Therapeutic Effects of Targeted PPARÉ£ Activation on Inflamed High-Risk Plaques Assessed by Serial Optical Imaging In Vivo.

Rationale: Atherosclerotic plaque is a chronic inflammatory disorder involving lipid accumulation within arterial walls. In particular, macrophages mediate plaque progression and rupture. While PPARγ agonist is known to have favorable pleiotropic effects on atherogenesis, its clinical application has been very limited due to undesirable systemic effects. We hypothesized that the specific delivery of a PPARγ agonist to inflamed plaques could reduce plaque burden and inflammation without systemic adverse effects. Methods: Herein, we newly developed a macrophage mannose receptor (MMR)-targeted biocompatible nanocarrier loaded with lobeglitazone (MMR-Lobe), which is able to specifically activate PPARγ pathways within inflamed high-risk plaques, and investigated its anti-atherogenic and anti-inflammatory effects both in in vitro and in vivo experiments. Results: MMR-Lobe had a high affinity to macrophage foam cells, and it could efficiently promote cholesterol efflux via LXRα-, ABCA1, and ABCG1 dependent pathways, and inhibit plaque protease expression. Using in vivo serial optical imaging of carotid artery, MMR-Lobe markedly reduced both plaque burden and inflammation in atherogenic mice without undesirable systemic effects. Comprehensive analysis of en face aorta by ex vivo imaging and immunostaining well corroborated the in vivo findings. Conclusion: MMR-Lobe was able to activate PPARγ pathways within high-risk plaques and effectively reduce both plaque burden and inflammation. This novel targetable PPARγ activation in macrophages could be a promising therapeutic strategy for high-risk plaques.

Processable high internal phase Pickering emulsions using depletion attraction.

High internal phase emulsions have been widely used as templates for various porous materials, but special strategies are required to form, in particular, particle-covered ones that have been more difficult to obtain. Here, we report a versatile strategy to produce a stable high internal phase Pickering emulsion by exploiting a depletion interaction between an emulsion droplet and a particle using water-soluble polymers as a depletant. This attractive interaction facilitating the adsorption of particles onto the droplet interface and simultaneously suppressing desorption once adsorbed. This technique can be universally applied to nearly any kind of particle to stabilize an interface with the help of various non- or weakly adsorbing polymers as a depletant, which can be solidified to provide porous materials for many applications.

High-speed time-resolved laser-scanning microscopy using the line-to-pixel referencing method.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to visualize photophysical characteristics of biological targets. However, conventional FLIM methods have some limitations that restrict obtaining high-precision images in real time. Here, we propose a high-speed time-resolved laser-scanning microscopy by incorporating a novel line-to-pixel referencing method into the previously suggested analog mean-delay (AMD) method. The AMD method dramatically enhances the photon accumulation speed for achieving the certain precision compared to the time-correlated single-photon counting (TCSPC) method while maintaining high photon efficiency. However, its imaging pixel rate can still be restricted by the rearm time of the digitizer when it is triggered by laser pulses. With our line-to-pixel referencing method, the pulse train repeats faster than the trigger rearm time can be utilized by generating a line trigger, which is phase-locked with only the first pulse in each horizontal line composing an image. Our proposed method has been tested with a pulsed laser with 40 MHz repetition rate and a commercial digitizer with a 500 ns trigger rearm time, and a frame rate of 3.73 fps with a pixel rate of 3.91 MHz was accomplished while maintaining the measurement precision under 20 ps.

Note: Development of a compact aperture-type XYθz positioning stage.

In this paper, we propose a new in-plane XYθz nano-positioning stage that utilizes piezoelectric actuators and flexure mechanisms. The proposed stage has an aperture and is compact, which facilitates its application in measurement equipment, especially those used for biological specimens. The stage has four piezoelectric actuators and four bridge-type flexure mechanisms, which are used to amplify the small motions produced by the piezoelectric actuators. This paper describes the modeling and design optimization of the stage, which has X- and Y-direction motion ranges of 300 µm and a θz-direction motion range of ±3.9 mrad. The stage measures 150 × 150 × 23 mm, and its aperture is 50 × 50 mm.

