Key Words: Fluorescence, TIRF
Definition:SPMFM enhances the fluorescence signal in TIRF microscopy and simultaneously reduces background noise.
TECHNOLOGY:
Surface plasmons are oscillations of free electrons at the surface of a metal film, which propagate along the surface to create an evanescent electromagnetic field. While surface plasmons cannot be excited directly with incident light from the same side, they can be excited on the opposite side with plane polarized light directed at the metal from the medium with the higher refractive index and at a precise angle (the surface plasmon angle). Excitation of surface plasmons is associated with a sharp decrease in the reflected light, a phenomenon used in Surface Plasmon Resonance (SPR) biosensors.
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The evanescent electromagnetic field associated with the surface plasmon is very similar to that created in TIRF. It has the same penetration depth for the same incident angle. However, the intensity of this field can be much greater in the presence of a metallic thin film when excited at the correct angle. As a result, fluorophores near the metallic surface receive much more light. The metallic thin film also changes fluorophore emission with collection efficiency varying with fluorophore-metal distance. For distances less than 10 nm, fluorescence is quenched. For distances greater that 150 nm, most emitted fluorescence is reflected by the metal (>90%). Detection efficiency is high for intermediate distances only (10-50 nm) and can reach values as high as 50% of the whole fluorescence emission (for silver thin film). The presence of the metallic thin film acts as a distance-dependent filter effectively selecting fluorophores at the correct distance for live membrane observation.
APPLICATIONS:
SPMFM can be used to enhance the sensitivity of TIRF applications (such as the observation of endo- and exocytosis, protein dynamics, cell-substrate interactions and signalling events close to the cell membrane).
IMAGE CAPTION:
Comparison image from Nikon Note 12. HEK cells transfected with mCherry on silver thin film (upper) and on a standard glass slide (lower) at a subcritical excitation angle (left) and at a supercritical angle corresponding to surface plasmon excitation of the silver (right). Image courtesy of Karla Balaa and Emmanuel Fort, Langevin Institute, ESPCI, Paris, France.
MICROSCOPE CONFIGURATION:
The imaging configuration is that for TIRF except that the coverglass is coated on one side with a nanometre film of metal (achieved by thermal evaporation under vacuum or purchased from commercial suppliers). Silver, gold or aluminium may be used although silver provides optimal results. The silver surface must be protected (e.g. using a self assembly polyethylene glycol (PEG) layer) before adding cells. As in TIRF, SPMFM requires a high numerical aperture (N.A.) objective (Nikon CFI Apochromat TIRF series 60x or 100x oil objectives (N.A. 1.49) to achieve required illumination angles.
Product links: Ti, CFI Apo TIRF 100X oil objective