FASPRI: a new method for increased spatial resolution in surface plasmon imaging of unlabelled living cells

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Surface plasmon resonance (SPR) can be used to monitor molecular events such as the binding of an antibody to its target. It is a highly sensitive optical method. In this method, a parallel beam of light is directed into a glass prism, coated on the hypotenuse face with a metal (often gold), so that it reflects off the metal film and emerges from the prism. At a certain angle of incidence, the light excites collective oscillations of free electrons knows as surface plasmons and the gold ceases to reflect light. This is seen as a sharp dip in the plot of reflected light intensity versus angle of incidence. The position and the depth of the dip change dramatically when an antibody binds to the antigen-coated gold, and the degree of binding can thus be measured accurately, even when the bound antibody layer is only one molecule thick. Attempts to form high-resolution microscope images with SPR have failed because the beam of light reflected is parallel, and such beams cannot form detailed images. We propose here to transform SPR into a high-resolution microscope method for imaging events involving small numbers of molecules in living cells without the need to label them with specific dyes. The reflected beam in the standard SPR method is essentially reflected by a metal mirror. It is a basic physical principle that in such a situation the incidence and reflected light interfere to produce a so-called standing wave, which has zero intensity at the mirror surface. It has been established since the 19th century that no light can be detected at the mirror surface. It may be useful to think of the standing waves that can be produced by hand in a skipping rope tethered to a wall at one end. At the wall no motion can be detected in the rope. We propose to place a thin fluorescent layer made of fluorescent organic dye or nanoscale crystals between the glass and the gold film. This will ordinarily not fluoresce because it is in the aforementioned zone of zero intensity. However, in any region where SPR occurs, the reflected beam will be substantially reduced in intensity (i.e. the mirror will cease to reflect) and instead of a standing wave, there will be an ordinary propagating wave passing into the gold layer, creating resonance. Fluorescence will then be excited. Since the fluorescence will radiate in all directions it will be ideal for imaging: we will use a microscope specifically optimised for high-resolution and sensitive imaging of light radiating from fluorescent particles. We call this approach FASPRI, which stands for Full-Aperture Surface Plasmon Resonance Imaging, since filling the full aperture of the lens is our key improvement over other attempts.First, we will sandwich a fluorescent layer between a gold film and a microscope coverslip. Next, we will check that the fluorescence appears at the plasmon resonance angle, which we will measure by reflectance. We will then adapt our existing fluorescence microscope and then perform FASPRI imaging of living algal and mammalian cells.