Intravascular optical imaging of high-risk plaques in vivo by targeting macrophage mannose receptors.

Macrophages mediate atheroma expansion and disruption, and denote high-risk arterial plaques. Therefore, they are substantially gaining importance as a diagnostic imaging target for the detection of rupture-prone plaques. Here, we developed an injectable near-infrared fluorescence (NIRF) probe by chemically conjugating thiolated glycol chitosan with cholesteryl chloroformate, NIRF dye (cyanine 5.5 or 7), and maleimide-polyethylene glycol-mannose as mannose receptor binding ligands to specifically target a subset of macrophages abundant in high-risk plaques. This probe showed high affinity to mannose receptors, low toxicity, and allowed the direct visualization of plaque macrophages in murine carotid atheroma. After the scale-up of the MMR-NIRF probe, the administration of the probe facilitated in vivo intravascular imaging of plaque inflammation in coronary-sized vessels of atheromatous rabbits using a custom-built dual-modal optical coherence tomography (OCT)-NIRF catheter-based imaging system. This novel imaging approach represents a potential imaging strategy enabling the identification of high-risk plaques in vivo and holds promise for future clinical implications.

High-speed 3-D measurement with a large field of view based on direct-view confocal microscope with an electrically tunable lens.

We propose a new structure of confocal imaging system based on a direct-view confocal microscope (DVCM) with an electrically tunable lens (ETL). Since it has no mechanical moving parts to scan both the lateral (x-y) and axial (z) directions, the DVCM with an ETL allows for high-speed 3-dimensional (3-D) imaging. Axial response and signal intensity of the DVCM were analyzed theoretically according to the pinhole characteristics. The system was designed to have an isotropic spatial resolution of 20 µm in both lateral and axial direction with a large field of view (FOV) of 10 × 10 mm. The FOV was maintained according to the various focal shifts as a result of an integrated design of an objective lens with the ETL. The developed system was calibrated to have linear focal shift over a range of 9 mm with an applied current to the ETL. The system performance of 3-D volume imaging was demonstrated using standard height specimens and a dental plaster.

Intracoronary dual-modal optical coherence tomography-near-infrared fluorescence structural-molecular imaging with a clinical dose of indocyanine green for the assessment of high-risk plaques and stent-associated inflammation in a beating coronary artery.

AIMS: Inflammation plays essential role in development of plaque disruption and coronary stent-associated complications. This study aimed to examine whether intracoronary dual-modal optical coherence tomography (OCT)-near-infrared fluorescence (NIRF) structural-molecular imaging with indocyanine green (ICG) can estimate inflammation in swine coronary artery. METHODS AND RESULTS: After administration of clinically approved NIRF-enhancing ICG (2.0 mg/kg) or saline, rapid coronary imaging (20 mm/s pullback speed) using a fully integrated OCT-NIRF catheter was safely performed in 12 atheromatous Yucatan minipigs and in 7 drug-eluting stent (DES)-implanted Yorkshire pigs. Stronger NIRF activity was identified in OCT-proven high-risk plaque compared to normal or saline-injected controls (P = 0.0016), which was validated on ex vivo fluorescence reflectance imaging. In vivo plaque target-to-background ratio (pTBR) was much higher in inflamed lipid-rich plaque compared to fibrous plaque (P < 0.0001). In vivo and ex vivo peak pTBRs correlated significantly (P < 0.0022). In vitro cellular ICG uptake and histological validations corroborated the OCT-NIRF findings in vivo. Indocyanine green colocalization with macrophages and lipids of human plaques was confirmed with autopsy atheroma specimens. Two weeks after DES deployment, OCT-NIRF imaging detected strong NIRF signals along stent struts, which was significantly higher than baseline (P = 0.0156). Histologically, NIRF signals in peri-strut tissue co-localized well with macrophages. CONCLUSION: The OCT-NIRF imaging with a clinical dose of ICG was feasible to accurately assess plaque inflammation and DES-related inflammation in a beating coronary artery. This highly translatable dual-modal molecular-structural imaging strategy could be relevant for clinical intracoronary estimation of high-risk plaques and DES biology.

