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
Immune system: right site at right moment
The multistep model of leukocyte recruitment Blood flow
Tethering and Rolling
Activation
Arrest
Mucins, selectins, integrins
Chemokines or Chemoattractans
Integrins
Stabilization Spreading Transmigration
Endothelial cells ms
Immunodeficiency, autoimmune diseases and cancer
s min
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
Bioflux facility in Laudanna’s Laboratory
Glass capillaries vs Bioflux microfluidics Glass capillary
Bioflux microfluidics
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
BeQuanti
(for Behavior Quantification)
The Automated Tool for Cell Behavior Quantification
Laboratory of Cell Trafficking and Signal Transduction
Carlo Laudanna
Roberto Marzotto
Lara Toffali
Michela Farenzena
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).
BeQuanti: licensing » Trial free license Feature
Limitation
Time period
30 days
Number of sequences per video
Max 2 sequences
Sequence length
Max 500 frames
Batch mode
Not included
Export results
Not included
» 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
[email protected]
BeQuanti The Automated Tool for Cell Behavior Quantification
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
BeQuanti The Automated Tool for Cell Behavior Quantification
1. New project
2. Analysis
3. Data
1.
New project and Movie import
1.
Sequence Selection
1.
Edit Analysis Parameters
1.
Adhesion parameters
Every single cell detected
Adhesive behaviors (events) analysis
Subrolling corresponds to very short cell movements detected between two distinct arrest events for a defined cell (adhesion stabilization)
Subrolling threshold: 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
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.
1.
Adhesion parameters
1.
Global Analysis vs Parametric Analysis
Incremental T. A. T. 0,04 s
3s
Frequency of 1/fps
Fixed T. A. T.
1.
Start analysis
BeQuanti supports multiprocessor and multi-core architectures
BeQuanti The Automated Tool for Cell Behavior Quantification
2. Analysis
2.
Play Mode: detections
2.
Play Mode: Tracking
2.
Play Mode: Events
2.
Play Mode: Arrested Types
BeQuanti The Automated Tool for Cell Behavior Quantification
3. Data
3.
Results report: Parametric Analysis
Total
3.
Results report: Parametric Analysis
Normalized
3.
Target an individual cell directly on the movie
3.
Target an individual cell directly on the movie
25
3.
Results report: Cell Morphology Analysis
3.
Results report: Cell Morphology Analysis
3.
Results report: Global Adhesion Analysis
0,04 s
3s
Incremental T. A. T.
3.
Results report: Global Adhesion Analysis
0,04 s
3s
Total
3.
Results report: Global Adhesion Analysis
0,04 s
Normalized
3s
3.
Export results (cvs format)
3.
Example: h-PMN with PTx CELL TYPE:
Human primary neutrophils isolated 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)
PMN control
0,4
0,21
0,08
0,02
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!
Laboratory of Cell Trafficking and Signal Transduction Department of Pathology and Diagnostic, Division of General Pathology University of Verona
Carlo Laudanna
Roberto Marzotto
[email protected]
[email protected]
+39 0458027689
+39 045 8949180
Lara Toffali
Michela Farenzena
[email protected]
[email protected]
+39 0458027145
+39 045 8949180
http://dp.univr.it/~laudanna/LCTST/index.html
www.embeddedvisionsystems.it
[email protected]
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