Measuring Cell Dynamics at the Substrate-Interface with Surface Plasmon Resonance Miscroscopy

In neuroelectronics the cell-electrode distance is one of the most critical parameters during cell recordings. Cardiomyocytes such as HL-1 are among the most popular model systems used for cell recordings because they periodically generate an action potential. This feature also leads to a cell contraction which affects the cell-electrode distance. Therefore a quantitative characterization of the membrane dynamics directly at the interface is crucial. Imaging said dynamics in vitro and label free is a great challenge.To achieve this we built a surface plasmon resonance microscope (SPRM).With gold coated sapphire chips as the substrate for the cell culture it is possible to excite plasmons (collective electron oscillations) in the gold layer by illuminating it under a specific angle.The resonance frequency of the plasmons depends strongly upon the dielectric constant of the gold's environment.In turn the angle spectrum of the reflected light depends upon said resonance frequencies.Due to these dependencies it is possible to deduce the cell-substrate distance.Our microscope is capable of imaging the interface in two different modes.The field of view in the live imaging mode is around 65 um x 65 um.Here we can observe cell dynamics qualitatively.The scanning mode uses localized surface plasmons to measure the cell-substrate distance. The resolution in z-direction lies in the nanometer range.This allows us to measure the movement of the cell membrane caused by the cell contraction.By scanning the region of interest we can characterize the cell dynamics at each scanning point with a time resolution of 150 ms

Characterization of the cell-substrate interface using surface plasmon resonance microscopy

In this thesis an improved surface plasmon resonance microscopy (SPRM) setup has been developed which combines a projector based SPRM widefield mode with several SPRM scanning modes for the investigation of the cell-substrate interface. Widefield SPRM can be used to image the cell-substrate adhesion areas qualitatively. Here, the resolution is strongly dependent on the light source. While coherent laser light gives rise to speckle noise, which frustrates the resolution of small cellular structures such as neuronal dendrites, using a projector as an incoherent light source allows for a high resolution imaging. Scanning SPRM can be used to determine the cell-substrate distance quantitatively. So far, the accuracy of these measurements was compromised by the assumption of a homogeneous refractive index (RI). In this thesis, it is shown that the RI can be extracted from the SPRM signal at each scanning point at the cell-substrate interface which allowed for an improvement of the distance accuracy by a factor of 25 compared to the standard analysis technique realizing a distance accuracy of up to 1.5 nm. The measurements of RI and distance were validated by several reference measurements. The RI of the cell gives interesting insights into the cellular structure and cellular processes. Scanning the cell-substrate interface, it could be shown that the RI profile of a cell can reveal the position of cell organelles and give quantitative values for their refractive indices while the scanning SPRM also allows for the reconstruction of the 3D structure of the basal cell membrane. New acquisition and analysis techniques facilitate the resolution of dynamic processes at the cell-substrate interface. Scanning one point at the interface of a periodically contracting cardiomyocyte over time with a simultaneous calcium imaging could resolve RI variations caused by the action potential as well as the dynamics of the cell membrane. Combining large numbers of these time-dependent measurements along the interface allowed for the reconstruction of the movement of the entire basal cell membrane. Additionally, SPRM was used to determine the effect of chemical fixation on the cell-substrate distance at the neuronal membrane. These measurements were correlated with focused ion beam sectioning (FIB) combined with electron microscopy of the cell-substrate interface allowing artefacts introduced by the cell preparation to be identified for the first time

Poster Award "Surface Plasmon Resonance Microscopy of the Cell-Chip Interface"

Poster: "Surface Plasmon Resonance Microscopy of the Cell-Chip Interface"Longterm investigation of neuronal networks require non-invasive recordings of the electrical signals. A good coupling between the biological and electronic system is crucial and depends particularly upon the cell-chip distance. The cell-chip distance is an important parameter towards a good sealing, with closer contact leading to a decreased signal dissipation in the cell-electrode cleft. We therefore try to optimize the contact geometry of said interface using protein and lipid coatings. In order to measure the distances between the cell membrane and the chip surface in vitro, we built a surface plasmon resonance microscope (SPRM). With gold coated sapphire chips as the substrate for the cell culture, it is possible to excite plasmons (collective electron oscillations) in the gold layer by illuminating it under a specific angle. The resonance frequency of the plasmons depends strongly upon the dielectric constant of the gold's environment. In turn the angle spectrum of the reflected light depends upon said resonance frequencies. Due to these dependencies it is possible to deduce the cell-substrate distance.Our microscope is capable of imaging the interface in two different modes. The field of view in the live imaging mode is around 65 um x 65 um.This is useful for determining the region of interest for the scanning mode. This mode uses localized surface plasmons to measure the cell-substrate distance. The resolution in z-direction lies in the nanometer range. This allows us to accurately characterize the cell-chip interface.Since SPRM is non-invasive and label free it is suited for longterm investigations.It is therefore possible to observe the development of neuronal networks over several week