Fiber-optic raster scanning two-photon endomicroscope using a tubular piezoelectric actuator.

A nonresonant, fiber-optic raster scanning endomicroscope was developed using a quarter-tubular piezoelectric (PZT) actuator. A fiber lever mechanism was utilized to enhance the small actuation range of the tubular PZT actuator and to increase its field-of-view. Finite element method simulation of the endoscopic probe was conducted for various conditions to maximize its scanning range. After fabricating the probe using a double clad fiber, we obtained two-photon fluorescence images using raster beam scanning of the fiber. The outer diameter of the probe was 3.5 mm and its rigid distal length was 30 mm including a high numerical aperture gradient index lens. These features are sufficient for input into the instrumental channel of a commercial colonoscope or gastroscope to obtain high resolution images in vivo.

Optimal design and experiment of a three-axis out-of-plane nano positioning stage using a new compact bridge-type displacement amplifier.

This paper presents the development of a new compact three-axis compliant stage employing piezoelectric actuators and a new flexure structure. A proposed stage works out-of-plane (Z, θx, θy) direction. The stage consists of 4 amplification flexures mounted piezoelectric actuators. New structure of flexure reduces height and enhances dynamic performance of stage. To certify excellent performance of the stage, comparison accomplished between conventional amplification flexure and new compact bridge type flexure. Modeling and optimal design of new type nano positioning stage performed. The optimal design is executed on the geometric parameters of the proposed flexure structure using Sequential Quadratic Programming. Experiments are carried out to verify the static and dynamic performance of the stage. The proposed out-of-plane nano-positioning stage has a Z-directional motion range 190 µm and a θx, θy-directional motion range ±2 mrad. The resolution of the stage is 4 nm, 40 nrad, and 40 nrad in the Z-, θx-, and θy-directional motions, respectively. The size of stage is 150 × 150 × 30 mm(3).

Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

Chromatic confocal microscopy with a novel wavelength detection method using transmittance.

Chromatic confocal microscopy (CCM) is a promising technology that enables high-speed three-dimensional surface profiling without mechanical depth scanning. However, the spectrometer, which measures depth information encoded by axial color, limits the speed of three-dimensional imaging. We present a novel method for chromatic confocal microscopy with transmittance detection. Depth information can be instantaneously obtained by the ratio of intensity signals from two photomultiplier tubes by detecting a peak wavelength using transmittance of a color filter. This non-destructive and high-speed surface profiling method might be useful in many fields, including the semiconductor and flat panel display industries, and in material science.

Design and demonstration of multimodal optical scanning microscopy for confocal and two-photon imaging.

We developed a multimodal microscopy based on an optical scanning system in order to obtain diverse optical information of the same area of a sample. Multimodal imaging researches have mostly depended on a commercial microscope platform, easy to use but restrictive to extend imaging modalities. In this work, the beam scanning optics, especially including a relay lens, was customized to transfer broadband (400-1000 nm) lights to a sample without any optical error or loss. The customized scanning optics guarantees the best performances of imaging techniques utilizing the lights within the design wavelength. Confocal reflection, confocal fluorescence, and two-photon excitation fluorescence images were obtained, through respective implemented imaging channels, to demonstrate imaging feasibility for near-UV, visible, near-IR continuous light, and pulsed light in the scanning optics. The imaging performances for spatial resolution and image contrast were verified experimentally; the results were satisfactory in comparison with theoretical results. The advantages of customization, containing low cost, outstanding combining ability and diverse applications, will contribute to vitalize multimodal imaging researches.

Design and optimization of voice coil actuator for six degree of freedom active vibration isolation system using Halbach magnet array.

