# Conclusions

In Chapter 2, surface plasmon resonance was used for immuno-sensing. Using a differential technique, immune reactions at the sensor surface could be followed by monitoring the shift of the surface plasmon excitation angle in time. The accuracy in the determination of the SPR excitation angle was very high, and additional information could be obtained by simultaneously measuring the reflectance and its second derivative with respect to the angle of incidence. In this way, position, depth and width of the reflectance minimum can be measured in real-time. As surface roughness is known to increase the width, this could yield some information on the dynamics of protein monolayer adsorption. Because the measurement is fast and the modulated beam can easily be scanned over the sensor surface, this method is suitable for multichannel measurements as well, as was demonstrated.

Laterally resolved sensor measurements were also carried out in an imaging system, where the angle of incidence of a wide, collimated laser beam was kept fixed, while imaging the reflecting light onto a video camera. This has the advantage of being able to actually observe the defined sensing areas on the sensor surface during the measurement, or even defining them after the measurement. Allthough we defined 16 sensing areas, in fact every CCD element acts as a separate detector that is operated in parallel, giving information on the sensing area that it receives the reflected light from. Therefore, the number of sensing areas might be much larger, as long as their size exceeds the propagation length of the SPs (typically $$\mu$$m’s). The signal to noise ratio decreases with the sensing area size, but the amount of light coupled in can be increased by several orders of magnitude.

To get an idea of the obtainable lateral resolution with SP imaging, and how to improve it, the propagation of SPs near an index step was studied in Chapter 3. A phenomenological model was developed and its results were compared to measurements for a number of wavelengths. All parameters in the model were derived from the layer structure and boundary conditions, leaving no parameters to be fitted. A good agreement between model and experimental values was found.

In Chapter 4 and 5 we have demonstrated the qualities of surface plasmon microscopy and the possibilty to measure local reflectance scans. Lipid phase-separated LB monolayers were succesfully prepared and characterized. Using a number of techniques to improve the acquisition of the images, the maximum lateral resolution as determined by diffraction was nearly attained. However, Brewster angle microscopy should ultimately be able to reach a similar resolution, and has the important advantage of the capability to study the monolayer at the air-water interface.

When a much higher lateral resolution is needed, purely optical means do not suffice, and atomic force microscopy can be used. This was demonstrated in Chapter 6, where together with the topography the chemical properties of the monolayer surface were used to image it, measuring the adhesive interaction between tip and sample. With this new adhesion force atomic force microscope the contrast can be high, because the adhesion depends only on the outer atoms of the surface, and not on the thickness of the layer under study. In this case, the contrast was seen to match that expected of CH2 and CH3 groups (16% difference in adhesion). The lateral resolution that was obtained was about 20 nm. It is expected that this form of microscopy can be used for the imaging and recognition of many other functional groups. It is especially well suited for monolayers, since these samples can be molecularly smooth by deposition on mica substrates, ensuring a constant interaction area between tip and sample.

Since surface plasmon microscopy measures ‘through’ the monolayer, and measures the layer thickness, while AFM only measures the outer surface of the monolayer, it would be interesting to combine both techniques (see Chapter 1). This is very well possible because the SPM measurement is carried out at the ‘substrate-side’ of the sample, while the AFM measurement is done on the outside of the sample. An SPM setup with the sample in the horizontal plane was designed especially for this purpose. It will be described in the appendix, since it was only recently completed, and combined measurements have not yet been done.