Non-invasive measurement of the refractive index of cell-organelles using surface plasmon resonance microscopy

The health of a eucariotic cell depends on the proper functioning of its cell organelles. Characterizing these nanometer to micrometer scaled specialized subunits without disturbing the cell is challenging but can also provide valuable insights regarding the state of a cell. We show, that objective-based scanning surface plasmon resonance microscopy can be used to analyze therefractive index of cell organelles quantitatively in a non-invasive and label-free manner with a lateral resolution at the diffraction limit

Measuring Cell Dynamics at the Substrate-Interface with Surface Plasmon Resonance Miscroscopy

In neuroelectronics the cell-electrode distance is one of the most critical parameters during cell recordings. Cardiomyocytes such as HL-1 are among the most popular model systems used for cell recordings because they periodically generate an action potential.This feature also leads to a cell contraction which affects the cell-electrode distance.Therefore a quantitative characterization of the membrane dynamics directly at the interface is crucial.Imaging said dynamics in vitro and label free is a great challenge.To achieve this we built a surface plasmon resonance microscope (SPRM).With gold coated sapphire chips as the substrate for the cell culture it is possible to excite plasmons (collective electron oscillations) in the gold layer by illuminating it under a specific angle.The resonance frequency of the plasmons depends strongly upon the dielectric constant of the gold's environment.In turn the angle spectrum of the reflected light depends upon said resonance frequencies.Due to these dependencies it is possible to deduce the cell-substrate distance. Our microscope is capable of imaging the interface in two different modes.The field of view in the live imaging mode is around 65 um x 65 um. Here we can observe cell dynamics qualitatively.The scanning mode uses localized surface plasmons to measure the cell-substrate distance.The resolution in z-direction lies in the nanometer range.This allows us to measure the movement of the cell membrane caused by the cell contraction.By scanning the region of interest we can characterize the cell dynamics at each scanning point with a time resolution of 150 ms

Surface Plasmon Resonance Microscopy of the Cell-Chip Interface

Longterm investigation of neuronal networks require non-invasive recordings of the electrical signals. A good coupling between the biological and electronic system is crucial and depends particularly upon the cell-chip distance. The cell-chip distance is an important parameter towards a good sealing, with closer contact leading to a decreased signal dissipation in the cell-electrode cleft. We therefore try to optimize the contact geometry of said interface using protein and lipid coatings. In order to measure the distances between the cell membrane and the chip surface in vitro, we built a surface plasmon resonance microscope (SPRM). With gold coated sapphire chips as the substrate for the cell culture, it is possible to excite plasmons (collective electron oscillations) in the gold layer by illuminating it under a specific angle. The resonance frequency of the plasmons depends strongly upon the dielectric constant of the gold's environment. In turn the angle spectrum of the reflected light depends upon said resonance frequencies. Due to these dependencies it is possible to deduce the cell-substrate distance.Our microscope is capable of imaging the interface in two different modes. The field of view in the live imaging mode is around 65 um x 65 um.This is useful for determining the region of interest for the scanning mode. This mode uses localized surface plasmons to measure the cell-substrate distance. The resolution in z-direction lies in the nanometer range. This allows us to accurately characterize the cell-chip interface.Since SPRM is non-invasive and label free it is suited for longterm investigations.It is therefore possible to observe the development of neuronal networks over several week

Nanometer-Resolved Mapping of Cell–Substrate Distances of Contracting Cardiomyocytes Using Surface Plasmon Resonance Microscopy

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

Correlating Surface Plasmon Resonance Microscopy of Living and Fixated Cells with Electron Microscopy Allows for Investigation of Potential Preparation Artifacts

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

Effective cell membrane tension is independent of polyacrylamide substrate stiffness

Most animal cells are surrounded by a cell membrane and an underlying actomyosin cortex. Both structures are linked, and they are under tension. In-plane 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. Peak forces in fibroblasts on hydrogels were about twice as high as those in neurons, indicating stronger membrane-cortex adhesion in fibroblasts. Steady state tether forces were generally higher in cells cultured on hydrogels than on glass, which we explain by a mechanical model. Our results provide new insights into the complex regulation of effective membrane tension and pave the way for a deeper understanding of the biological processes it instructs