Thermally Managed, Injectable Optoelectronic Probe with Heat Dissipation Guide for Photodynamic Therapy.

The development of fabrication technologies and appearance of new materials has resulted in dramatic increase in the performance of electronic devices, while the overall size has decreased. Recent electronic devices made of micro/nano-size components show high efficiency and outstanding performance with compact size, but these devices have revealed several fatal problems. In particular, the isolated heat that is generated by numerous components concentrated in a limited small area at high density, such as bio-integrated devices, is an issue that needs to be urgently addressed, because it is closely related to the performance and lifetime of electronic devices. To solve these problems, the microscale light emitting diode (µLED)-based neural probe is introduced on an injectable heat dissipation guide. The heat dissipation guide is made of boron nitride (BN) nanomaterials with high thermal conductivity. The heat management noticeably improves the optical output performance of the µLEDs, in which BN effectively dissipates heat, and allows enhanced lighting from the LEDs to be transmitted through brain tissue without thermal damage. Moreover, it shows remarkable improvement in the therapeutic effect of photodynamic therapy of mouse cancer cells.

Antioxidant and antibacterial effects of medicinal plants and their stick-type medicinal concentrated beverages.

Nine medicinal plants and their stick-type medicinal concentrated beverages (SMCB-I and SMCB-II) with different combination ratio were evaluated on antioxidant, nitric oxide (NO) inhibitory, and antibacterial effects against pathogenic bacteria involved in respiratory system illnesses. Antioxidant activity was high in Syzygium aromaticum, Pueraria lobata, Plantago asiatica, and Kalopanax pictus which have higher contents of total phenolics and total flavonoids. The NO inhibitory activity was high in Syzygium aromaticum, Plantago asiatica, and Glycyrrhiza uralensis. Syzygium aromaticum, Plantago asiatica, Kalopanax pictus and Glycyrrhiza uralensis showed higher antibacterial activity than the other five medicinal plants against Staphylococcus aureus, Corynebacterium diphtheriae, and Mycobacterium sp. SMCB-II exhibited higher antioxidant, NO inhibitory, and antibacterial effects than SMCB-I, since Syzygium aromaticum, Pueraria lobata, and Kalopanax pictus were only used for the production of SMCB-II. The SMCBs would be expected to contribute to an easy-to-carry, easy-to-consume, and high value-added health beverage for the modern people.

Rapid and Selective Targeting of Heterogeneous Pancreatic Neuroendocrine Tumors.

Design of tissue-specific contrast agents to delineate tumors from background tissues is a major unmet clinical need for ultimate surgical interventions. Bioconjugation of fluorophore(s) to a ligand has been mainly used to target overexpressed receptors on tumors. However, the size of the final targeted ligand can be large, >20 kDa, and cannot readily cross the microvasculature to meet the specific tissue, resulting in low targetability with a high background. Here, we report a small and hydrophilic phenoxazine with high targetability and retention to pancreatic neuroendocrine tumor. This bioengineered fluorophore permits sensitive detection of ultrasmall (<0.5 mm) ectopic tumors within a few seconds after a single bolus injection, highlighting every tumor in the pancreas from the surrounding healthy tissues with reasonable half-life. The knowledge-based approach and validation used to develop structure-inherent tumor-targeted fluorophores have a tremendous potential to improve treatment outcome by providing definite tumor margins for image-guided surgery.

Microsphere-based interferometric optical probe.

Fluorescent optical probes have rapidly transformed our understanding of complex biological systems by providing specific information on biological targets in the natural living state. However, their utility is often limited by insufficient brightness, photostability, and multiplexing capacity. Here, we report a conceptually new optical probe, termed 'reflectophore', which is based on the spectral interference from a dielectric microsphere. Reflectophores are orders-of-magnitudes brighter than conventional fluorophores and are free from photobleaching, enabling practically unlimited readout at high fidelity. They also offer high-degree multiplexing, encoded in their optical size, which can be readily decoded through interferometric detection with nanoscale accuracy, even in turbid biological media. Furthermore, we showcase their biological applications in cellular barcoding and microenvironmental sensing of a target protein and local electric field.

Optical lens-microneedle array for percutaneous light delivery.

