10) where R(pCa) represents the column of the measured ratios, and
23. Images suffering from high background signal can be pro- cessed by defining a background ROI (region of interest) fol-
24. In addition image stacks can be presented spatially using the plugins “3D Viewer” or “Volume Viewer”. A minimum over- lap between images should exist for the software to calculate a continuum of structures, i.e., the z-distance between images should be sufficiently low. For the calculation of 3D represen- tations the signal intensity levels have to be relatively high.
Unfortunately, unlike in fluorescence images, in SHG images from collagen networks from cultured MSCs provide only rela- tively low intensities. Therefore SHG image quality has to be optimized.
Cell Viability Assessment by 2-Channel Multiphoton Imaging
Acknowledgments
This work was supported by the Emerging Fields Initiative (EFI) of the University of Erlangen-Nürnberg (project TOPbiomat) and the Erlangen Graduate School in Advanced Optical Technologies (SAOT) within the German Excellence Initiative. We want to thank Dana Kralisch and Nadine Hessler (JeNaCell GmbH) for providing BNC fleeces.
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Chapter 20
Average Rheological Quantities of Cells in Monolayers
Haider Dakhil and Andreas Wierschem
Abstract
Measuring rheological properties of cells in monolayers enables quantifying average cell properties in single experimental runs despite large cell-to-cell variations. Here, we describe how to modify a commercial rotational rheometer to accomplish the necessary precision for a monolayer rheometer and we delineate the steps for setting up experiments detecting average viscoelastic cell properties.
Key words Cell rheology, Linear viscoelasticity, Narrow-gap rheometry, Cell monolayer
1 Introduction
Rheology is the study of deformation of matter under the influence of stress. Rheological studies enable quantification of material properties such as viscosity of fluids or viscoelasticity of solids and fluids [1, 2]. Rheological properties of biological cells are closely related to their physiological activities and have vital functional implications such as mechanical stability, adjustment to environmental load, migration, proliferation, phagocytosis, or contraction. For single cell studies, different techniques like optical and magnetic tweezers, atomic force microscopy, magnetic twisting cytometry, micropipettes, microplates, cell poking, and particle tracking micro-rheology have been employed [3, 4]. Depending for instance on cell identity, life cycle, shape, structure, and level of proteins [5], cells show variations in stiffness and dynamic moduli by an order of magnitude [6]. Hence, to quantify the impact of drugs, aging, or diseases on the viscoelastic cellular properties, it is of great importance to determine average viscoelastic cell properties.
Averaging over a large number of cells in a single experimental run can be achieved by studying cells in a monolayer between rheometer disks in the parallel-disk configuration [7] and detecting the cell coverage [6].
To study average rheological properties of cells in a monolayer, the cells have to be adhered between two transparent plates with a
258
gap-width variation to be a fraction of the average cell height. This is beyond the scope of commercial rheometers that usually have a systematic error in the gap width of about ±25 μ m and more [8–10]. To overcome this limitation and meet the requirements for cell monolayers, we developed a setup for commercial rota- tional rheometers significantly reducing the variation to less than
±1 μ m [4, 6].
2 Materials
Cells in suspension like, for instance, HeLa cells or fibroblasts that can be adhered to solid substrates with proteins like, for instance, fibroblasts [11]. Use the following materials:
1. Cultural medium for 3 T6 Swiss Albino murine fibroblasts: