BeQuanti webinar

January 22, 2018 | Author: Anonymous | Category: Science, Health Science, Immunology
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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

View more...

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