A Funnel Type PVDF Underwater Energy Harvester with Spiral Structure Mounted on the Harvester Support.

For the purpose of stably supplying electric power to the underwater wireless sensor, the energy harvesting technology in which a voltage is obtained by generating displacement in a piezoelectric material using flow-induced vibration is one of the most attractive research fields. The funnel type energy harvester (FTEH) with PVDF proposed in this study is an energy harvester in which the inlet has a larger cross-sectional area than the outlet and a spiral structure is inserted to generate a vortex flow at the inlet. Based on numerical analysis, when PVDF with L = 100 mm and t = 1 mm was used, the electric power of 39 µW was generated at flow velocity of 0.25 m/s. In experiment the average RMS voltage of FTEH increased by 0.0209 V when the flow velocity increased by 1 m/s. When measured at 0.25 m/s flow velocity for 25 s, it was shown that voltage doubler rectifier (VDR) generated a voltage of 133.4 mV, 2.25 times larger than that of full bridge rectifier (FBR), and the energy charged in the capacitor was 44.3 nJ, 14% higher in VDR than that of the FBR. In addition, the VDR can deliver power of 17.75 µW for 1 kΩ load. It is shown that if the voltage generated by the FTEH using the flow velocity is stored using the VDR electric circuit, it will greatly contribute to the stable power supply of the underwater wireless sensor.

Quantitative two-photon microscopy imaging analysis of human skin to evaluate enhanced transdermal delivery by hybrid-type multi-lamellar nanostructure: retraction.

[This retracts the article on p. 3974 in vol. 9, PMID: 30338168.].

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.

In vivo longitudinal depth-wise visualization of tumorigenesis by needle-shaped side-view confocal endomicroscopy.

In vivo, longitudinal observation of tumorigenesis in a live mouse model over an extended time period has been actively pursued to obtain a better understanding of the cellular and molecular mechanism in a highly complex tumor microenvironment. However, common intravital imaging approaches based on a conventional laser scanning confocal or a two-photon microscope have been mostly limited to the observation of superficial parts of the solid tumor tissue. In this work, we implemented a small diameter needle-shaped side-view confocal endomicroscope that can be directly inserted into a solid tumor in a minimally-invasive manner in vivo. By inserting the side-view endomicroscope into the breast tumor from the surface, we achieved in vivo depth-wise cellular-level visualization of microvasculature and fluorescently labeled tumor cells located deeply inside the tumor. In addition, we successfully performed longitudinal depth-wise visualization of a growing breast tumor over three weeks in a live mouse model, which revealed dynamic changes in microvasculature such as a decreasing amount of intratumoral blood vessels over time.

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.

Neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury.

The lung is highly vulnerable during sepsis, yet its functional deterioration accompanied by disturbances in the pulmonary microcirculation is poorly understood. This study aimed to investigate how the pulmonary microcirculation is distorted in sepsis-induced acute lung injury (ALI) and reveal the underlying cellular pathophysiologic mechanism.Using a custom-made intravital lung microscopic imaging system in a murine model of sepsis-induced ALI, we achieved direct real-time visualisation of the pulmonary microcirculation and circulating cells in vivo We derived the functional capillary ratio (FCR) as a quantitative parameter for assessing the fraction of functional microvasculature in the pulmonary microcirculation and dead space.We identified that the FCR rapidly decreases in the early stage of sepsis-induced ALI. The intravital imaging revealed that this decrease resulted from the generation of dead space, which was induced by prolonged neutrophil entrapment within the capillaries. We further showed that the neutrophils had an extended sequestration time and an arrest-like dynamic behaviour, both of which triggered neutrophil aggregates inside the capillaries and arterioles. Finally, we found that Mac-1 (CD11b/CD18) was upregulated in the sequestered neutrophils and that a Mac-1 inhibitor restored the FCR and improved hypoxaemia.Using the intravital lung imaging system, we observed that Mac-1-upregulated neutrophil aggregates led to the generation of dead space in the pulmonary microcirculation that was recovered by a Mac-1 inhibitor in sepsis-induced ALI.

Quantitative two-photon microscopy imaging analysis of human skin to evaluate enhanced transdermal delivery by hybrid-type multi-lamellar nanostructure.

