Lissajous scanning structured illumination microscopy.

High-resolution fluorescent microscopic imaging techniques are in high demand to observe detailed structures or dynamic mechanisms of biological samples. Structured illumination microscopy (SIM) has grabbed much attention in super-resolution imaging due to simple configuration, high compatibility with common fluorescent molecules, and fast image acquisition. Here, we report Lissajous scanning SIM (LS-SIM) by using a high fill-factor Lissajous scanning micromirror and laser beam modulation. The LS-SIM was realized by a Lissajous scanned structured illumination module, relay optics, and a conventional fluorescent microscope. The micromirror comprises an inner mirror and an outer frame, which are scanned at pseudo-resonance with electrostatic actuation. The biaxial scanning frequencies are selected by the frequency selection rule for high fill-factor (> 80%) Lissajous scanning. Structured illumination (SI) was then realized by modulating the intensity of a laser beam at the least common multiple (LCM) of the scanning frequencies. A compact Lissajous scanned SI module containing a fiber-optic collimator and Lissajous micromirror has been fully packaged and coupled with relay optics and a fiber-based diode pumped solid state (DPSS) laser including acousto-optic-modulator (AOM). Various structured images were obtained by shifting the phase and orientation of the illumination patterns and finally mounted with a conventional fluorescent microscope. The LS-SIM has experimentally demonstrated high-resolution fluorescent microscopic imaging of reference targets and human lung cancer cell PC-9 cells. The LS-SIM exhibits the observable region in spatial frequency space over 2x, the line-edge sharpness over 1.5x, and the peak-to-valley (P-V) ratio over 2x, compared to widefield fluorescent microscopy. This method can provide a new route for advanced high-resolution fluorescent microscopic imaging.

Lissajous scanned variable structured illumination for dynamic stereo depth map.

Structured illumination plays an important role in advanced photographic and microscopic imaging applications. Here we report variable structured illumination (VSI) using Lissajous scanning techniques. The variable structured illumination module comprises Lissajous scanning micromirror and fiber-based diode pumped solid state (DPSS) laser with intensity modulation, combined with a stereo camera for dynamic stereo depth map. The micromirror projects static and discrete patterns by modulating the intensity of a laser beam at the least common multiple (LCM) of two scanning frequencies. The pattern density is increased by either decreasing the greatest common divisor (GCD) of scanning frequencies or decreasing the duty rate of the laser modulation. The scanning amplitude also controls the field-of-view (FOV) for the exact illumination of a target object for dynamic stereo depth map. The variable structured illumination module provides a new route for advanced imaging applications such as high-quality depth map, super-resolution, or motion recognition.

Handheld endomicroscope using a fiber-optic harmonograph enables real-time and in vivo confocal imaging of living cell morphology and capillary perfusion.

Confocal laser endomicroscopy provides high potential for noninvasive and in vivo optical biopsy at the cellular level. Here, we report a fully packaged handheld confocal endomicroscopic system for real-time, high-resolution, and in vivo cellular imaging using a Lissajous scanning fiber-optic harmonograph. The endomicroscopic system features an endomicroscopic probe with a fiber-optic harmonograph, a confocal microscope unit, and an image signal processor. The fiber-optic harmonograph contains a single mode fiber coupled with a quadrupole piezoelectric tube, which resonantly scans both axes at ~ 1 kHz to obtain a Lissajous pattern. The fiber-optic harmonograph was fully packaged into an endomicroscopic probe with an objective lens. The endomicroscopic probe was hygienically packaged for waterproofing and disinfection of medical instruments within a 2.6-mm outer diameter stainless tube capable of being inserted through the working channel of a clinical endoscope. The probe was further combined with the confocal microscope unit for indocyanine green imaging and the image signal processor for high frame rate and high density Lissajous scanning. The signal processing unit delivers driving signals for probe actuation and reconstructs confocal images using the auto phase matching process of Lissajous fiber scanners. The confocal endomicroscopic system was used to successfully obtain human in vitro fluorescent images and real-time ex vivo and in vivo fluorescent images of the living cell morphology and capillary perfusion inside a single mouse.

Lissajous Scanning Two-photon Endomicroscope for In vivo Tissue Imaging.

