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

info@evsys.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

lara.toffali@univr.it

+39 0458027145

http://dp.univr.it/~laudanna/LCTST/index.html

Carlo Laudanna

carlo.laudanna@univr.it

+39 0458027689

Roberto Marzotto

roberto.marzotto@evsys.it

+39 045 8949180

Michela Farenzena

michela.farenzena@evsys.it

+39 045 8949180

www.embeddedvisionsystems.it

info@evsys.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