Surface plasmon resonance microscopy

Surface plasmon resonance microscopy (SPRM), also called surface plasmon resonance imaging (SPRI), is a label free analytical tool that combines the surface plasmon resonance of metallic surfaces with imaging of the metallic surface. The heterogeneity of the refractive index of the metallic surface imparts high contrast images, caused by the shift in the resonance angle. SPRM can achieve a sub-nanometer thickness sensitivity and lateral resolution achieves values of micrometer scale. SPRM is used to characterize surfaces such as self-assembled monolayers, multilayer films, metal nanoparticles, oligonucleotide arrays, and binding and reduction reactions. Surface plasmon polaritons are surface electromagnetic waves coupled to oscillating free electrons of a metallic surface that propagate along a metal/dielectric interface. Since polaritons are highly sensitive to small changes in the refractive index of the metallic material, it can be used as a biosensing tool that does not require labeling. SPRM measurements can be made in real-time, such as measuring binding kinetics of membrane proteins in single cells, or DNA hybridization.

History The concept of classical SPR has been since 1968 but the SPR imaging technique was introduced in 1988 by Rothenhäusler and Knoll. Capturing a high resolution image of low contrast samples for optical measuring techniques is a near impossible task until the introduction of SPRM technique that came into existence in the year 1988. In SPRM technique, plasmon surface polariton (PSP) waves are used for illumination. In simple words, SPRI technology is an advanced version of classical SPR analysis, where the sample is monitored without label through the use of a CCD camera. The SPRI technology with the aid of CCD camera gives advantage of recording the sensograms and SPR images, and simultaneously analyzes hundreds of interactions.

Principles Surface plasmons or surface plasmon polaritons are generated by coupling of electrical field with free electrons in a metal. SPR waves propagate along the interface between dielectrics and a conducting layer rich in free electrons. As shown in Figure 2, when light passes from a medium of high refractive index to a second medium with a lower refractive index, the light is totally reflected under certain conditions. In order to get total internal reflection (TIR), the θ1 and θ2 should be within a certain range that can be explained through the Snell's law. When light passes through a high refractive index media to a lower refractive media, it is reflected at an angle θ2, which is defined in Equation 1.

In the TIR process some portion of the reflected light leaks a small portion of electrical field intensity into medium 2 (η1 > η2). The light leaked into the medium 2 penetrates as an evanescent wave. The intensity and penetration depth of the evanescent wave can be calculated according to Equations 2 and 3, respectively.

Figure 3 shows a schematic representation of surface plasmons coupled to electron density oscillations. The light wave is trapped on the surface of the metal layer by collective coupling to the electrons of the metal surface. When the electron's plasma and the electric field of the wave light couple their frequency oscillations they enters into resonance.

Recently, the leakage light inside of the metal surface had been imaged. Radiation of different wavelengths (green, red and blue) was converted into surface plasmon polaritons, through the interaction of the photons at the metal/dielectric interface. Two different metal surfaces were used; gold and silver. The propagation length of the SPP along the x-y plane (metal plane) in each metal and photon wavelength were compared. The propagation length is defined as the distance traveled by the SPP along the metal before its intensity decreases by a factor of 1/e, as defined in Equation 4. Figure 4 shows the leakage light captured by a color CCD camera, of the green, red and blue photons in gold (a) and silver (b) films. In part c) of Figure 4, the intensity of the surface plasmon polaritons with the distance is shown. It was determined that the leakage light intensity is proportional to the intensity in the waveguide.

where δSPP is the propagation length; ε'm and ε″m are the relative permittivity of the metal and λ0 is the free space wavelength. The metallic film is capable of absorbing light due to the coherent oscillation of the conduction band electrons induced by the interaction with an electromagnetic field. Electrons in the conduction band induce polarization after interaction with the electric field of the radiation. A net charge difference is created in the surface of the metal film, creating a collective dipolar oscillation of electrons with the same phase. When the electron motion matches the frequency of the electromagnetic field, the absorption of incident radiation occurs. The oscillation frequency of gold surface plasmons is found in the visible region of the electromagnetic spectrum, giving a red color while silver gives yellow color. Nanorods exhibit two absorption peaks in the UV-vis region due to longitudinal and transversal oscillation, for gold nanorods the transverse oscillation generates a peak at 520 nm, while the longitudinal oscillation generates absorption at longer wavelengths, within a range of 600 to 800 nm. Silver nanoparticles shift their light absorption wavelengths to higher energy levels, where the blue shifting goes from 408 nm to 380 nm, and 372 nm, when they change from sphere to rod and wire, respectively. The absorption intensity and wavelength of gold and silver depends on the size and shape of the particles. In Figure 5, the size and shape of silver nanoparticles influenced the intensity of the scattered light and maximum wavelength of silver nanoparticles. The triangular shaped particles appear red with a maximum scattered light at 670–680 nm, the pentagonal particles appear in green (620–630 nm) and the spherical particles have higher absorption energies (440–450 nm), appear in blue.

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