1. Depth Intensity Correction of Biofilm Volume Data From CLSM Karsten Rodenacker1, Martina Hausner2, Martin Kühn2, Stefan Wuertz2, Sumitra Purkayastha31GSF-IBB, 2TU München, Germany3ISI, India

2. Content • Introduction • Material and Methods • Results • Summary and Discussion

3. Y X Z Introduction Biofilm • Biofilm

4. Introduction Biofilm Histogram of a volume block

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

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

8. Introduction Observations • Intensity decay frequency Intensity depth

9. Introduction Observations • Intensity decayundermorph.openingandexpon.fitting after opening 1 after opening 2 after opening 3

10. Introduction Idea • Correction by the decay function for reliable segmentationProblem: Estimation Limitations

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

12. Introduction Partial Solutions • Iterative methodsusing 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 µm3=.25x.25x1.(µm)3)

14. 01 07 13 19 MaterialFlowchannel • Position ofprobe Z X Y ... Direction of flow

15. Methods • ModelDecay caused by - the surrounding medium - occlusion (shadowing)

16. Methods Model

17. Methods Model

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

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