Measurement of SPR evanescent wave (EW) penetration depth by geometric relationship of measured radii of polymer microspheres. A) Bright-field and SPR images (for 5 wavelengths) of a polymethylmethacrylate (PMMA) microsphere in water. B) Diagram of a microsphere at the SPR sensor interface showing that only a fraction of the bead lies within the EW. The equation shows the relationship between the EW penetration depth (d) and the radius of the sphere measured by bright field (r
) and SPRI (r
). The overlay shows a layer model of the interface; the water layer decreases thickness as the bead moves toward the surface and into the EW. C) SPR image and insert of a microsphere used to illustrate the image analysis procedure for measuring the value of r
, the SPRI detected radius. The standard deviation (σ) of background intensity is determined by the annulus-shaped ROI in the primary image, and in the image insert a value of 3σ is set as the threshold with pixels values above threshold colored red; the radius of the bead (r
in blue) is determined from the area of circle (green dashed circle) computed from the threshold. D) Exponential decay from surface into media of the SPR generated EW calculated as field intensity versus distance from surface (nm). Two values are labeled: the 1/e decay at 37% field strength commonly referred to as the “penetration depth”, and the 5% field strength value, which is the theoretical detection limit. E) Extent of the EW field depth (Lp) measured for several polymer microspheres as a function of excitation wavelength, along with theoretical values of the penetration depth (1/e) and detection limit (5% field strength). Within the standard deviation of the measurement error, the decay values agree for all microspheres and the calculated penetration depth at 1/e field decay.