Intravital microscopic interrogation of peripheral taste sensation

Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo

The Effect of Static Stretch on Elastin Degradation in Arteries

Previously we have shown that gradual changes in the structure of elastin during an elastase treatment can lead to important transition stages in the mechanical behavior of arteries [1]. However, in vivo arteries are constantly being loaded due to systolic and diastolic pressures and so understanding the effects of loading on the enzymatic degradation of elastin in arteries is important. With biaxial tensile testing, we measured the mechanical behavior of porcine thoracic aortas digested with a mild solution of purified elastase (5 U/mL) in the presence of a static stretch. Arterial mechanical properties and biochemical composition were analyzed to assess the effects of mechanical stretch on elastin degradation. As elastin is being removed, the dimensions of the artery increase by more than 20% in both the longitude and circumference directions. Elastin assays indicate a faster rate of degradation when stretch was present during the digestion. A simple exponential decay fitting confirms the time constant for digestion with stretch (0.11±0.04 h−1) is almost twice that of digestion without stretch (0.069±0.028 h−1). The transition from J-shaped to S-shaped stress vs. strain behavior in the longitudinal direction generally occurs when elastin content is reduced by about 60%. Multiphoton image analysis confirms the removal/fragmentation of elastin and also shows that the collagen fibers are closely intertwined with the elastin lamellae in the medial layer. After removal of elastin, the collagen fibers are no longer constrained and become disordered. Release of amorphous elastin during the fragmentation of the lamellae layers is observed and provides insights into the process of elastin degradation. Overall this study reveals several interesting microstructural changes in the extracellular matrix that could explain the resulting mechanical behavior of arteries with elastin degradation

Light-guiding hydrogels for cell-based sensing and optogenetic synthesis in vivo

Polymer hydrogels are widely used as cell scaffolds for biomedical applications. While the biochemical and biophysical properties of hydrogels have been extensively investigated, little attention has been paid to their potential photonic functionalities. Here, we report cell-integrated polyethylene glycol-based hydrogels for in-vivo optical sensing and therapy applications. Hydrogel patches containing cells were implanted in awake, freely moving mice for several days and shown to offer long-term transparency, biocompatibility, cell-viability, and light-guiding properties (loss: <1 dB/cm). Using optogenetic, glucagon-like peptide-1 (GLP-1) secreting cells, we conducted light-controlled therapy using the hydrogel in a mouse model with type-2 diabetes and attained improved glucose homeostasis. Furthermore, real-time optical readout of encapsulated heat-shock-protein-coupled fluorescent reporter cells made it possible to measure the nanotoxicity of cadmium-based bare and shelled quantum dots (CdTe; CdSe/ZnS) in vivo

Toward biomaterial-based implantable photonic devices

Optical technologies are essential for the rapid and efficient delivery of health care to patients. Efforts have begun to implement these technologies in miniature devices that are implantable in patients for continuous or chronic uses. In this review, we discuss guidelines for biomaterials suitable for use in vivo. Basic optical functions such as focusing, reflection, and diffraction have been realized with biopolymers. Biocompatible optical fibers can deliver sensing or therapeutic-inducing light into tissues and enable optical communications with implanted photonic devices. Wirelessly powered, light-emitting diodes (LEDs) and miniature lasers made of biocompatible materials may offer new approaches in optical sensing and therapy. Advances in biotechnologies, such as optogenetics, enable more sophisticated photonic devices with a high level of integration with neurological or physiological circuits. With further innovations and translational development, implantable photonic devices offer a pathway to improve health monitoring, diagnostics, and light-activated therapies. Keywords: biomaterials; biocompatible; biodegradable; optics; photonicsUnited States. Department of Defense (Award FA9550-13-1-0068)National Institutes of Health (U.S.) (Award P41-EB015903)National Institutes of Health (U.S.) (Award R01-CA192878)National Science Foundation (U.S.) (Award CBET-1264356)National Science Foundation (U.S.) (Award ECCS-1505569

On the near-wall accumulation of injectable particles in the microcirculation: smaller is not better

Although most nanofabrication techniques can control nano/micro particle (NMP) size over a wide range, the majority of NMPs for biomedical applications exhibits a diameter of ~100 nm. Here, the vascular distribution of spherical particles, from 10 to 1,000 nm in diameter, is studied using intravital microscopy and computational modeling. Small NMPs (≤100 nm) are observed to move with Red Blood Cells (RBCs), presenting an uniform radial distribution and limited near-wall accumulation. Larger NMPs tend to preferentially accumulate next to the vessel walls, in a size-dependent manner (~70% for 1,000 nm NMPs). RBC-NMP geometrical interference only is responsible for this behavior. In a capillary flow, the effective radial dispersion coefficient of 1,000 nm particles is ~3-fold larger than Brownian diffusion. This suggests that sub-micron particles could deposit within diseased vascular districts more efficiently than conventional nanoparticles

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

A novel repeat sequence-based PCR (rep-PCR) using specific repeat sequences of Mycobacterium intracellulare as a DNA fingerprinting

Repetitive sequence-based PCR (rep-PCR) is a potential epidemiological technique that can provide high-throughput genotype fingerprints of heterogeneous Mycobacterium strains rapidly. Previously published rep-PCR primers, which are based on nucleotide sequences of Gram-negative bacteria may have low specificity for mycobacteria. Moreover, it was difficult to ensure the continuity of the study after the commercial rep-PCR kit was discontinued. Here, we designed a novel rep-PCR for Mycobacterium intracellulare, a major cause of nontuberculous mycobacterial pulmonary disease with frequent recurrence. We screened the 7,645 repeat sequences for 200 fragments from the genome of M. intracellulare ATCC 13950 in silico, finally generating five primers with more than 90% identity for a total of 226 loci in the genome. The five primers could make different band patterns depending on the genome of three different M. intracellulare strains using an in silico test. The novel rep-PCR with the five primers was conducted using 34 bacterial samples of 7 species containing 25 M. intracellulare clinical isolates, compared with previous published rep-PCRs. This shows distinguished patterns depending on species and blotting assay for 6 species implied the sequence specificity of the five primers. The Designed rep-PCR had a 95–98% of similarity value in the reproducibility test and showed 7 groups of fingerprints in M. intracellulare strains. Designed rep-PCR had a correlation value of 0.814 with VNTR, reference epidemiological method. This study provides a promising genotype fingerprinting method for tracing the recurrence of heterogeneous M. intracellulare

Bioabsorbable polymer optical waveguides for deep-tissue photomedicine

Advances in photonics have stimulated significant progress in medicine, with many techniques now in routine clinical use. However, the finite depth of light penetration in tissue is a serious constraint to clinical utility. Here we show implantable light-delivery devices made of bio-derived or biocompatible, and biodegradable polymers. In contrast to conventional optical fibres, which must be removed from the body soon after use, the biodegradable and biocompatible waveguides may be used for long-term light delivery and need not be removed as they are gradually resorbed by the tissue. As proof of concept, we demonstrate this paradigm-shifting approach for photochemical tissue bonding (PTB). Using comb-shaped planar waveguides, we achieve a full thickness (>10 mm) wound closure of porcine skin, which represents ∼10-fold extension of the tissue area achieved with conventional PTB. The results point to a new direction in photomedicine for using light in deep tissues

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