The limited penetration depth of light in skin tissues is a practical bottleneck in dermatologic applications of light-induced therapies, including anti-microbial blue light therapy and photodynamic skin cancer therapy. Here, we demonstrate a novel device, termed optical microneedle array (OMNA), for percutaneous light delivery. A prototype device with a 11 by 11 array of needles at a spacing of 1 mm and a length of 1.6 mm was fabricated by press-molding poly-(lactic acid) (PLA) polymers. The device also incorporates a matched microlens array that focuses the light through the needle tips at specific points to achieve an optimal intensity profile in the tissue. In experiments done with bovine tissues, the OMNA enabled us to deliver a total of 7.5% of the input photons at a wavelength of 491 nm, compared to only 0.85% without the device. This 9-fold enhancement of light delivery was close to the prediction of 10.8 dB by ray-tracing simulation and is expected to increase the effective treatment depth of anti-microbial blue light therapy significantly from 1.3 to 2.5 mm in human skin.

Two-photon excited photoconversion of cyanine-based dyes.

The advent of phototransformable fluorescent proteins has led to significant advances in optical imaging, including the unambiguous tracking of cells over large spatiotemporal scales. However, these proteins typically require activating light in the UV-blue spectrum, which limits their in vivo applicability due to poor light penetration and associated phototoxicity on cells and tissue. We report that cyanine-based, organic dyes can be efficiently photoconverted by nonlinear excitation at the near infrared (NIR) window. Photoconversion likely involves singlet-oxygen mediated photochemical cleavage, yielding blue-shifted fluorescent products. Using SYTO62, a biocompatible and cell-permeable dye, we demonstrate photoconversion in a variety of cell lines, including depth-resolved labeling of cells in 3D culture. Two-photon photoconversion of cyanine-based dyes offer several advantages over existing photoconvertible proteins, including use of minimally toxic NIR light, labeling without need for genetic intervention, rapid kinetics, remote subsurface targeting, and long persistence of photoconverted signal. These findings are expected to be useful for applications involving rapid labeling of cells deep in tissue.

Selective two-photon collagen crosslinking in situ measured by Brillouin microscopy.

Two-photon polymerization has enabled precise microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, and cellular scaffolds. We present two-photon collagen crosslinking (2P-CXL) of intact corneal tissue using riboflavin and femtosecond laser irradiation. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Measurement of local changes in longitudinal mechanical moduli with confocal Brillouin microscopy enabled the visualization of the cross-linked pattern without perturbation of the surrounding non-irradiated regions. 2P-CXL induced stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high resolution with broad implications in ophthalmology, laser surgery, and tissue engineering.

Bioorthogonal Click Chemistry-Based Synthetic Cell Glue.

Artificial methods of cell adhesion can be effective in building functional cell complexes in vitro, but methods for in vivo use are currently lacking. Here, a chemical cell glue based on bioorthogonal click chemistry with high stability and robustness is introduced. Tetrazine (Tz) and trans-cyclooctene (TCO) conjugated to the cell surface form covalent bonds between cells within 10 min in aqueous conditions. Glued, homogeneous, or heterogeneous cell pairs remain viable and stably attached in a microfluidic flow channel at a shear stress of 20 dyn cm(-2) . Upon intravenous injection of assembled Jurkat T cells into live mice, fluorescence microscopy shows the trafficking of cell pairs in circulation and their infiltration into lung tissues. These results demonstrate the promising potential of chemically glued cell pairs for various applications ranging from delivering therapeutic cells to studying cell-cell interactions in vivo.

In vivo imaging of Lgr5-positive cell populations using confocal laser endomicroscopy during early colon tumorigenesis.

BACKGROUND AND STUDY AIMS: A diagnostic molecular marker for pre-neoplastic lesions, particularly before polyposis, is still lacking. Lgr5 has been broadly accepted as a marker for intestinal cancer stem cells. The aim of this study was to investigate the monitoring of Lgr5( + ) cells as a useful tool for the early diagnosis of premalignant lesions before polyp formation. METHODS: In vivo molecular imaging was performed to examine colon tumorigenesis in Lgr5-EGFP mice treated with azoxymethane and dextran sodium sulfate. eGFP( +) Lgr5( +) regions in the descending colon were longitudinally monitored using side-view confocal endomicroscopy. Based on the eGFP signal intensity on the luminal surface, polyps were classified into two groups - Lgr5-high and Lgr5-low. White light colonoscopy was used to monitor polyp formation. RESULTS: Approximately 75 % of the polyps originated from foci containing Lgr5-eGFP( +) cells, whereas 25 % of the polyps emerged from Lgr5( -) foci. Among eGFP( +) foci, Lgr5-high foci grew faster than Lgr5-low foci. CONCLUSIONS: Polyps developed at Lgr5( +) regions. Luminal Lgr5 expression was correlated with the growth rate of early-stage adenomas. Lgr5 is a promising molecular marker for the early diagnosis of colon tumors.