An endomicroscope opens new frontiers of non-invasive biopsy for in vivo imaging applications. Here we report two-photon laser scanning endomicroscope for in vivo cellular and tissue imaging using a Lissajous fiber scanner. The fiber scanner consists of a piezoelectric (PZT) tube, a single double-clad fiber (DCF) with high fluorescence collection, and a micro-tethered-silicon-oscillator (MTSO) for the separation of biaxial resonant scanning frequencies. The endomicroscopic imaging exhibits 5 frames/s with 99% in scanning density by using the selection rule of scanning frequencies. The endomicroscopic scanner was compactly packaged within a stainless tube of 2.6 mm in diameter with a high NA gradient-index (GRIN) lens, which can be easily inserted into the working channel of a conventional laparoscope. The lateral and axial resolutions of the endomicroscope are 0.70 µm and 7.6 µm, respectively. Two-photon fluorescence images of a stained kidney section and miscellaneous ex vivo and in vivo organs from wild type and green fluorescent protein transgenic (GFP-TG) mice were successfully obtained by using the endomicroscope. The endomicroscope also obtained label free images including autofluorescence and second-harmonic generation of an ear tissue of Thy1-GCaMP6 (GP5.17) mouse. The Lissajous scanning two-photon endomicroscope can provide a compact handheld platform for in vivo tissue imaging or optical biopsy applications.

Antireflective structures on highly flexible and large area elastomer membrane for tunable liquid-filled endoscopic lens.

The antireflection coating on an optical lens plays a key role not only in minimizing specular reflection but also in maximizing light transmission. Antireflective structures (ARS) found in the eyes of moths have the same role as conventional antireflection coating, and they also provide many intriguing features such as broadband coverage, self-antireflection, and strong water repellence. In particular, ARS on a highly flexible membrane can effectively deliver antireflective properties, which are rarely achieved by conventional hard coating. Herein, we report antireflective structures on a highly flexible elastomeric lens membrane for tunable endoscopic imaging applications. A thin antireflective membrane was fabricated using a large-area nanohole template and replica molding. The thin membrane increases light transmittance up to 96.8% in the visible region while enhancing the image contrast up to 56%, higher than that of a smooth membrane during lens deformation. This antireflective, tunable, liquid-filled lens offers new opportunities for compact endoscopic laparoscopy enabling maximum transmittance and variable zoom imaging in narrow and low-illumination environments.

Scanning MEMS Mirror for High Definition and High Frame Rate Lissajous Patterns.

Scanning MEMS (micro-electro-mechanical system) mirrors are attractive given their potential use in a diverse array of laser scanning display and imaging applications. Here we report on an electrostatic MEMS mirror for high definition and high frame rate (HDHF) Lissajous scanning. The MEMS mirror comprised a low Q-factor inner mirror and frame mirror, which provided two-dimensional scanning at two similar resonant scanning frequencies with high mechanical stability. The low Q inner mirror enabled a broad frequency selection range. The high definition and high frame rate (HDHF) Lissajous scanning of the MEMS mirror was achieved by selecting a set of scanning frequencies near its resonance with a high greatest common divisor (GCD) and a high total lobe number. The MEMS mirror had resonant scanning frequencies at 5402 Hz and 6702 Hz in x and y directions, respectively. The selected pseudo-resonant frequencies of 5450 Hz and 6700 Hz for HDHF scanning provided 50 frames per second with 94% fill factor in 256 × 256 pixels. This Lissajous MEMS mirror could be utilized for assorted HDHF laser scanning imaging and display applications.

1.65 mm diameter forward-viewing confocal endomicroscopic catheter using a flip-chip bonded electrothermal MEMS fiber scanner.

We report a 1.65 mm diameter forward-viewing confocal endomicroscopic catheter using a flip-chip bonded electrothermal MEMS fiber scanner. Lissajous scanning was implemented by the electrothermal MEMS fiber scanner. The Lissajous scanned MEMS fiber scanner was precisely fabricated to facilitate flip-chip connection, and bonded with a printed circuit board. The scanner was successfully combined with a fiber-based confocal imaging system. A two-dimensional reflectance image of the metal pattern 'OPTICS' was successfully obtained with the scanner. The flip-chip bonded scanner minimizes electrical packaging dimensions. The inner diameter of the flip-chip bonded MEMS fiber scanner is 1.3 mm. The flip-chip bonded MEMS fiber scanner is fully packaged with a 1.65 mm diameter housing tube, 1 mm diameter GRIN lens, and a single mode optical fiber. The packaged confocal endomicroscopic catheter can provide a new breakthrough for diverse in-vivo endomicroscopic applications.

Frequency selection rule for high definition and high frame rate Lissajous scanning.