Transdermal skin delivery is a method to transport various topical formulations to a deeper skin layer non-invasively. Permeability analysis of many delivering agents has been mostly conducted by a simple tape stripping method. However, it cannot reveal a detailed depth-dependent distribution profile of transdermally delivered agents in the skin. In this work, we achieved a cellular-level depth-defined visualization of fluorophore-labelled human epidermal growth factor (EGF) transdermally delivered to human skin by using encapsulation with common liposomes and newly fabricated multi-lamellar nanostructures using a custom-design two-photon microscopy system. It was able to generate 3D reconstructed images displaying the distribution of human EGF inside the human skin sample with high-resolution. Based on a depthwise fluorescence intensity profile showing the permeation of human EGF, a quantitative analysis was performed to assess the transdermal delivery efficacy achieved by each formulation, showing a significant improvement of the efficacy with the utilization of multi-lamellar nanostructure.

Nanoparticle-Assisted Transcutaneous Delivery of a Signal Transducer and Activator of Transcription 3-Inhibiting Peptide Ameliorates Psoriasis-like Skin Inflammation.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in psoriatic skin inflammation and acts as a key player in the pathogenesis and progression of this autoimmune disease. Although numerous inhibitors that intervene in STAT3-associated pathways have been tested, an effective, highly specific inhibitor of STAT3 has yet to be identified. Here, we evaluated the in vitro and in vivo biological activity and therapeutic efficacy of a high-affinity peptide specific for STAT3 (APTstat3) after topical treatment via intradermal and transcutaneous delivery. Using a preclinical model of psoriasis, we show that intradermal injection of APTstat3 tagged with a 9-arginine cell-penetrating peptide (APTstat3-9R) reduced disease progression and modulated psoriasis-related cytokine signaling through inhibition of STAT3 phosphorylation. Furthermore, by complexing APTstat3-9R with specific lipid formulations led to formation of discoidal lipid nanoparticles (DLNPs), we were able to achieve efficient skin penetration of the STAT3-inhibiting peptide after transcutaneous administration, thereby effectively inhibiting psoriatic skin inflammation. Collectively, these findings suggest that DLNP-assisted transcutaneous delivery of a STAT3-inhibiting peptide could be a promising strategy for treating psoriatic skin inflammation without causing adverse systemic events. Moreover, the DLNP system could be used for transdermal delivery of other therapeutic peptides.

Intravital imaging of a pulmonary endothelial surface layer in a murine sepsis model.

Direct intravital imaging of an endothelial surface layer (ESL) in pulmonary microcirculation could be a valuable approach to investigate the role of a vascular endothelial barrier in various pathological conditions. Despite its importance as a marker of endothelial cell damage and impairment of the vascular system, in vivo visualization of ESL has remained a challenging technical issue. In this work, we implemented a pulmonary microcirculation imaging system integrated to a custom-design video-rate laser scanning confocal microscopy platform. Using the system, a real-time cellular-level microscopic imaging of the lung was successfully performed, which facilitated a clear identification of individual flowing erythrocytes in pulmonary capillaries. Subcellular level pulmonary ESL was identified in vivo by fluorescence angiography using a dextran conjugated fluorophore to label blood plasma and the red blood cell (RBC) exclusion imaging analysis. Degradation of ESL width was directly evaluated in a murine sepsis model in vivo, suggesting an impairment of pulmonary vascular endothelium and endothelial barrier dysfunction.

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.

In vivo cellular-level real-time pharmacokinetic imaging of free-form and liposomal indocyanine green in liver.

Indocyanine green (ICG) is a near-infrared fluorophore approved for human use which has been widely used for various clinical applications. Despite the well-established clinical usage, our understanding about the microscopic in vivo pharmacokinetics of systemically administered ICG has been relatively limited. In this work, we successfully visualized real-time in vivo pharmacokinetic dynamics of the intravenously injected free-form and liposomal ICG in cellular resolution by utilizing a custom-built video-rate near infrared laser-scanning confocal microscopy system. Initial perfusion and clearance from blood stream, diffusion into perisinusoidal space, and subsequent absorption into hepatocyte were directly visualized in vivo. The quantification analysis utilizing the real-time image sequences revealed distinct dynamic in vivo pharmacokinetic behavior of free-form and liposomal ICG.

In vivo longitudinal cellular imaging of small intestine by side-view endomicroscopy.