This paper describes the design, modeling, optimization, and validation of an active vibration isolation system using a voice coil motor. The active vibration isolating method was constructed with a passive isolator and an active isolator. A spring was used for passive isolating; an actuator was used for active isolating. The proposed active vibration isolation system (AVIS) can isolate disturbances for many kinds of instruments. Until now, developed AVIS were able to isolate a six degree-of-freedom disturbance effectively. This paper proposes the realization of such a six degree-of-freedom active vibration isolation system that can work as a bench top device for precision measuring machines such as atomic force microscope, scanning probe microscope, etc.

Spectrally resolved fluorescence lifetime imaging microscope using tunable bandpass filters.

A simple structure of spectral fluorescence lifetime imaging microscope (SLIM) is designed with the use of tunable bandpass filter, a kind of Fabry-perot filter that transmission wavelength is varying according to incident angle of light. Feasibility tests of this angle-tuned bandpass filter (ATBF) are performed and it shows high transmission and constant spectral bandwidth (20 nm) with respect to angle of incidence. Furthermore, using two ATBFs in series, spectral bandwidth can be adjustable down to 4 nm. In this paper, dual ATBFs are implemented to the detection part of fluorescence lifetime imaging microscope (FLIM) system so that we obtained spectrally resolved FLIM images. We compare these SLIM images with an original FLIM image and confirm that the former case provides high accuracy to analyze lifetime distribution as well as high contrast of images. The proposed SLIM microscope with good wavelength selectivity has many opportunities to utilize to other applications such as FLIM-Föster resonant energy transfer and autofluorescence imaging.

An integrated allele-specific polymerase chain reaction-microarray chip for multiplex single nucleotide polymorphism typing.

An integrated allele-specific polymerase chain reaction (AS PCR) and microarray chip has been developed for multiplex single nucleotide polymorphism (SNP) typing on a portable genetic analyzer instrumentation. We applied the integrated PCR-microarray system for on-site Hanwoo (Korean indigenous beef cattle) identification. Eleven sets of primers were designed, among which ten sets of primers targeted ten SNP loci to discriminate Hanwoo from the imported beef cattle and one primer set was used as a positive PCR control. The AS PCR for multiplex SNP typing was conducted on a glass-based microchip consisting of four layers: a microchannel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for micropump and microvalve function. The resultant AS PCR products were mixed with a hybridization buffer in a micromixer channel through the micropumping operation, and then the microarray assay was performed in the downstream process. Eleven duplicate probes were spotted in a glass slide, which was connected at the end of the micromixer channel unit. When the mixed solution was injected into the disposable microarray chip, pneumatically actuated micropumping was executed to speed up the hybridization process by inducing the convective flow. The fluorescence signals on each spot were monitored by a miniaturized fluorescence scanner, and the Hanwoo was verified by detecting the number of fluorescent spots with three or fewer among eleven. An integrated portable PCR-microarray genetic analysis microsystem was first demonstrated for rapid, accurate, and on-site multiplex SNP typing to differentiate animal species.

Development of a compact and long range XYθ(z) nano-positioning stage.

In this study, we describe the development of a novel, compact, and long range in-plane XYθ(z) nano-positioning stage with piezoelectric actuator and flexure mechanism. The stage is composed of an X-directional motion part and a Y, θ(z)-directional motion part, which are linked serially. The stage consists of a bridge-type amplifying mechanism for the amplification of deformation of the piezoelectric actuator, a double compound guide mechanism for performing only desired motion, and a circular hinge mechanism that permits rotational motion in the Y and θ(z)-stages. To set the design variables of the stage, optimal design is carried out. To verify the results of the optimal design process and the performance of the stage, the FEM simulation and experiment are carried out. The proposed XYθ(z) nano-positioning stage has a translational motion range of 700 µm and a rotational motion range of 0.3°; it has a closed-loop resolution of 5 nm, 5 nm, and 0.025 arcsec in the X-, Y-, and θ(z)-directional motions, respectively. The proposed stage is a novelty in that it has a compact size of 200 × 200 × 30 mm(3), and decoupled kinematic design.

Design and analysis of multi-color confocal microscopy with a wavelength scanning detector.

