## 4 Applications of PCEM
### PCEM for Imaging of Nanoparticle and Protein-Protein Binding  
Because the PC surface structure restricts lateral propagation of light at the resonant wavelength, it is possible to create spatial maps of the resonant wavelength and the resonant damping that allow high spatial resolution imaging of small-size biomaterials distributed across the surface. Particles smaller than the pixel size (e.g., 600\({}^{2}\) nm\({}^{2}\) for our current PCEM) are very challenging to visualize and identify. However, it is possible to detect the presence of individual particles when the PWS induced by each particle is higher than the detection sensitivity limit of PCEM at each pixel location (the noise-induced PWS need to be considered as well). It is noteworthy that the PWV image for a particle is always within a diffraction-limited distance of up to five (or more) adjacent pixels and, hence, it is not expected to observe a PWS of only one pixel when a sub-micron nanoparticle attaches to the PC. As shown in Figure 4F, a PWV image is acquired for a 3 \(\times\) 3 polystyrene particle array that is printed by thermal Dip-Pen Nanolithography (tDPN) [97,98] with heated atomic force microscopy (AFM) tips. Each particle has the dimension of \(\sim\)540\({}^{2}\times\) 40 nm\({}^{3}\) and \(\sim\)5 \(\upmu\)m gaps in between (Figure 4E). Figure 4G demonstrates two acquired spectra (one from a pixel at particle location, and one from  
Figure 5: Wavelength-sensitive live cell image from instrument 3–PCEM. (**A**) Brightfield and (**B**) PWV images of Panc-1 cells attached to the PC surface. Lamellipodial extensions are visible, especially from cell 2, demonstrating the ability of PCEM to resolve regional differences in single-cell attachment; (**C**) Representative spectra (normalized) from background regions and regions with cellular attachment. Selected areas of the PWV image from beneath a cell show the PWS of a typical Panc-1 cell is \(\sim\)1.0 nm; (**D**) Time-lapse PWS images of cellular attachment of dental stem cells (mHAT9a) (Reprinted in part with permission from [45], © 2013 RSC Publishing). background location) and each printed particle can cause \(\sim\)0.5 nm PWS, which can be easily detected and visualized using the PCEM system. Not only dielectric nanoparticles (as optical scatters) but also metal nanoparticles (as optical absorbers) as small as \(\sim\)100 nm can be detected via PIV-shift images using PCEM [48]. Single nanoparticles allowing direct visualization in PCEM can be applied as biosensing tags to detect protein-protein binding for multiple events on a large sensor surface synchronously. This detection and imaging capability may be used in high-throughput screening during extended periods while avoiding photobleaching issues that are inherent for fluorescence dye tags. Furthermore, the resonance wavelength of nanoparticles can be conveniently tuned through the incident angle of the illumination light [44], the dimension of the PC biosensor [80], and the size or geometry of the nanoparticle [48, 99, 100]. An example is plotted in Figure 6 for PCEM detection of a target protein molecule (e.g., Rabbit Immunoglobulin G (IgG)) binding with the immobilized capture antibodies (e.g., anti-Rabbit IgG) using gold nanorods (AuNR) as tags [48]. The aspect ratio of the AuNR (dimension of \(\sim\)65\({}^{2}\)\(\times\) 30 nm\({}^{3}\)) was tuned such that its localized surface plasmon resonance (LSPR) matched the resonant wavelength of the PC biosensor, and thus further improved the signal-to-noise ratio performance of the imaging system. Figure 6: PCEM detection of protein-protein binding. (**A**) Schematic illustration of the PCEM detection of protein-protein binding on the PC biosensor surface; (**B**) SEM images of AuNR-IgG (AuNR conjugated with SH-PEG-IgG) attached to the PC biosensor surface.