn 2, Stefan Wuertz 2, Sumitra Purkayastha 3 1 GSF-IBB, 2 TU München, Germany 3 ISI, India

2. Content Introduction Material and Methods Results Summary and Discussion

3. Introduction Biofilm Biofilm X Y Z

4. Introduction Biofilm Histogram of a volume block

5. Introduction Biofilm Biofilm (projection in Y) Surface Intensity Decay in depth X Z Y

6. 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

7. Introduction First observations Tissue section (projection in Y) Surface Intensity decay X Z Y

8. Introduction Observations Intensity decay depth frequency Intensity

9. Introduction Observations Intensity decay under morph. opening and expon. fitting after opening 1 after opening 2 after opening 3

10. Introduction Idea Correction by the decay function for reliable segmentation Problem: Estimation Limitations

11. Introduction Goals Reliable segmentation for - Volume estimation and - estimation of local relationship of objects and of objects in clusters

12. Introduction Partial Solutions Iterative methods using approximated correction after Visser et al. and Roerdink et al. Depth table correction (Rigaut & Vassy).

13. Material Hybridized biofilm - FISH (Fluor. In Situ Hybridization) - EYFP (Enh. Yellow Fluorescent Protein) Image (volume) acquisition with Zeiss LSM410 (voxelsize.0625 µm 3 =.25x.25x1. (µm) 3 )

14. Material Flowchannel Position of probe Direction of flow 01070713 1919 X Y Z...

15. Methods Model Decay caused by - the surrounding medium - occlusion (shadowing)

16. Methods Model

18. Methods Depth - histograms - quantiles (p=.999) - offset by saturation - intensity decay fit Frequency Depth Intensity

19. Results Measurements Volume: RED Volume: GREEN Volume: GREEN in RED Volume: GREEN in dilated(RED) Volumes in Clusters gen. from RED

20. Results Measurements Volumes in voxel

21. Results Measurements Gain by correction

22. Summary and Discussion Disadvantages Advantages

23. Discussion Disadvantages Scaling problems Verification of correction Possible errors by deviations from the assumptions

24. 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

25. Discussion Advantages Simplicity multiplicative correction (table operation) Little computational effort a histogram above all data 255 quantiles