The aim of the research project described in this thesis was the study of surface plasmon microscopy (SPM) and the application of this method to biological monolayers. The emphasis was more on the development of methods than on the study of the samples themselves.

Two types of biological monolayers have been studied. The first one is a protein monolayer, and more specifically; layers of antibodies and antigens. Improving the knowledge about detection of the immune reactions that can take place between these compounds is important for the development of diagnostic assays, biomaterials, and protein purification methods. We will study this interaction using surface plasmon resonance (SPR) sensor methods.

The second type is a lipid monolayer, which has been used as a model system for a biological cell membrane. Lipid monolayers can be spread and studied at the air-water interface, but can also be transferred to a solid substrate. This is necessary for the two techniques that were applied to lipid monolayers in this work. Surface plasmon microscopy was applied to a phase-separated lipid monolayer that was deposited on a substrate using the Langmuir-Blodgett (LB) technique. The different phases could be microscopically observed since they coexist in separate domains and have different optical properties. To study these layers at a lateral scale smaller than possible with optical methods, a new atomic force microscopy (AFM) method was used. With this microscope the adhesive properties of the layer, which depend on the lipid phase, are used for imaging.

This thesis will start with a short overview of methods for the characterization of surface layers. After an introduction into Fresnel theory, we will discuss ellipsometry, Brewster angle microscopy (BAM) and surface plasmon resonance (SPR) methods. The Fresnel equations satisfactorily describe these methods at lateral scales much larger than the wavelength of the used light. This theory can therefore be used to determine system parameters by fitting the measurements.

In the second chapter we will see how SPR was used to develop an accurate, differential immunosensor, with a number of advantages over present SPR immunosensors. It will also be demonstrated that SPR is well suited for use in multichannel immunosensors. The first multichannel measurements will be presented with one as well as twodimensional arrays of sensor surfaces.

Going towards higher lateral resolution, plasmon propagation effects will make the Fresnel theory invalid at the microscopic scale. We have developed a phenomenological model to describe the reflectance near index steps at the microscopic scale. All experimental parameters used for the calculations have been derived from the layer system under investigation (not from fitting). A systematical comparison of theoretical and experimental values was made for 11 wavelengths in the visible region and will be presented in the third chapter.

In the fourth chapter a surface plasmon microscopy (SPM) setup will be described. We will show that there is a trade-off relationship between thickness resolution (or contrast) and lateral resolution with respect to the choice of wavelength. We have chosen for a short wavelength and tackled the problem of an extremely low contrast by developing some image acquisition methods. In this way the maximum diffraction limited lateral resolution can be obtained.

A short introduction to Langmuir-Blodgett (LB) monolayers will be given in the fifth chapter. After that we will describe the application of the SPM to the imaging of these monolayers, using the techniques described in the previous chapter.

The sixth chapter will cover another new technique that was applied for the characterization of lipid LB monolayers: adhesion mode atomic force microscopy. It will be demonstrated that the orientation of molecules in the monolayer strongly influences the adhesion of the tip to the surface. This allows these monolayers to be imaged with superresolution (20 nm) and high contrast. We will argue that this technique holds a promise for functional group identification.

The thesis will end with a general discussion of the results obtained in this work, and an appendix where a new surface plasmon microscope that was developed (just before completion of this project) is described.