## 2 Principles of Modern PCEM
### PCEM Imaging Modality and Operating Principle  
As shown in the schematic diagram (Figure 4B), the PCEM detection instrument uses a linear scanning approach and is built upon the body of an inverted microscope (Carl Zeiss Axio Observer Z1). In addition to ordinary brightfield imaging, a second illumination path is provided from a fiber-coupled broadband LED, which is incident on the PC from below. The unpolarized LED output light passes a polarized beam splitter (PBS) to illuminate the PC with light polarized with its axis perpendicular to the grating lines (e.g., \(y\) direction), representing the transverse magnetic (TM) mode. Since the resonant wavelength of a 1D PC surface is only sensitive to the incident angle in one angular dimension (perpendicular to the grating) (\(y\) direction), the light can be focused in the orthogonal angular dimension (parallel to the grating) (e.g., \(x\) direction) to strengthen the incident intensity. Therefore, the light passing through the PBS is focused in one axis (\(x\) direction) by a cylindrical lens, while the light remains collimated in the other angular dimension (\(y\) direction). The linear beam (collimated direction) is focused on the back focal plane of the objective lens of the microscope. The light emerging from the objective lens (upwards) is thus incident on the PC, so it is collimated in the direction perpendicular to the PC grating lines (\(y\) direction) and, thus, all the light reaching the PC with the TM polarization has the same angle of incidence. The reflected light beam passes through the objective lens in the opposite direction (downwards), after which it is projected onto an imaging spectrometer through a narrow entrance slit. The imaging spectrometer contains a diffraction grating that disperses the wavelength components of the PC-reflected light. Once the spectrometer is determined, the dimension of one imaged pixel of the PC in the direction parallel to the grating lines (\(x\) direction) is determined by the magnification of the objective lens and the dimension of pixelswithin the charge-coupled device (CCD) camera (Photometrics Cascade, 512\({}^{2}\) pixels). A motorized stage (Applied Scientific Instruments, MS2000) linearly translates the PC in the perpendicular direction to the grating (\(y\) direction). The step-size of the stage (together with the magnifications of the objective lens) determines the pixel size of the PCEM imaging system in the \(y\) direction. Therefore, a large area can be scanned in a line-by-line fashion by translating the PC sensor in steps perpendicular to the linear grating direction (\(y\) direction). For example, with a 10 \(\times\) objective lens of the microscope, a 16 \(\upmu\)m\({}^{2}\) pixel size of the CCD camera, and a 0.6 \(\upmu\)m step size of the motorized stage, a final acquired image with 0.6\({}^{2}\)\(\upmu\)m\({}^{2}\) pixel size can be measured in PCEM (with an acquisition speed of \(\sim\)10 s per frame for a scanning area of 300\({}^{2}\)\(\upmu\)m\({}^{2}\)).  
For PCEM data acquisition, the linear light beam reflected from the PC that contains the resonant biosensing signal produces a spatially resolved spectrum for each point along the line with a narrow bandwidth (e.g., \(\Delta\lambda\sim\) 4 nm) and forms a 2D spectrograph (e.g., 512\({}^{2}\) pixels) across the line (Figure 4D). After line-by-line scanning, a 3D spectrum data (e.g., 512\({}^{3}\) cube) can be acquired (Figure 4D inset) and the signal/image processing can be performed with computational software (Matlab, MathWorks). Specifically, the spectrum signal can be mathematically fit with a second-order polynomial or Lorentz function for each pixel to extract the peak wavelength and intensity values. With a background image acquired beforehand, shifts in the peak wavelength value (PWV) or shifts in the local peak intensity value (PIV) can be calculated at each pixel location to measure the redistribution of the attached biomaterials.