Hyejun Ra

Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two- dimensional scanning mirror

Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce two-photon imaging based on microelectromechanical systems (MEMS) scanners. Single crystalline silicon scanning mirrors that are 0.75 mm x 0.75 mm in size and driven in two dimensions by microfabricated vertical comb electrostatic actuators can provide optical deflection angles through a range of approximately16 degrees . Using such scanners we demonstrated two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.52 kHz.

Two-Dimensional MEMS Scanner for Dual-Axes Confocal Microscopy

In this paper, we present a novel 2-D microelectromechanical systems (MEMS) scanner that enables dual-axes confocal microscopy. Dual-axes confocal microscopy provides high resolution and long working distance, while also being well suited for miniaturization and integration into endoscopes for in vivo imaging. The gimbaled MEMS scanner is fabricated on a double silicon-on-insulator (SOI) wafer (a silicon wafer bonded on a SOI wafer) and is actuated by self-aligned vertical electrostatic combdrives. Maximum optical deflections of plusmn4.8deg and plusmn5.5deg are achieved in static mode for the outer and inner axes, respectively. Torsional resonant frequencies are at 500 Hz and 2.9 kHz for the outer and inner axes, respectively. The imaging capability of the MEMS scanner is successfully demonstrated in a breadboard setup. Reflectance images with a field of view of are achieved at 8 frames/s. The transverse resolutions are 3.94 mum and 6.68 mum for the horizontal and vertical dimensions, respectively.

In vivo brain imaging using a portable 2.9 g two-photon microscope based on a microelectromechanical systems scanning mirror

We present a two-photon microscope that is approximately 2.9 g in mass and 2.0 x 1.9 x 1.1 cm(3) in size and based on a microelectromechanical systems (MEMS) laser-scanning mirror. The microscope has a focusing motor and a micro-optical assembly composed of four gradient refractive index lenses and a dichroic microprism. Fluorescence is captured without the detected emissions reflecting off the MEMS mirror, by use of separate optical fibers for fluorescence collection and delivery of ultrashort excitation pulses. Using this microscope we imaged neocortical microvasculature and tracked the flow of erythrocytes in live mice.

Fiber-optic confocal microscope using a MEMS scanner and miniature objective lens

We designed and constructed a single-fiber-optic confocal microscope (SFCM) with a microelectromechanical system (MEMS) scanner and a miniature objective lens. Axial and lateral resolution values for the system were experimentally measured to be 9.55 mum and 0.83 mum respectively, in good agreement with theoretical predictions. Reflectance images were acquired at a rate of 8 frames per second, over a 140 mum x 70 mum field-of-view. In anticipation of future applications in oral cancer detection, we imaged ex vivo and in vivo human oral tissue with the SFCM, demonstrating the ability of the system to resolve cellular detail.

Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner

The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5-7 microm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 x 100 microm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy.

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

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.

In vivo near-infrared dual-axis confocal microendoscopy in the human lower gastrointestinal tract

Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5 frames/sec with a field of view of 362×212 μm(2) and a maximum imaging depth of 140 μm. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.

siRNA silencing of keratinocyte-specific GFP expression in a transgenic mouse skin model

Small interfering RNAs (siRNAs) can be designed to specifically and potently target and silence a mutant allele, with little or no effect on the corresponding wild-type allele expression, presenting an opportunity for therapeutic intervention. Although several siRNAs have entered clinical trials, the development of siRNA therapeutics as a new drug class will require the development of improved delivery technologies. In this study, a reporter mouse model (transgenic click beetle luciferase/humanized monster green fluorescent protein) was developed to enable the study of siRNA delivery to skin; in this transgenic mouse, green fluorescent protein reporter gene expression is confined to the epidermis. Intradermal injection of siRNAs targeting the reporter gene resulted in marked reduction of green fluorescent protein expression in the localized treatment areas as measured by histology, real-time quantitative polymerase chain reaction and intravital imaging using a dual-axes confocal fluorescence microscope. These results indicate that this transgenic mouse skin model, coupled with in vivo imaging, will be useful for development of efficient and ‘patient-friendly’ siRNA delivery techniques and should facilitate the translation of siRNA-based therapeutics to the clinic for treatment of skin disorders.

Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery

A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500x500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections.

Single fiber confocal microscope with a two-axis gimbaled MEMS scanner for cellular imaging

We present a single fiber reflectance confocal microscope with a two dimensional MEMS gimbaled scanner. Achieved lateral and axial resolutions are 0.82 mum and 13 mum, respectively. The field of view is 140 x 100 mum at 8 frames/second. Images and videos of cell phantoms and tissue are presented with sub-cellular resolution.