Three-dimensional in vivo imaging by a handheld dual-axes confocal microscope

Department of Electrical Engineering, Stanford University, Stanford, CA 94305, USA.
Optics Express (Impact Factor: 3.49). 06/2008; 16(10):7224-32. DOI: 10.1364/OE.16.007224
Source: PubMed


We present a handheld dual-axes confocal microscope that is based on a two-dimensional microelectromechanical systems (MEMS) scanner. It performs reflectance and fluorescence imaging at 488 nm wavelength, with three-dimensional imaging capability. The fully packaged microscope has a diameter of 10 mm and acquires images at 4 Hz frame rate with a maximum field of view of 400 microm x 260 microm. The transverse and axial resolutions of the handheld probe are 1.7 microm and 5.8 microm, respectively. Capability to perform real time small animal imaging is demonstrated in vivo in transgenic mice.

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Available from: Christopher H Contag, Nov 26, 2015
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    • "The specifications of the handheld DAC microscope system for in vivo imaging have been previously reported (Ra et al., 2008; Piyawattanametha et al., 2009). The microscope operates at the NIR wavelength of 785 nm, which allows for noninvasive imaging in skin down to a depth of 200 ×… m. "
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    ABSTRACT: Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics.
    Full-text · Article · Dec 2010 · Journal of Investigative Dermatology
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    • "Endoscopic confocal microscopes with MEMS actuators have been limited to low NA (0.25-0.4) imaging due to size constraints, making them unsuited for skin imaging where NA > 0.7 is desired [15]. Dual axis confocal configurations provide increased axial resolution over single axis counterparts, but suffer from decreased collection efficiency due to low NA collection optics [16][17]. High NA objective lenses for endoscopic confocal microscopes have been developed [18][19], but they are designed for use with coherent fiber bundles to relay scanned light to the sample. "
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    ABSTRACT: This paper describes a handheld laser scanning confocal microscope for skin microscopy. Beam scanning is accomplished with an electromagnetic MEMS bi-axial micromirror developed for pico projector applications, providing 800 x 600 (SVGA) resolution at 56 frames per second. The design uses commercial objective lenses with an optional hemisphere front lens, operating with a range of numerical aperture from NA=0.35 to NA=1.1 and corresponding diagonal field of view ranging from 653 microm to 216 microm. Using NA=1.1 and a laser wavelength of 830 nm we measured the axial response to be 1.14 mum full width at half maximum, with a corresponding 10%-90% lateral edge response of 0.39 mum. Image examples showing both epidermal and dermal features including capillary blood flow are provided. These images represent the highest resolution and frame rate yet achieved for tissue imaging with a MEMS bi-axial scan mirror.
    Full-text · Article · Feb 2010 · Optics Express
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    • "The scaling laws in optics enable miniaturized imaging probes with functionality that cannot practically be achieved with traditional technologies (Solgaard 2008). Micromirrors powered by electrostatic vertical comb drives (Dooyoung et al. 2004; Hyuck et al. 2007; Krishnamoorthy et al. 2003) provide the favorable optical characteristics, large deflection angles and fast response times required for real-time imaging, and have been employed in optical coherence tomography (Aguirre et al. 2007; Woonggyu et al. 2005; Kumar et al. 2008a), confocal (Maitland et al. 2006; Ra et al. 2008; Kumar et al. 2008b) and multi-photon (Hoy et al. 2008; Piyawattanametha et al. 2006; Fu et al. 2006) microscopies. Unfortunately, the micromirrors used in these studies have been fabricated by complicated micromachining processes, resulting in low wafer yield, non-linear transformation between input voltage and mechanical scan angle and unstable scanning characteristics due to a significant tendency to exhibit the pull-in phenomenon and cease scanning operation. "
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    ABSTRACT: A handheld, forward-imaging, laser-scanning confocal microscope (LSCM) demonstrating optical sectioning comparable with microtome slice thicknesses in conventional histology, targeted towards interventional imaging, is reported. Fast raster scanning (approximately 2.5 kHz line scan rate, 3.0-5.0 frames per second) was provided by a 2-axis microelectromechanical system (MEMS) scanning mirror fabricated by a method compatible with complementary metal-oxide-semiconductor (CMOS) processing. Cost-effective rapid-prototyped packaging combined the MEMS mirror with micro-optical components into a probe with 18 mm outer diameter and 54 mm rigid length. ZEMAX optical design simulations indicate the ability of the handheld optical system to obtain lateral resolution of 0.31 and axial resolution of 2.85 microm. Lateral and axial resolutions are experimentally measured at 0.5 microm and 4.2 microm respectively, with field of view of 200 x 125 microm. Results of reflectance imaging of ex vivo swine liver, and fluorescence imaging of the expression of cytokeratin and mammaglobin tumor biomarkers in epithelial human breast tissue from metastatic breast cancer patients are presented. The results indicate that inexpensive, portable handheld optical microscopy tools based on silicon micromirror technologies could be important in interventional imaging, complementing existing coarse-resolution techniques to improve the efficacy of disease diagnosis, image-guided excisional microsurgery, and monitored photodynamic therapy.
    Full-text · Article · Dec 2009 · Biomedical Microdevices
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