Be Quanti : software for automatic detection and analysis of cell adhesion underflow

1. BeQuanti: software for automatic detection and analysis of cell adhesion underflow  Lara Toffali1, Michela Farenzena2, Roberto Marzotto2 and Carlo Laudanna1 1 Laboratory of Cell Trafficking and Signal Transduction Department of Pathology and Diagnostic, Division of General Pathology, University of Verona 2 eVS embedded Vision Systems, Verona

2. Immune system: right site at right moment

3. The multistep model of leukocyte recruitment Blood flow Stabilization Spreading Transmigration Arrest Integrins Tethering and Rolling Mucins, selectins, integrins Activation Chemokines or Chemoattractans Leukocyte Endothelial cells ms s  min Immunodeficiency, autoimmune diseases and cancer

4. Bioflux facility in Laudanna’s Laboratory Laboratory of Cell Trafficking and Signal Transduction Department of Pathology and Diagnostic, Division of General Pathology University of Verona

5. Bioflux facility in Laudanna’s Laboratory

6. Glass capillaries vs Bioflux microfluidics Bioflux microfluidics Glass capillary

7. Underflow adhesion assays with Bioflux Leukocytes adhesion uderflow at 2 dyne/cm2 Selectin ICAM-1 Chemokines Recording: 25 frames/s Movie length: 5-10 minutes = Total frames: 7500-15000 Manually performed movie analysis Limited Time consuming Prone to errors Tedious

8. BeQuanti (for Behavior Quantification) The Automated Tool for Cell Behavior Quantification Laboratory of Cell Trafficking and Signal Transduction Roberto Marzotto Michela Farenzena Carlo Laudanna Lara Toffali

9. BeQuanti comparison with other packages Leucotrak: Willenbrock, F. et al., Biophys J. 105, 1110-22 (2013). ImageJ: Meyer dos Santos, S. et al. Platelets.21, 60-6 (2010). ImagoQuant: Giegold, O. et al., Exp Dermatol.18, 238-45 (2009).

10. BeQuanti: licensing • Trial free license • Full license • No features limitations • Node lock license • Updates included up to the next major release • Unlimited technical support eVS embedded Vision Systems Srl www.embeddedvisionsystems.it info@evsys.it

11. The Automated Tool for Cell Behavior Quantification BeQuanti DV, MPEG, Mov and AVI For each frame a list of cell positions (x, y) A list of cell, for each cell the frames and position in which it appears Tethering, Rolling, Subrolling Arrest and Unclassified Round, Polarized, Spread and Unknown

12. BeQuanti The Automated Tool for Cell Behavior Quantification 1. New project 2. Analysis 3. Data

13. 1. New project and Movie import

14. 1. Sequence Selection

15. 1. Edit Analysis Parameters

16. 1. Adhesion parameters Subrolling corresponds to very short cell movements detected between two distinct arrest events for a defined cell (adhesion stabilization) Every single cell detected Adhesive behaviors (events) analysis Spatial Arrest Threshold S.A.T. maximal tolerated small cell movements distinguishing arrest versus rolling Temporal Arrest Threshold T.A.T. temporal cutoff defining arrest versus rolling Subrolling threshold: maximal tolerated small cell movements (between two distinct arrest events) distinguishing subrolling versus rolling Cell arrest is fully defined by the combination of S.A.T. and T.A.T.

17. 1. Adhesion parameters

18. 1. Global Analysis vs Parametric Analysis Incremental T. A. T. Fixed T. A. T. 0,04 s 3 s Frequency of 1/fps

19. 1. Start analysis BeQuanti supports multiprocessor and multi-core architectures

20. BeQuanti The Automated Tool for Cell Behavior Quantification 2. Analysis

21. 2. Play Mode: detections

22. 2. Play Mode: Tracking

23. 2. Play Mode: Events

24. 2. Play Mode: Arrested Types

25. BeQuanti The Automated Tool for Cell Behavior Quantification 3. Data

26. 3. Results report: Parametric Analysis Total

27. 3. Results report: Parametric Analysis Normalized

28. 3. Target an individual cell directly on the movie

29. 3. Target an individual cell directly on the movie 25

30. 3. Results report: Cell Morphology Analysis

31. 3. Results report: Cell Morphology Analysis

32. 3. Results report: Global Adhesion Analysis 0,04 s 3 s Incremental T. A. T.

33. 3. Results report: Global Adhesion Analysis 0,04 s Total 3 s

34. 3. Results report: Global Adhesion Analysis 0,04 s Normalized 3 s

35. 3. Export results (cvs format)

36. 3. Example: h-PMN with PTx PMN control CELL TYPE: Human primary neutrophilsisolated from healthy volunteers by standard gradient centrifugation (Ficoll). Contaminating erythrocytes were removed by dextran-500 sedimentation followed by hypotonic lysis. TREATMENT: Primary neutrophils were treated or not with 2 μg/ml Pertussis Toxin (PTx) for 3 h at 37°C COATING: 1 ug/ml P-Selectin, 5 μg/ml ICAM-1 and 25 nM FMLP PARAMETERS: S.A.T. 5 μm – T.A.T. 2s – Sub-rolling threshold 20 μm PMN treated with Pertussis Toxin (2 μg/ml) 0,4 0,21 0,02 0,08

37. Future developments • Detection of different kind of cells • Analysis of cell behavior on different backgrounds, including adhesion: • - on endothelial cells • - in different experimental settings (Intravital microscopy or glass capillary) • Detection of cell motility underflow, such as cell crawling and transmigration BeQuanti was developed and tested to analyze underflow experiments performed with BioFlux system at 20x phase contrast magnification BeQuanti is fully customizable upon request!

38. Laboratory of Cell Trafficking and Signal Transduction Department of Pathology and Diagnostic, Division of General Pathology University of Verona Roberto Marzotto roberto.marzotto@evsys.it +39 045 8949180 Carlo Laudanna carlo.laudanna@univr.it +39 0458027689 Lara Toffali lara.toffali@univr.it +39 0458027145 Michela Farenzena michela.farenzena@evsys.it +39 045 8949180 www.embeddedvisionsystems.it http://dp.univr.it/~laudanna/LCTST/index.html info@evsys.it

39. Image processing details • Cells Detection: cells classification by a discriminative classifier trained on cells images, based on gradient orientation • Cells Tracking: global optimal matching • Events Classification: exhaustive recursive search • Cells Morphology Classification: shape and color based classification