It has been shown that quantitative measurements of the cell-substrate distance of steady cells are possible with scanning surface plasmon resonance microscopy setups in combination with an angle resolved analysis. However, the accuracy of the determined cell-substrate distances as well as the capabilities for the investigation of cell dynamics remained limited due to the assumption of a homogeneous refractive index of the cytosol. Strong spatial or temporal deviations between the local refractive index and the average value can result in errors in the calculated cell-substrate distance of around 100 nm, while the average accuracy was determined to 37 nm. Here, we present a combination of acquisition and analysis techniques that enables the measurement of the cell-substrate distance of contractile cells as well as the study of intracellular processes through changes in the refractive index at the diffraction limit. By decoupling the measurement of the cell-substrate distance and the refractive index of the cytoplasm, we could increase the accuracy of the distance measurement on average by a factor of 25 reaching 1.5 nm under ideal conditions. We show a temporal and spatial mapping of changes in the refractive index and the cell-substrate distance which strongly correlate with the action potentials and reconstruct the three-dimensional profile of the basal cell membrane and its dynamics, while we reached an actual measurement accuracy of 2.3 nm.
Most animal cells are surrounded by a cell membrane and an underlying actomyosin cortex. Both structures are linked with each other, and they are under tension. Membrane tension and cortical tension both influence many cellular processes, including cell migration, division, and endocytosis. However, while actomyosin tension is regulated by substrate stiffness, how membrane tension responds to mechanical substrate properties is currently poorly understood. Here, we probed the effective membrane tension of neurons and fibroblasts cultured on glass and polyacrylamide substrates of varying stiffness using optical tweezers. In contrast to actomyosin-based traction forces, both peak forces and steady state tether forces of cells cultured on hydrogels were independent of substrate stiffness and did not change after blocking myosin II activity using blebbistatin, indicating that tether and traction forces are not directly linked with each other. Peak forces on hydrogels were about twice as high in fibroblasts if compared to neurons, indicating stronger membrane-cortex adhesion in fibroblasts. Finally, tether forces were generally higher in cells cultured on hydrogels compared to cells cultured on glass, which we attribute to substrate-dependent alterations of the actomyosin cortex and an inverse relationship between tension along stress fibres and cortical tension. Our results provide new insights into the complex regulation of membrane tension, and they pave the way for a deeper understanding of biological processes instructed by it.
The investigation of the cell–substrate interface is of great importance for a broad spectrum of areas such as biomedical engineering, brain‐chip interfacing, and fundamental research. Due to its unique resolution and the prevalence of instruments, electron microscopy (EM) is used as one of the standard techniques for the analysis of the cell–substrate interface. However, possible artifacts that might be introduced by the required sample preparation have been the subject of speculation for decades. Due to recent advances in surface plasmon resonance microscopy (SPRM), the technique now offers a label‐free alternative for the interface characterization with nanometer resolution in axial direction. In contrast to EM, SPRM studies do not require fixation and can therefore be performed on living cells. Here, a workflow that allows for the quantification of the impact of chemical fixation on the cell–substrate interface is presented. These measurements confirm that chemical fixation preserves the average cell–substrate distances in the majority of studied cells. Furthermore, it is possible to correlate the SPRM measurements with EM images of the cell–substrate interface of the exact same cells, thus identifying regions of good agreement between the two methods and revealing artifacts introduced during further sample preparation.
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