Card9 mediates intestinal epithelial cell restitution, T-helper 17 responses, and control of bacterial infection in mice.

BACKGROUND & AIMS: Caspase recruitment domain 9 (CARD9) is an adaptor protein that integrates signals downstream of pattern recognition receptors. CARD9 has been associated with autoinflammatory disorders, and loss-of-function mutations have been associated with chronic mucocutaneous candidiasis, but the role of CARD9 in intestinal inflammation is unknown. We characterized the role of Card9 in mucosal immune responses to intestinal epithelial injury and infection. METHODS: We induced intestinal inflammation in Card9-null mice by administration of dextran sulfate sodium (DSS) or Citrobacter rodentium. We analyzed body weight, assessed inflammation by histology, and measured levels of cytokines and chemokines using quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Cell populations were compared between wild-type and Card9-null mice by flow cytometry analysis. RESULTS: Colon tissues and mesenteric lymph nodes of Card9-null mice had reduced levels of interleukin (IL)-6, interferon-γ, and T-helper (Th)17 cytokines after administration of DSS, compared with wild-type mice. IL-17A and IL-22 expression were reduced in the recovery phase after DSS administration, coincident with decreased expression of antimicrobial peptides and the chemokine (C-C motif) ligand 20 (Ccl20). Although Card9-null mice had more intestinal fungi based on 18S analysis, their Th17 responses remained defective even when an antifungal agent was administered throughout DSS exposure. Moreover, Card9-null mice had impaired immune responses to C rodentium, characterized by decreased levels of colonic IL-6, IL-17A, IL-22, and regenerating islet-derived 3 gamma (RegIIIγ), as well as fewer IL-22-producing innate lymphoid cells (ILCs) in colon lamina propria. CONCLUSIONS: The adaptor protein CARD9 coordinates Th17- and innate lymphoid cell-mediated intestinal immune responses after epithelial injury in mice.

In vivo femtosecond endosurgery: an intestinal epithelial regeneration-after-injury model.

Regeneration of the intestinal epithelium after injury or during pathogenesis is a dynamic cellular process critical for host immunity. However, current epithelial injury models provide poor spatial control, complicating the study of precise cellular responses. Here we developed endoscopic femtosecond-laser surgery capable of generating acute tissue injury. A side-view probe provides a convenient access to the distal colon in the mouse in vivo and allows real-time intraoperative monitoring as well as pre- and post-surgery examinations via multiphoton imaging. The photo-induced damage showed a nonlinear dependence on laser intensity. At an optical power of 200 mW (2.5 nJ per pulse), scanning the beam focus over 300x300 µm(2) area in the colonic mucosa generated substantial vascular damages within 30 s. We confirmed the localized tissue damage and the physiologic regeneration of the disrupted epithelium by in situ barrier function assays, validating the animal model for epithelial regeneration following injury. The femtosecond endosurgery technique is applicable to various experimental models based on laser-induced perturbations.

Fabrication and operation of GRIN probes for in vivo fluorescence cellular imaging of internal organs in small animals.

Intravital fluorescence microscopy has emerged as a powerful technique to visualize cellular processes in vivo. However, owing to their size, the objective lenses required have limited physical accessibility to various tissue sites in the internal organs of small animals. The use of small-diameter probes using graded-index (GRIN) lenses expands the capabilities of conventional intravital microscopes to minimally invasive imaging of internal organs. In this protocol, we describe the detailed steps for the fabrication of front- and side-view GRIN probes and the integration and operation of the probes in a confocal microscope to enable visualization of fluorescent cells and microvasculature in various mouse organs. Some experience in building an optical setup is required to complete the protocol. We also present longitudinal imaging of immune cells in renal allografts and tumor development in the colon. Fabrication and integration can be completed in 5-7 h, and a typical in vivo imaging session takes 1-2 h.

The ß-glucan receptor Dectin-1 activates the integrin Mac-1 in neutrophils via Vav protein signaling to promote Candida albicans clearance.

