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Depth Intensity Correction of Biofilm Volume Data From CLSM

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. Content. Introduction Material and Methods Results Summary and Discussion. Y. X.

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Depth Intensity Correction of Biofilm Volume Data From CLSM

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  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

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