Lissajous microscanners are very attractive in compact laser scanning applications such as endomicroscopy or pro-projection display owing to high mechanical stability and low operating voltages. The scanning frequency serves as a critical factor for determining the scanning imaging quality. Here we report the selection rule of scanning frequencies that can realize high definition and high frame-rate (HDHF) full-repeated Lissajous scanning imaging. The fill factor (FF) monotonically increases with the total lobe number of a Lissajous curve, i.e., the sum of scanning frequencies divided by the great common divisor (GCD) of bi-axial scanning frequencies. The frames per second (FPS), called the pattern repeated rate or the frame rate, linearly increases with GCD. HDHF Lissajous scanning is achieved at the bi-axial scanning frequencies, where the GCD has the maximum value among various sets of the scanning frequencies satisfying the total lobe number for a target FF. Based on this selection rule, the experimental results clearly demonstrate that conventional Lissajous scanners substantially increase both FF and FPS by slightly modulating the scanning frequencies at near the resonance within the resonance bandwidth of a Lissajous scanner. This selection rule provides a new guideline for HDHF Lissajous scanning in compact laser scanning systems.

Electrothermal MEMS parallel plate rotation for single-imager stereoscopic endoscopes.

This work reports electrothermal MEMS parallel plate-rotation (PPR) for a single-imager based stereoscopic endoscope. A thin optical plate was directly connected to an electrothermal MEMS microactuator with bimorph structures of thin silicon and aluminum layers. The fabricated MEMS PPR device precisely rotates an transparent optical plate up to 37° prior to an endoscopic camera and creates the binocular disparities, comparable to those from binocular cameras with a baseline distance over 100 µm. The anaglyph 3D images and disparity maps were successfully achieved by extracting the local binocular disparities from two optical images captured at the relative positions. The physical volume of MEMS PPR is well fit in 3.4 mm x 3.3 mm x 1 mm. This method provides a new direction for compact stereoscopic 3D endoscopic imaging systems.

Electrothermal MEMS fiber scanner for optical endomicroscopy.

We report a novel MEMS fiber scanner with an electrothermal silicon microactuator and a directly mounted optical fiber. The microactuator comprises double hot arm and cold arm structures with a linking bridge and an optical fiber is aligned along a silicon fiber groove. The unique feature induces separation of resonant scanning frequencies of a single optical fiber in lateral and vertical directions, which realizes Lissajous scanning during the resonant motion. The footprint dimension of microactuator is 1.28 x 7 x 0.44 mm3. The resonant scanning frequencies of a 20 mm long optical fiber are 239.4 Hz and 218.4 Hz in lateral and vertical directions, respectively. The full scanned area indicates 451 µm x 558 µm under a 16 Vpp pulse train. This novel laser scanner can provide many opportunities for laser scanning endomicroscopic applications.

Micromachined tethered silicon oscillator for an endomicroscopic Lissajous fiber scanner.

This work reports micromachined tethered silicon oscillators (MTSOs) for endoscopic Lissajous fiber scanners. An MTSO comprises an offset silicon spring for stiffness modulation of a scanning fiber and additional mass for modulation of resonant scanning frequency in one body. MTSOs were assembled with a resonant fiber scanner and enhanced scanning reliability of the scanner by eliminating mechanical cross coupling. The fiber scanner with MTSOs was fully packaged as an endomicroscopic catheter and coupled with a conventional laparoscope and spectral domain OCT system. The endomicroscope was maneuvered with the integrated laparoscope and in vivo swine tissue OCT imaging was successfully demonstrated during open surgery. This new component serves as an important element inside an endoscopic Lissajous fiber scanner for early cancer detection or on-demand minimum lesional margin decision during noninvasive endoscopic biopsy.

Lissajous fiber scanning for forward viewing optical endomicroscopy using asymmetric stiffness modulation.

We report a fully packaged and compact forward viewing endomicroscope by using a resonant fiber scanner with two dimensional Lissajous trajectories. The fiber scanner comprises a single mode fiber with additional microstructures mounted inside a piezoelectric tube with quartered electrodes. The mechanical cross-coupling between the transverse axes of a resonant fiber with a circular cross-section was completely eliminated by asymmetrically modulating the stiffness of the fiber cantilever with silicon microstructures and an off-set fiber fragment. The Lissajous fiber scanner was fully packaged as endomicroscopic catheter passing through the accessory channel of a clinical endoscope and combined with spectral domain optical coherence tomography (SD-OCT). Ex-vivo 3D OCT images were successfully reconstructed along Lissajous trajectory. The preview imaging capability of the Lissajous scanning enables rapid 3D imaging with high temporal resolution. This endoscopic catheter provides many opportunities for on-demand and non-invasive optical biopsy inside a gastrointestinal endoscope.