Resistance to fungal infections is attributed to engagement of host pattern-recognition receptors, notably the ß-glucan receptor Dectin-1 and the integrin Mac-1, which induce phagocytosis and antifungal immunity. However, the mechanisms by which these receptors coordinate fungal clearance are unknown. We show that upon ligand binding, Dectin-1 activates Mac-1 to also recognize fungal components, and this stepwise process is critical for neutrophil cytotoxic responses. Both Mac-1 activation and Dectin-1- and Mac-1-induced neutrophil effector functions require Vav1 and Vav3, exchange factors for RhoGTPases. Mac-1- or Vav1,3-deficient mice have increased susceptibility to systemic candidiasis that is not due to impaired neutrophil recruitment but defective intracellular killing of C. albicans yeast forms, and Mac-1 or Vav1,3 reconstitution in hematopoietic cells restores resistance. Our results demonstrate that antifungal immunity depends on Dectin-1-induced activation of Mac-1 functions that is coordinated by Vav proteins, a pathway that may localize cytotoxic responses of circulating neutrophils to infected tissues.

Blockade of vascular endothelial growth factor sensitizes tumor-associated vasculatures to angiolytic therapy with a high-frequency ultrashort pulsed laser.

Because of high spatial resolution and superior tissue penetration, a femtosecond laser of the near-infrared spectrum has great potential to improve the efficacy of conventional photodynamic therapy; however, the lack of suitable photosensitizers has so far limited its bedside applications. Recently, our group reported that a brief irradiation by femtosecond lasers in the absence of exogenous probes can modulate various cellular behaviors in vitro and in vivo. Here, we demonstrate that targeted irradiation by a femtosecond laser disrupted tumor-associated blood vessels, and the inhibition of vascular endothelial growth factor signaling augmented the efficacy of laser-induced angiolysis. Further, we show that reactive oxygen species (ROS) are generated in response to laser irradiation, and reducing the intracellular levels of ROS rendered endothelial cells resistant to laser-induced cytotoxicity. Collectively, these results indicate that a femtosecond laser can be used as a vascular-disrupting therapeutic modality for cancer treatment, especially when used in combination with conventional anti-angiogenic therapies.

Dynamic fluorescence imaging for multiparametric measurement of tumor vasculature.

Angiogenesis is essential for tumor growth and a promising target for cancer therapy. Blood vessel monitoring is an indispensable tool for evaluation and development of anti-angiogenic drugs. Here, we report a new noninvasive in vivo imaging tool, named dynamic fluorescence imaging (DyFI), for the simultaneous measurement of multiple vascular parameters including vascular density, perfusion rate, and permeability using spatiotemporal profiles of indocyanine green. Using DyFI in a tumor xenograft model, we quantitatively measured multiple vascular parameters in tumors and normal tissues with high spatial resolution. The multimodality of this method allowed us to find negative spatial correlations between perfusion and permeability. Moreover, DyFI was effective for revealing the early effects of an anti-angiogenic drug. We suggest that DyFI could be a useful tool for the preclinical development of anti-angiogenic drugs.

Quantitative analysis of peripheral tissue perfusion using spatiotemporal molecular dynamics.

BACKGROUND: Accurate measurement of peripheral tissue perfusion is challenging but necessary to diagnose peripheral vascular insufficiency. Because near infrared (NIR) radiation can penetrate relatively deep into tissue, significant attention has been given to intravital NIR fluorescence imaging. METHODOLOGY/PRINCIPAL FINDINGS: We developed a new optical imaging-based strategy for quantitative measurement of peripheral tissue perfusion by time-series analysis of local pharmacokinetics of the NIR fluorophore, indocyanine green (ICG). Time-series NIR fluorescence images were obtained after injecting ICG intravenously in a murine hindlimb ischemia model. Mathematical modeling and computational simulations were used for translating time-series ICG images into quantitative pixel perfusion rates and a perfusion map. We could successfully predict the prognosis of ischemic hindlimbs based on the perfusion profiles obtained immediately after surgery, which were dependent on the preexisting collaterals. This method also reflected increases in perfusion and improvements in prognosis of ischemic hindlimbs induced by treatment with vascular endothelial growth factor and COMP-angiopoietin-1. CONCLUSIONS/SIGNIFICANCE: We propose that this novel NIR-imaging-based strategy is a powerful tool for biomedical studies related to the evaluation of therapeutic interventions directed at stimulating angiogenesis.