Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells

Abstract

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. Three dimensional-Magnetic Twisting Cytometry (3D-MTC) is a technique for applying local mechanical stresses on living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real time acquisition of a living cell’s mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC – microscopy platform takes around 20 days to construct and the experimental procedures require ~4 days when carried out by a life sciences graduate student.

Understanding the extracellular forces that determine cell fate and maintenance

“…A major goal of 2D protocols that exploit externally applied or intracellularly generated forces is to improve stem cell maturation and lineage purity, which may be counterintuitive to the expansion of 3D systems in the field over the past decade (Burridge et al, 2014;Maffioletti et al, 2015;Patsch et al, 2015;Si-Tayeb et al, 2010), but which has clear practical advantages. Thus, our emphasis on these well-defined protocols is consistent with both a drive towards standardization of stem cell protocols and to a demand for more refined biomechanical tools (Dahl et al, 2005;Engler et al, 2007;Yim and Sheetz, 2012;Zhang et al, 2017), global force sensors (Zhou et al, 2015), and molecular strain and tension sensors (Grashoff et al, 2010). In the next section of this Review, we turn our attention to how such tools can be used to understand the cellular effects of physical inputs in a defined and reductionist way.…”

Receptor-mediated cell mechanosensing

“…, 1993 ). Moreover, when MTC was combined with fluorescence microscopy ( Zhang et al. , 2017 ), various cell signaling events following the application of torque on cadherin-expressing cells ( le Duc et al.…”

Force-Mediating Magnetic Nanoparticles to Engineer Neuronal Cell Function

“…or to induce tensile stretch on mammalian cells to stimulate ion channels and cell communication (Lee J. et al, 2014 ). Recently, the torque approach has been used in conjunction with confocal microscopy to image force responses in living cells (Zhang et al, 2017 ). The approach has been further expanded upon by Chen et al through the integration of a multi-pole electromagnet that allows for control of both the twisting direction as well as the magnetic strength (Chen et al, 2016 ).…”