Standardisation of methods to probe cell and tissue elasticity with AFM
Duration: months 3-40.
Lead beneficiary: UB
Objectives: The main goal of this WP is to define a method to probe cell and tissue elasticity using AFM assuring robustness and reproducibility for clinical use.
This WP will be carried out on cell lines, freshly prepared or artificial tissue samples. Implementation on clinical samples is the subject of WP6.
Task 2.1. Determination of the most reliable and applicable method to calibrate the deflection sensitivity and spring constant of cantilevers.
In particular, contact-less strategies for spring constant calibration will be considered, including the shared use of interferometer within the network (i.e. through Vmicro), or the development of one to be built in each
lab (see Task 5.4 in WP5). (Lead UB, INSERM, UMIL, O11,Vmicro)
Task 2.2. Determination of the optimal tip geometry and calibration of relevant geometrical parameters.
The characterisation of the tip geometry (open angle, radius...) will be carried out using optical and electron micrographs and/or standardisation grids. (Lead UMIL, IFJPAN, CEA-IBS, UB,Vmicro)
Task 2.3. Establishment of the experimental conditions of piezo velocity, indentation depth, mapping area, number of measurements and samples for reliable probing of cell elasticity.
Experimental conditions will be determined after a systematic use of various piezo velocities and indentation depths, different cell areas including junctions, nuclear, peri-nuclear and peripheral regions, and statistical tests using different number of measurements and samples. (Lead IBEC,CNRS, IFJPAN, CEA-IBS, UMIL, UB)
Task 2.4. Definition of the most robust and reliable data analysis method to determine cell elasticity (Young’s modulus).
The data acquired in task 1.3 will be analysed using different elastic contact model (pyramidal, spherical, bottom correction...), both approach and/or retract curves, point-by-point analysis, various contact point determination strategies to define the most reliable and robust approach. (Lead UB, IBEC, CNRS, IFJPAN, CEA-IBS, INSERM,UMIL, CHUGR, BioMeca)
Task 2.5. Assessment of the standardisation procedure.
The established standard procedure will be assessed by applying it on a same cell line distributed to at least five different laboratories. The reliability and robustness of the results will
be then evaluated to determine the accuracy of mechanical tests. (Lead WWU, ALL partners)
Task 2.6. Transfer of the standardised procedure to probe tissue elasticity.
Similar tasks will be developed in parallel to define the standardised method to probe tissue and extracellular matrix mechanics for clinical use. Connection with
WP6. (Lead WWU, IBEC, CNRS, IFJPAN, UMIL, UB, CHUGR)