Standardized microgel beads as elastic cell mechanical probes

Abstract

Cell mechanical measurements are gaining increasing interest in biological and biomedical studies. However, there are no standardized calibration particles available that permit the cross-comparison of different measurement techniques operating at different stresses and time-scales. Here we present the rational design, production, and comprehensive characterization of poly-acylamide (PAAm) microgel beads mimicking biological cells. We produced mono-disperse beads at rates of 20 -60 kHz by means of a microfluidic droplet generator, where the pre-gel composition was adjusted to tune the beads' elasticity in the range of cell and tissue relevant mechanical properties. We verified bead homogeneity by optical diffraction tomography and Brillouin microscopy. Consistent elastic behavior of microgel beads at different shear rates was confirmed by AFM-enabled nanoindentation and real-time deformability cytometry (RT-DC). The remaining inherent variability in elastic modulus was rationalized using polymer theory and effectively reduced by sorting based on forward-scattering using conventional flow cytometry. Our results show that PAAm microgel beads can be standardized as mechanical probes, to serve not only for validation and calibration of cell mechanical measurements, but also as cell-scale stress sensors. Significance StatementOften vastly different cell mechanical properties are reported even for the same cell type when employing different measurement techniques. This discrepancy shows the urgent need for standardized calibration particles to cross-compare and validate techniques. Microgel beads can serve this purpose, but they have to fulfil specific requirements such as homogeneity, sizes and elasticities in the range of the cells, and they have to provide comparable results independent of the method applied. Here we demonstrate the standardized production of polyacrylamide microgel beads with all the features an elastic cell-mimic should have. These can not only be used as method calibration particles, but can also serve as cell-scale sensors to quantify normal and shear stresses exerted by other cells and inside tissues, enabling many new applications.

Microparticle traction force microscopy reveals subcellular force exertion patterns in immune cell–target interactions

“…However, current technologies have lacked the resolution to identify contributions from individual subcellular force transmitting structures. Moreover, synthesis of suitable hydrogel MPs for such applications can be rather complex, often requiring specialized expertise in microfluidics 17,18 . Particle properties, especially their size and mechanical properties, must also be optimized for accurate traction force measurements 18 .…”

Accurate evaluation of size and refractive index for spherical objects in quantitative phase imaging

“…[35,23,32,31,27]). Finally, complementing the optical analysis of microgel beads as presented here with a mechanical description using, for instance, atomic force microscopy, will allow to establish a well-characterized microgel bead toolbox [40] which would be an invaluable reference for the optomechanical analysis of cells using techniques such as Brillouin microscopy or the optical stretcher. Thus, the optical characterization of spheres is fundamentally important to address topical questions in cell biology and biophysics.…”

Using real-time fluorescence and deformability cytometry and deep learning to transfer molecular specificity to label-free sorting

“…First, to demonstrate FACS-like capabilities of soRT-FDC to perform fluorescence-activated sorting, we mixed polyacrylamide microgel beads labeled with AlexaFluor488 and unlabeled ones in a 1:5 ratio (beads were produced in house 46 , see Online Methods for details) and sorted for fluorescence intensity (Figure 1d, upper panel). The percentage of fluorescence-positive beads contained in the sorting gate (FL-1 intensity between 1,000 and 10,000 a.u.)…”

“…Another image-derived parameter that is of high interest for sorting, as it gives access to mechanical properties of cells 48,49 , is deformation. To demonstrate sorting for deformation, we utilized a mixture of two polyacrylamide microgel bead populations of different stiffness 46 . We sorted for the beads with low deformation values of 0.000 -0.016 contained within the size range of 95 -105 µm 2 (Figure 1d, lower panel).…”

“…Figure 1 and 2b) and sorting based on size (Figure 2d, Polyacrylamide bead production. Polyacrylamide hydrogel beads were produced in house using PDMS-based flow-focusing microfluidic device as described elsewhere 46 . For the preparation of beads with different stiffnesses (Figure 1d, lower panel, Figure 2a) total monomer concentrations (molar ratio of bis-acrylamide to acrylamide 1:61.5; #146072 and #A3553, Sigma-Aldrich) of 7.9%, 9.9% and 13.8% were used.…”