Surface Plasmon Resonance is the versatile optical method to detect changes of refractive index in the close vicinity to the sensor surface. Resonance occurs when the polarization and wave-vector of excitation light matches those of the surface plasmon-polariton (SPP). In these conditions the energy of the impinging light couples to SPP and a sharp decrease of the reflected light is observed. The resonance conditions are influenced only by changes of the refractive index in the volume of exponentially decaying evanescent field with penetration depth of about 150-200 nm.

Sub-micron and nanosized particles on the sensor surface disturb propagation conditions of the SPP. This results in the intense scattering of the SPP at those obstructions and to the change of the intensity of the reflected light. The observed change is proportional to the intensity of SPP, size of particle and its distance to the sensor surface. Besides it depends on the refraction index difference between the particle and surrounding media. In most cases the signal caused by the adsorption of single nanoparticles is rather small and requires a very careful design of SPR microscopy setup.

This phenomenon is similar to the stone in the water (or the rain droplets). While the stone itself might be too small to be seen directly, its presence is revealed upon the interaction with the surface waves. This interaction results in the concentric secondary waves with sizes larger than the stones or droplets themselves. Thus it is possible to detect each otherwise invisible single stone and droplet, as long as they interact with the water surface.

High resolution surface plasmon resonance microscopy instrument is being developed in the frame of NANODETECTOR project. It allows label-free visualization and ultrasensitive detection of single engineered and biological objects of sub-wavelength size such as viruses or bacteriophages. In our first experiments at LUAS this technique was used to observe binding of polymeric and inorganic nanoparticles from aqueous solutions to gold surface. The example of video images acquired and processed in real time are shown on the figure below. The binding of engineered nanopartciles to the SPR sensor surface is influenced by many types of interactions, what allows discrimination of nanoparticles based on their particular properties - e.g. surface potential and surface chemistry. Development of corresponding receptor layers for identification of engineered nanoparticles is in-progress.

One of the early examples of the differential SPR imaging of 100-nm Au NP's, binding to the sensor surface (only the part of the imaged surface is shown). Each frame corresponds to 2 seconds of real time. Black and white spots denote the attachment and detachment of single Au NP's to the surface.