Fig. 1

Surface Plasmon Resonance Microscopy (SPRM) of pancreatic beta-cells (a) Schematic presenting the experimental setup for SPRM, depicting the layered structure of gold thin film on glass substrate with cells adhered to the gold surface in Hanks’ balanced salt solution (HBSS). A fiber-coupled laser (690 nm) is collimated before focusing on the back focal plane (BFP) of a high numerical aperture oil immersion objective to produce a collimated beam at the sample. The angle of illumination is varied by laterally scanning the focus on the BFP. The sample is imaged using a 2D CMOS pixelated detector. (b) A magnified view of the SPR sensor and cell interface showing the interface layers (glass, Au thin film of 50 nm, medium (HBSS), cell membrane of c.7 nm thickness, and cytosol), with an illustration of the penetration depth of SPs in both metal and dielectric media. This indicates sensitivity to the cell membrane and the proximal intra- and extracellular spaces. (c) SPR curves presenting the reflection coefficient for various angles of incidence, simulated for bare gold with HBSS and for the gold-cell interface respectively. (d) The corresponding first derivative of reflectivity with respect to the angle of incidence, showing the variations in the sensitivity of the measurement for optimising the angle of illumination. (e) (i). Brightfield microscopy image of live MIN6 beta-cells cultured on PLL-modified Au thin film. e (ii), e (iii), and e (iv) are the corresponding SPRM images at different angles of illumination. The angle of incidence is selected in region iii, although this gives reduced sensitivity, it allows simultaneous tracking of cells and the extracellular regions where cells are not present on the sensor.