## 3 History of PCEM Development
### Instrument 2--Transmission Acquisition Mode with Upright Microscopy and Laser Source  
To improve spatial resolution, an upright microscope (Olympus BX-51 WI) was integrated into the PC imaging system in 2009 [76] and the resulting system was named the \"Photonic Crystal Enhanced Microscope\" [44]. Instead of measuring reflection efficiency as a function of wavelength from the bottom of the PC surface, the second generation PCEM measured transmission efficiency as a function of incident angle, using a fixed illumination wavelength from a beam-expanded laser (Instrument 2, Figure 3A). This instrument was designed as a wide-field imaging system with collimated angle-tunable laser illumination, which allowed imaging of a PC surface using the same illumination source and imaging optics for both enhanced fluorescence (EF) and label-free (LF) modalities. As shown in Figure 3A, the light beam generated from a HeNe laser passes through a half-wave plate (for polarization control), a variable neutral density filter, a rotating diffuser (to reduce speckle and fringes), a beam expander, an aperture, and a motorized angle-tunable mirror before passing through the PC (which is positioned beneath the microscope objective lens). The gimbal-mounted motorized mirror sits on top of a motorized linear stage in order to maintain a constant illumination area on the PC device (as the mirror rotates) and provide selective light coupling to the PC. Using this approach, high spatial-resolution and high sensitivity LF and EF images (Figure 3B) can be accurately registered with each other since a common beam-path is shared for both imagingmodes [76]. An electron-multiplying (EM) CCD camera was used to acquire high resolution and large-area images, and thus enable high-throughput analysis. Moreover, images can be simultaneously acquired with other imaging techniques available on the EF/LF microscope, including reflected brightfield (BF) and differential interference contrast (DIC) images that can be overlaid with EF and LF images.  
This transmission-based PC imaging modality that was capable of simultaneous label-free and enhanced fluorescence imaging (EF/LF) was further developed and utilized in several follow-up publications [20,41,42,44,76,81,84]. One of the main applications envisioned for the instrument was for performing DNA and protein microarray analysis, in which the label-free image of immobilized capture spots could be used to verify correct microarray printing and uniform spot density, while the enhanced fluorescence imaging modality would be used after hybridization of the target molecules from a test sample that carries fluorescent tags. Optimization of the imaging spatial resolution was reported in [81]. Microplate, microfluidic channel, and spot-based affinity capture assays were also demonstrated with this detection platform [84]. Figure 3C shows an example of a label-free image acquired with a tunable resonant angle for a DNA microarray immobilized on the biosensor surface [37]. Figure 3D shows a line profile through a row (red line in Figure 3C) containing 4 blank spots followed by 12 probe spots. It can be clearly observed that areas where the probe DNA has been immobilized produce a measurable increase in the resonant angle.