Spectral (or multi-color) microscopy has the ability to detect the fluorescent light of biological specimens with a broad range of wavelengths. Currently, the acousto-optic tunable filter (AOTF) is widely used in spectral microscopy as a substitute for a multiple-dichroic mirror to divide excitation and emission signals while maintaining sufficient light efficiency. In addition, systems which utilize an AOTF have a very fast switching speed and high resolution for wavelength selection. In this paper, confocal-spectral microscopy is proposed with a particular spectrometer design with a wavelength-scanning galvano-mirror. This enables the detection of broadband (480-700 nm) fluorescence signals by a single point detector (photomultiplier tube) instead of a CCD pixel array. For this purpose, a number of optical elements were applicably designed. A prism is used to amplify the dispersion angle, and the design of the relay optics matches the signals to the diameter of the wavelength-scanning galvano-mirror. Also, a birefringent material known as calcite is used to offset the displacement error at the image plane depending on the polarization states. The proposed multi-color confocal microscopy with the unique detection body has many advantages in comparison with commercial devices. In terms of the detection method, it can be easily applied to other imaging modalities.

Development of a novel 3-degrees of freedom flexure based positioning system.

Flexure mechanisms have been widely used for nanometer positioning systems. This article presents a novel conceptual design of an ultra-precision 3-degrees of freedom (XYθ(Z)) positioning system with nanometer precision. The main purpose of this novel stage design is for the application of measurement equipment, in particular biological specimens. The stage was designed as a hollow type and with a compact size for the inverted microscope. This stage includes piezoelectric transducer actuators, double compound amplification mechanisms, moving plate, and capacitor sensors. The double compound amplification mechanism was designed using a mathematical model and analyzed by the finite element method. Since the relationship between the variables of the hinge parameters and system performances are complicated, an optimization procedure was used to obtain the optimal design parameters, which maximized the system bandwidth. Based on the solution of the optimization problem, the design of the stage and FEM simulation results are presented. Finally, the stage was manufactured and tested.

Integrated allele-specific polymerase chain reaction-capillary electrophoresis microdevice for single nucleotide polymorphism genotyping.

An integrated allele-specific (AS) polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for multiplex single nucleotide polymorphism (SNP) genotyping on a portable instrumentation, which was applied for on-site identification of HANWOO (Korean indigenous beef cattle). Twelve sets of primers were designed for targeting beef cattle's eleven SNP loci for HANWOO verification and one primer set for a positive PCR control, and the success rate for identification of HANWOO was demonstrated statistically. The AS PCR and CE separation for multiplex SNP typing was carried out on a glass-based microchip consisting of four layers: a microchannel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for microvalve function. The operation of the sample loading, AS PCR, microvalve, and CE on a chip was automated with a portable genetic analyzer, and the laser-induced fluorescence detection was performed on a miniaturized fluorescence detector. The blind samples were correctly identified as a HANWOO by showing one or two amplicon peaks in the electropherogram, while the imported beef cattle revealed more than five peaks. Our genetic analysis platform provides rapid, accurate, and on-site multiplex SNP typing.

Development of flexure based 6-degrees of freedom parallel nano-positioning system with large displacement.

This paper details the development of a novel flexure jointed precision parallel nano-positioning system in combination with piezo-electric stepping motor for the application of precise optics alignment. The characteristics of the developed system are evaluated in this paper by the simulation and experiments. Based on the precision piezo-electric stepping motor and flexure joints, a high precision motion is obtained. Results of this paper include that of a translation resolution of 15 nm and a rotational resolution of 0.14 arc sec being achieved. In addition, the piezo-electric stepping motors provide a power-off hold characteristic to the system. Meanwhile, the parallel structure provides the high dynamic bandwidth of the lowest resonant frequency of 396.1 Hz. The symmetric structure is advantageous for thermal variation. To increase the motion range of the system, all of flexure joints are designed specially and the coupled workspace of ±2 mm × ±2 mm × ±2 mm × ±2° × ±2° × ±2° is achieved. The overall size of the designed system is Φ350 mm × 120 mm.