10.1117/2.1200705.0742 Microtechnology enables endoscopic confocal microscopy Hyejun Ra, Wibool Piyawattanametha, Yoshihiro Taguchi, and Olav Solgaard A new approach in optical architecture promises small-scale, high- resolution in vivo imaging for medical applications. Single-axis confocal microscopes are commonly used to obtain high-resolution images of biological tissue. Traditionally, excised tissue samples are fixed with preservative chemicals and in- spected under a tabletop microscope. However, there is now rapidly growing interest in in vivo imaging, which will permit observation of biological processes that were previously inacces- sible. The biggest challenge in realizing this goal is miniaturiz- ing the microscope while maintaining high resolution and image quality. Extended efforts toward constructing small versions of single- axis confocal microscopes have involved either shifting the op- tical fiber movement or using the lens to scan the sample. But the results are neither fast enough for real-time imag- ing nor sufficiently practical to scale down to endoscopic sizes. Microelectromechanical systems (MEMS) scanners pro- vide a scalable approach for miniaturizing single-axis confo- cal scanning endoscopes. 1, 2 Yet the high numerical aperture (NA) lens required to ensure high resolution has led to mi- croscopes with limited working distance (WD) and field of view (FOV). Our approach decouples resolution and WD by using a dual-axes confocal architecture, 3 and shrinks the sys- tem while maximizing imaging performance with a new MEMS scanner. Dual-axes confocal microscopy uses two low-NA objectives with the illumination and collection axes crossed at an angle, θ, from the midline, as shown in Figure 1. This design offers ad- vantages over conventional single-axis architecture for in vivo imaging. First, subcellular resolution can be achieved in both transverse and axial dimensions using low-NA objectives that facilitate miniaturization. Second, a long WD allows for post- objective scanning, which minimizes aberrations. Third, scat- tered light along the illuminated beam has a low probability of being collected outside the focus, thus increasing the detection Figure 1. Schematic of the dual-axes confocal microscope architecture. sensitivity and dynamic range. The 2D MEMS scanner is the key optical element that enables post-objective scanning in a diminu- tive package while maximizing FOV. We designed and fabricated a 2D MEMS scanner, 4 as shown in Figure 2. It is made from double silicon-on-insulator (SOI) wafers and is actuated by self-aligned vertical combs for large scanning angles. The scanner is designed to be integrated in a 5mm-diameter endoscope, and achieves maximum DC opti- cal deflections of ±4.8 and ±5.5 ◦ for the outer and inner axes, respectively. The corresponding torsional resonant frequencies are 500 and 2.9kHz. These mirror characteristics allow real-time imaging with a large FOV. The scanner is also metallized with a 10nm-thick aluminum layer to increase mirror reflectivity. 5 The imaging capability of the 2D scanner in the dual-axes con- figuration was first demonstrated in a breadboard setup for both reflectance and fluorescence modes. A schematic of the imaging arrangement is shown in Figure 3. The target sample is illumi- nated by a 488nm-wavelength laser beam. The signal from the volume where the beams overlap is collected by a single-mode fiber and acquired by a computer. For fluorescence imaging, a long-pass optical filter is inserted into the collection path to se- lectively transmit the desired fluorescent signal. Continued on next page