BeQuanti: software for automatic
detection and analysis of cell
adhesion underflow
Laboratory of Cell Trafficking and Signal TransductionDepartment of Pathology and Diagnostic,
Division of General Pathology, University of Verona
Lara Toffali1, Michela Farenzena2, Roberto Marzotto2 and Carlo Laudanna1
eVS embedded Vision Systems, Verona
1
2
Immune system: right site at right moment
Tethering and Rolling Mucins, selectins, integrins
ActivationChemokines or Chemoattractans
ArrestIntegrins
StabilizationSpreading
Transmigration
LeukocyteLeukocyte
Blood flow
Endothelial cells
The multistep model of leukocyte recruitment
ms s min
Immunodeficiency, autoimmune diseases and cancer
Laboratory of Cell Trafficking and Signal TransductionDepartment of Pathology and Diagnostic, Division of General Pathology
University of Verona
Bioflux facility in Laudanna’s Laboratory
Bioflux facility in Laudanna’s Laboratory
Glass capillaries vs Bioflux microfluidics
Glass capillary Bioflux microfluidics
Leukocytes adhesion uderflow at 2 dyne/cm2
Underflow adhesion assays with Bioflux
Recording: 25 frames/sMovie length: 5-10 minutes = Total frames: 7500-15000
Selectin ICAM-1
Chemokines
Manually performed movie analysis
LimitedTime consumingProne to errors
Tedious
Roberto Marzotto
Michela Farenzena
Carlo Laudanna
Lara Toffali
Laboratory of Cell Trafficking and Signal Transduction
BeQuantiThe Automated Tool for Cell Behavior Quantification
(for Behavior Quantification)
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 comparison with other packages
BeQuanti: licensing
» Trial free license
» Full license» No features limitations
» Node lock license
» Updates included up to the next major release
» Unlimited technical support
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
eVS embedded Vision Systems Srl
www.embeddedvisionsystems.it
BeQuantiThe Automated Tool for Cell Behavior Quantification
DV, MPEG, Mov and AVI
Tethering, Rolling, SubrollingArrest and Unclassified
Round, Polarized,Spread and Unknown
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
1. New project 2. Analysis 3. Data
BeQuantiThe Automated Tool for Cell Behavior Quantification
New project and Movie import1.
Sequence Selection1.
Edit Analysis Parameters1.
Adhesion parameters1.
Every single cell detected
Adhesive behaviors (events) analysis
Spatial Arrest ThresholdS.A.T.
maximal tolerated small cell movements distinguishing
arrest versus rolling
Temporal Arrest Threshold T.A.T.
temporal cutoff defining arrest versus rolling
Cell arrest is fully defined by the combination of S.A.T. and T.A.T.
Subrolling threshold:
maximal tolerated small cell movements (between two
distinct arrest events)distinguishing subrolling
versus rolling
Subrolling corresponds to very short cell movements
detected between two distinct arrest events for a
defined cell (adhesion stabilization)
Adhesion parameters1.
Global Analysis vs Parametric Analysis1.
Incremental T. A. T.
0,04 s 3 s
Fixed T. A. T.
Frequency of 1/fps
Start analysis1.
BeQuanti supports multiprocessor and
multi-core architectures
2. Analysis
BeQuantiThe Automated Tool for Cell Behavior Quantification
Play Mode: detections2.
Play Mode: Tracking2.
Play Mode: Events2.
Play Mode: Arrested Types2.
3. Data
BeQuantiThe Automated Tool for Cell Behavior Quantification
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 Analysis3.
Results report: Cell Morphology Analysis3.
Results report: Global Adhesion Analysis
0,04 s
3 s
Incremental T. A. T.
3.
Results report: Global Adhesion Analysis
0,04 s
3 s
3.
Total
Results report: Global Adhesion Analysis
0,04 s
3 s
3.
Normalized
Export results (cvs format)3.
PMN control PMN treated with Pertussis Toxin (2 μg/ml)
Human primary neutrophils isolated from healthy volunteers by standard gradient centrifugation (Ficoll). Contaminating erythrocytes were removed by dextran-500 sedimentation followed by hypotonic lysis.
1 ug/ml P-Selectin, 5 μg/ml ICAM-1 and 25 nM FMLP
Primary neutrophils were treated or not with 2 μg/ml Pertussis Toxin (PTx) for 3 h at 37°C
CELL TYPE:
TREATMENT:
COATING:
Example: h-PMN with PTx3.
0,21 0,4
0,08 0,02
PARAMETERS: S.A.T. 5 μm – T.A.T. 2s – Sub-rolling threshold 20 μm
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
Lara Toffali
+39 0458027145
http://dp.univr.it/~laudanna/LCTST/index.html
Carlo Laudanna
+39 0458027689
Roberto Marzotto
+39 045 8949180
Michela Farenzena
+39 045 8949180
www.embeddedvisionsystems.it
» 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
Image processing details