Abstract

Cell adhesion and chemotaxis are two key factors determining cell behaviour and differentiation which are currently analysed by microscopic examination of the cell or membrane-associated fluorescence labels. These analyses are often slow, labour intensive and of limited informational content. This thesis describes the physical theory and experimental aspects of an optical method suitable for monitoring cell contact, adhesion to a surface and chemotaxis beyond the conventional limit of optical microscopy by means of a device that utilises both a plain bare surface and arrays of apertures nanolithographically-produced in the surface of a Surface Plasmon Resonance (SPR) sensor structure. Any minute vertical movement of the cell, within the near-field of the SPR active surface or actual cell/surface contact, creates intensity fluctuations, detectable in the far-field. This was demonstrated during experiments with non-apertured devices. (A video demonstrating the biological features of the device accompanies this thesis and may be obtained by contacting University of Plymouth's LRC.) The light scattered by each nanolithographically-produced aperture also fluctuates as a consequence of the cell approaching to within a few hundred nanometres of the aperture bearing surface and demonstrated detection of minute vertical movement on the surface of the apertured device. The combination of apertured and non-apertured detection results in a highly spatially-sensitive 3-dimensional sensor. Digitising the output from a CCD camera allows patterns of intensity fluctuation to be correlated with the contact and adhesion of individual cells on the active surface over a short period of time (2-3 minutes). Initial trials of an apertured device (diameter (^) « wavelength of incident light ( X ) ) carried out by our collaborating partners Drs R. Carr and S. Al-shukri at the Centre for Applied Microbiology and Research, Porton Down demonstrated that the use of apertures etched in a SPR metal surface produced a highly sensitive dielectric monitor, i.e. sensitive to very small changes in the refractive index of the micro-environment adjacent to the aperture. This was proposed as being of potentially great value in the development of extremely sensitive probes of dielectric particulates of sub-micron dimensions, i.e. biological macromolecules and supramolecular structures. Characterisation of the associated radiative and non-radiative evanescent fields on the surface of the device was conducted in order to gain a greater knowledge of the mechanisms by which the interactions between the cells adjacent to and in direct contact with the apertures and evanescent fields produced such significant intensity fluctuations in the results at CAMR. A combinational Scanning Probe Microscope was developed and used in Scanning Nearfield Optical Microscope and Photon Scanning Tunnelling Microscope modes of operation to detect the evanescent and radiative fields respectively. Detailed mapping of the radiative pattern in the near-field of the large apertures {

Document Type

Thesis

Publication Date

1997

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