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Biomedical Optics & Medical Imaging
Microtechnology enables endoscopic confocal microscopy
A new approach in optical architecture promises small-scale, high-resolution in vivo imaging for medical applications.
29 May 2007, SPIE Newsroom. DOI: 10.1117/2.1200705.0742
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 inspected 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 inaccessible. The biggest challenge in realizing this goal is miniaturizing 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 optical fiber movement or using the lens to scan the sample. But the results are neither fast enough for real-time imaging nor sufficiently practical to scale down to endoscopic sizes. Microelectromechanical systems (MEMS) scanners provide a scalable approach for miniaturizing single-axis confocal scanning endoscopes.1,2 Yet the high numerical aperture (NA) lens required to ensure high resolution has led to microscopes 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 system while maximizing imaging performance with a new MEMS scanner.
Figure 1. Schematic of the dual-axes confocal microscope architecture.
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 advantages 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, scattered light along the illuminated beam has a low probability of being collected outside the focus, thus increasing the detection sensitivity and dynamic range. The 2D MEMS scanner is the key optical element that enables post-objective scanning in a diminutive 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 optical 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
Figure 2. Scanning electron micrograph of the 2D microelectromechanical system (MEMS) scanner includes two mirrors: one for illumination and one for collection.
Figure 3. The macroscopic dual-axes breadboard imaging setup. PMT: Photomultiplier tube.
The imaging capability of the 2D scanner in the dual-axes configuration 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 illuminated 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 selectively transmit the desired fluorescent signal.
Figure 4. Reflectance image of ex vivo human colon tissue from the breadboard system taken at eight frames per second. (The scale bar is 30μm.)
Figure 5. Fluorescence image of muscle tissue removed from a mouse that produces green fluorescent protein. The image was obtained with the handheld probe prototype. (The scale bar is 100μm.)
Results from the breadboard setup4,5 demonstrate the feasibility of high-resolution real-time imaging with a 2D MEMS scanner. A reflectance image of excised human colon tissue is shown in Figure 4. In the image, we can discern crypt structures with lumen and surrounding colonocytes. The measured full-width-half-maximum (FWHM) transverse resolution is 3.9 and 6.7μm for the horizontal and vertical dimensions, respectively.
We are now downsizing the breadboard system to a 10mm-diameter handheld probe using 488nm-wavelength light. Figure 5 shows a fluorescence image from the first probe prototype. The results of the efforts described here hold great promise for high-resolution real-time imaging that will enable functional in vivo studies on small animal models of human disease and early diagnosis of cancer for patients in the clinic.
The authors would like to thank Daesung Lee, Gordon S. Kino, Michael J. Mandella, Thomas D. Wang, and Christopher H. Contag for useful discussions, and Pei-Lin Hsiung and Jonathan T. C. Liu for technical assistance. This research is supported by the National Institutes of Health under grant U54 CA105296.
Hyejun Ra and Olav Solgaard
Stanford, CA, USA
Hyejun Ra received her BS degree in electrical engineering from Seoul National University, Korea, in 2001, and her MS degree in electrical engineering from Stanford University, CA, in 2004. She is currently working toward a PhD degree in electrical engineering at Stanford University. Her research interests include developing MEMS and optical systems for biomedical imaging and sensing applications.
Olav Solgaard received his BS degree in electrical engineering from the Norwegian Institute of Technology. He obtained his MS and PhD degrees in electrical engineering from Stanford University, where he is currently an associate professor of electrical engineering. His research interests are micro-optical and nano-optical devices that combine MEMS, photonic crystals, integrated optics, and free-space optics. He has authored more than 180 technical publications and holds 25 patents.
Stanford, CA, USA
National Electronics and Computer Technology Center, Thailand
Wibool Piyawattanametha received his MS and PhD degrees in electrical engineering from the University of California, Los Angeles, CA, in 1999 and 2004, respectively. Prior to that, he obtained a BEng in electronics engineering from King Mongkut's Institute of Technology Ladkrabang, Thailand in 1994. He is currently with the National Electronics and Computer Technology Center (NECTEC), Thailand, and Stanford University as a research scientist. His interests include MEMS and biomedical imaging systems.
Yoshihiro Taguchi received his BS, MS, and PhD degrees in mechanical engineering from Keio University, Japan, in 1999, 2001, and 2004, respectively. He was a postdoctoral researcher at Stanford University from 2004 to 2006. He is currently with Keio University, Japan, as an instructor of system design engineering. His interests are in optical MEMS devices using near-field optics, near-field fluorescence, thermoreflectance, and molecular vibration spectroscopy for property measurements.
4. H. Ra, Y. Taguchi, D. Lee, W. Piyawattanametha, O. Solgaard, "Two-dimensional MEMS scanner for dual-axes confocal in vivo microscopy," in IEEE Int. Conf. Microelectromech. Sys., Istanbul, Turkey, pp. 862-865, 2006.