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| Home > Publications database > Surface Plasmon Resonance Microscopy for the Characterization of Cell-Substrate Distances |
| Book/Dissertation / PhD Thesis | FZJ-2025-02992 |
2025
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 978-3-95806-830-8
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Please use a persistent id in citations: urn:nbn:de:0001-2602031113021.788680827696 doi:10.34734/FZJ-2025-02992
Abstract: Cell-substrate distances play an important role in many biomedical applications, including cancer therapies, drug development, implants, and fundamental neuroscience research. Several microscopic approaches have been established to measure cell-substrate distances, including surface plasmon resonance microscopy (SPRM). (Amplitude-scanning) SPRM, like other leading methods, provides nanometer-precise imaging of the cell-substrate distance with diffraction-limited lateral resolution. Also, it has the advantage of being both non-invasive and label-free. In addition to cell-substrate distances, the employed variant of SPRM provides simultaneously the cytoplasmic RI, which can be seen as a complement and a necessity. One goal of this work is to make SPRM more accessible and reproducible through the development of an automated and robust data analysis, thereby reducing existing barriers for users and requirements for sample and measurement data. Related objectives are to determine the resulting measurement uncertainties, demonstrate the achievable, analyzable fraction of measurements, and to discuss measurement results in a method comparison. In addition to primary measurements on an SPRM prototype, X-ray reflectometry (XRR), optical microscopy and atomic force microscopy (AFM) are used as secondary methods for verification and substrate-quality assessment. In particular, XRR offers untapped potential for the characterization of SPRM sensors. The results of this work include the development and publication of a new data analysis software and the successful implementation and evaluation of a sphere-substrate distance measurement to validate the setup and data analysis. The latter allows the experimental determination of statistical and systematic measurement errors, rounded up to ±5.8nm (stat) −5.2nm (syst) on Au substrates and ±8.9nm (stat) +6.3nm (syst) on SiO2 substrates. A data set of 68 SPRM measurements of HEK293 cell-substrate distances on SiO2-coated substrates is then presented, comparing six common biochemical cell adhesion coatings with untreated SiO2 surfaces. SPRM and XRR sensor characterizations are in agreement within measurement uncertainties. Analyzed cell-substrate distances are compared with previously published values measured by electron microscopy (TEM). It is noticeable that SPRM cell-substrate distances below 50nm are rather rare (8.9 %) in contrast to TEM measurements (44.5 %). Another series of 25 neuron-substrate distance measurements on bare Au substrates, with or without one of two common cell adhesion coatings, is also presented. For those who are familiar with the challenges of amplitude-scanning SPRM, it should be emphasized that the presented series of cell-substrate distance measurements could be fully evaluated.
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