Visualization of cellular dynamics in the gastrointestinal tract of living mouse model to investigate the pathophysiology has been a long-pursuing goal. Especially, for chronic disease such as Crohn's disease, a longitudinal observation of the luminal surface of the small intestine in the single mouse is highly desirable to investigate the complex pathogenesis in sequential time points. In this work, by utilizing a micro-GRIN lens based side-view endomicroscope integrated into a video-rate confocal microscopy system, we successfully performed minimally-invasive in vivo cellular-level visualization of various fluorescent cells and microvasculature in the small intestinal villi. Also, with a transgenic mouse universally expressing photoconvertible protein, Kaede, we demonstrated repetitive cellular-level confocal endoscopic visualization of same area in the small intestinal lumen of a single mouse, which revealed the continuous homeostatic renewal of the small intestinal epithelium.

Optical clearing based cellular-level 3D visualization of intact lymph node cortex.

Lymph node (LN) is an important immune organ that controls adaptive immune responses against foreign pathogens and abnormal cells. To facilitate efficient immune function, LN has highly organized 3D cellular structures, vascular and lymphatic system. Unfortunately, conventional histological analysis relying on thin-sliced tissue has limitations in 3D cellular analysis due to structural disruption and tissue loss in the processes of fixation and tissue slicing. Optical sectioning confocal microscopy has been utilized to analyze 3D structure of intact LN tissue without physical tissue slicing. However, light scattering within biological tissues limits the imaging depth only to superficial portion of LN cortex. Recently, optical clearing techniques have shown enhancement of imaging depth in various biological tissues, but their efficacy for LN are remained to be investigated. In this work, we established optical clearing procedure for LN and achieved 3D volumetric visualization of the whole cortex of LN. More than 4 times improvement in imaging depth was confirmed by using LN obtained from H2B-GFP/actin-DsRed double reporter transgenic mouse. With adoptive transfer of GFP expressing B cells and DsRed expressing T cells and fluorescent vascular labeling by anti-CD31 and anti-LYVE-1 antibody conjugates, we successfully visualized major cellular-level structures such as T-cell zone, B-cell follicle and germinal center. Further, we visualized the GFP expressing metastatic melanoma cell colony, vasculature and lymphatic vessels in the LN cortex.

In vivo analysis of THz wave irradiation induced acute inflammatory response in skin by laser-scanning confocal microscopy.

The recent development of THz sources in a wide range of THz frequencies and power levels has led to greatly increased interest in potential biomedical applications such as cancer and burn wound diagnosis. However, despite its importance in realizing THz wave based applications, our knowledge of how THz wave irradiation can affect a live tissue at the cellular level is very limited. In this study, an acute inflammatory response caused by pulsed THz wave irradiation on the skin of a live mouse was analyzed at the cellular level using intravital laser-scanning confocal microscopy. Pulsed THz wave (2.7 THz, 4 µs pulsewidth, 61.4 µJ per pulse, 3Hz repetition), generated using compact FEL, was used to irradiate an anesthetized mouse's ear skin with an average power of 260 mW/cm(2) for 30 minutes using a high-precision focused THz wave irradiation setup. In contrast to in vitro analysis using cultured cells at similar power levels of CW THz wave irradiation, no temperature change at the surface of the ear skin was observed when skin was examined with an IR camera. To monitor any potential inflammatory response, resident neutrophils in the same area of ear skin were repeatedly visualized before and after THz wave irradiation using a custom-built laser-scanning confocal microscopy system optimized for in vivo visualization. While non-irradiated control skin area showed no changes in the number of resident neutrophils, a massive recruitment of newly infiltrated neutrophils was observed in the THz wave irradiated skin area after 6 hours, which suggests an induction of acute inflammatory response by the pulsed THz wave irradiation on the skin via a non-thermal process.

Gradient index lens based combined two-photon microscopy and optical coherence tomography.

We report a miniaturized probe-based combined two-photon microscopy (TPM) and optical coherence tomography (OCT) system. This system is to study the colorectal cancer in mouse models by visualizing both cellular and structural information of the colon in 3D with TPM and OCT respectively. The probe consisted of gradient index (GRIN) lenses and a 90° reflecting prism at its distal end for side-viewing, and it was added onto an objective lens-based TPM and OCT system. The probe was 2.2 mm in diameter and 60 mm in length. TPM imaging was performed by raster scanning of the excitation focus at the imaging speed of 15.4 frames/s. OCT imaging was performed by combining the linear sample translation and probe rotation along its axis. This miniaturized probe based dual-modal system was characterized with tissue phantoms containing fluorescent microspheres, and applied to image mouse colonic tissues ex vivo as a demonstration. As OCT and TPM provided structural and cellular information of the tissues respectively, this probe based multi-modal imaging system can be helpful for in vivo studies of preclinical animal models such as mouse colonic tumorigenesis.