Confocal reference free traction force microscopy.

Martin Bergert,Stephan J P Kress,Aldo Ferrari,David K Kim,Dimos Poulikakos,Olga Sorkine-Hornung,Manuel Zündel,Tobias Lendenmann,David J Norris,Alexander E Ehret,Daniele Panozzo,Edoardo Mazza,Patrizia Richner

doi:10.1038/ncomms12814

Abstract

The mechanical wiring between cells and their surroundings is fundamental to the regulation of complex biological processes during tissue development, repair or pathology. Traction force microscopy (TFM) enables determination of the actuating forces. Despite progress, important limitations with intrusion effects in low resolution 2D pillar-based methods or disruptive intermediate steps of cell removal and substrate relaxation in high-resolution continuum TFM methods need to be overcome. Here we introduce a novel method allowing a one-shot (live) acquisition of continuous in- and out-of-plane traction fields with high sensitivity. The method is based on electrohydrodynamic nanodrip-printing of quantum dots into confocal monocrystalline arrays, rendering individually identifiable point light sources on compliant substrates. We demonstrate the undisrupted reference-free acquisition and quantification of high-resolution continuous force fields, and the simultaneous capability of this method to correlatively overlap traction forces with spatial localization of proteins revealed using immunofluorescence methods.

Highlights

The mechanical wiring between cells and their surroundings is fundamental to the regulation of complex biological processes during tissue development, repair or pathology
The cTFM set-up in Fig. 1a–c is comprised of a typical, 170 mm glass coverslip spin-coated with a E30 mm thick layer of a highly deformable CY52-276 silicone (Dow Corning)[22,23,24,25]
Our results demonstrate that the cTFM platform is capable of separate detection of in-plane and out-of-plane components of cell tractions with high resolution

Summary

Introduction

The mechanical wiring between cells and their surroundings is fundamental to the regulation of complex biological processes during tissue development, repair or pathology. Continuum TFM methods exploit elastic substrates containing randomly dispersed fluorescent beads[11,12,13] These approaches yield high-resolution in- and out-of-plane force maps but require the additional acquisition of a reference (load-free) image, typically captured on cell removal and destruction, which markedly complicates experimental procedures and precludes post-processing such as the colocalization with immunofluorescence staining. Attempts to bypass these shortcomings, by applying micro-patterning of adhesive islands[14,15] or lithographic photoresist into ordered arrays[16], are hampered by major drawbacks such as the poor spatial resolution or the introduction of intrusive topographical features. We use highly precise electrohydrodynamic nanodripprinting of quantum dots (QDs)[18,19,20,21] into monocrystalline, confocal arrays on elastomeric substrates and introduce a highresolution and reference-free method (called confocal TFM or cTFM), capable of in- and out-of-plane force detection, which takes advantage of many assets of previously developed approaches while significantly advancing the landscape of reference-free force detection in cell biology and medicine

Methods

Results

Conclusion